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Developing a porcine slaughterhouse model for normothermic regional perfusion of donor kidneys Student Vera Tichelaar Faculty supervisor prof dr HGD Leuvenink Daily supervisor LH Venema University Medical Center Groningen Department of Surgery ndash Surgical Research Laboratory 01-09-2016
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Abstractsamenvatting Objectives To decrease waiting time for a kidney transplant donor kidneys of inferior quality
are increasingly being accepted Normothermic regional perfusion (NRP) in donation after
circulatory death restores abdominal circulation with autologous blood and improves organ
quality The use of blood in machine perfusion can cause inflammation injury tissues damage
and cell death Probably it is better to replace the blood in NRP with an artificial preservation
solution To test the different solutions a normothermic machine perfusion model for an
isolated kidney has to be designed The aim of this study is to design a NMP model with
porcine slaughterhouse kidneys to test kidney viability
Methods Porcine kidneys and autologous whole blood were obtained from the
slaughterhouse Kidneys were transported to our lab and reperfused for 4 hours using the
NMP set-up Seven groups were created (n=1-4) Kidneys were transported differently using
cold storage subnormothermic machineperfusion oxygenated- or non-oxygenated
hypothermic machine perfusion Warm and cold ischeamic times differ between groups In
one group mannitol insulin nutrients and dexamethson was added during NMP (NMP+)
Hemodynamics were monitored and perfusate and urine samples were taken regularly
Biopsies were taken to asses renal histology
Results A stable NMP was established There was a significant difference between groups
Kidneys preserved using oxygenated hypothermic machine perfusion and reperfused with
NMP+ performed significantly better These kidneys showed better creatinine clearance
sodium reabsorption and urine production than the control group
Conclusion The model created using oxygenated hypothermic machine perfusion and NMP+
likely is useful for testing different perfusion solutions However more research is required to
optimize this model
Doelstellingen Om wachttijd voor een niertransplantatie te verkorten worden donornieren
van mindere kwaliteit in toenemende mate geaccepteerd Normotherme regionale perfusie
(NRP) na donatie na hartdood hersteld de abdominale circulatie met autoloog bloed en
verbeterd de orgaan kwaliteit Het gebruik van bloed in machine perfusie kan inflammatoire
schade weefselschade en celdood veroorzaken Waarschijnlijk is het beter om bloed
tevervangen door een artificieumlle preservatie vloeistof Om verschillende vloeistoffen te
kunnen testen moet er een normotherm machineperfusie (NMP) model ontworpen worden
Het doel van deze studie is het ontwerpen van een NMP model met varkens nieren uit het
slachthuis om de kwaliteit van de nieren te testen
Methode Varkens nieren en autoloog bloed werden verzameld in het slachthuis De nieren
werden vervoerd naar ons lab en 4 uur op de NMP opstelling geperfundeerd 7 groepen zijn
getest met 1-4 nieren per groep Er zijn verschillende manieren van transport gebruikt cold
storage subnormotherme machineperfusie geoxygeneerde of niet ge-oxygeneerde
hypotherme machineperfusie Warme en koude ischemie tijden verschillen per groep In een
groep is tijdens NMP mannitol insuline voedingsstoffen en dexamethason toegevoegd
(NMP+) De hemodynamica werd gemonitord en perfusaat en urine monsters werden
regelmatig genomen Biopsieeumln werden genomen om renale histologie te beoordelen
Resultaten Een stabiel NMP systeem werd verkregen De groep waarbij geoxygeneerde
hypotherme machineperfusie en NMP+ gebruikt werd presteerde significant beter dan de
controle groep Er was betere creatinine klaring natrium reabsorptie en urine productie
Conclusie Het gecreeumlerde model waarbij gebruik gemaakt wordt van geoxygeneerde
hypotherme machineperfusie en NMP+ is bruibaar om verschillende vloeistoffen voor
perfusiedoeleinden te testen Verder onderzoek is nodig om de nierfunctie tijden de NMP
periode te verbeteren
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table of contents Abstractsamenvatting 2
Introduction 4
Organ shortage 4
Normothermic regional perfusion 5
Perfusion solutions in NRP 6
Machine perfusion 7
Study objectives 8
Material and methods 9
Experimental design 9
Organ and blood retrieval 9
Kidney transport 10
Perfusion 11
Urine and perfusate analysis 13
Statistical analysis 13
Results 14
Stabilizing the NMP system 14
Renal hemodynamics 16
Renal function 18
Renal Histology 22
Discussion 23
Considerations 23
Study strengths and limitations 24
Recommendations for future research 25
Bibliography 26
Acknowledgements 29
Appendices 30
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Introduction
Organ shortage Kidneys are the most frequently transplanted solid organs Patients suffering from end-stage
renal disease kidney transplantation offers improved quality of life and better life expectancy
when compared to dialysis The persisting organ shortage represents a severe problem in
transplant medicine1 (Table 1) Transplant waiting lists are increasing at a greater rate than the
availability of donors Declining numbers of the classical donation after brain death (DBD)
donors are available due improvements in the management of severe neurologic injuries2
This has triggered interest in marginal donors as an additional organ source Expanded criteria
donors (ECD) and donation after circulatory death (DCD) are examples of such marginal
donors Marginal donors that normally would have been declined in the past are now
considered for transplantation to decrease organ shortage3
Therefore the number of organ
donors has remained more of less stable However on the other hand a decreasing number of
patients become eligible for organ transplants due to obesity excessive alcohol consumption
poorly controlled hypertension and diabetes4 Thereby maintaining the long waiting time for a
kidney transplant Kidney transplantation outcome is negatively affected by this waiting time
with poorer outcome for patients subjected to prolonged dialysis5
A possible solution for organ shortage is the use of donation after circulatory death donors
The different types of DCD may be categorized using the Maastricht criteria6 (Table 2) The
controlled DCD- or Maastricht category III donors are most used in the Netherlands and
are those who have suffered massive brain injury but do not meet the criteria of brain death A
decision to withdraw supportive treatment is made independently of donor status Kidneys
from these patients undergo a period of warm ischemia between asystole and organ retrievel
leading to poorer transplant outcomes with higher incidence of primary non-function (PNF)
and increased complications rates7 In a porcine study an increasing warm ischemic time
(WIT) leads to a proportional impairment of early renal function associated with greater
severity of underlying oxidative tissue injury Kidneys sustaining less warm ischemia
demonstrated better function represented by creatinine clearance urine output renal
hemodynamics and oxygen consumption8
Table 1 Kidney graft shortage in US and Eurotransplant region
United states
(National Kidney
Foundation)
Eurotransplant region
(Eurotransplant
international foundation
statistics report)
Patients on kidney
transplant waiting list
100791 (January 2016) 10282 (March 2016)
Deceased donor kidneys
transplanted
11570 (2014) 1827 (2015)
Median waiting time to
deceased donor kidney
transplant
Up to 5 years Up to 4 years
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Table 2 Maastricht Categories of Donation after Circulatory Death
Category Description
I Dead on arrival at the hospital
II Unsuccessful resuscitation at the hospital
III Withdrawal of supportive treatment
IV Cardiac arrest following establishment of brain death
Patients who have circulatory arrest in relatively uncontrolled situations may also become
cardiac death donors These ldquouncontrolled DCD-rdquo or Maastricht categories I en II donors
experience a longer period of warm ischemia than controlled DCD which results in even
higher incidences of PNF and delayed graft function (DGF)7
Normothermic regional perfusion Organ procurement from DCD donors is associated with a higher rate of organ injury and
discards most likely due to the haste of removing the organs to minimize the WIT9
Normothermic regional perfusion (NRP) is a new and advanced technique which can be
utilized in DCD donors It restores the abdominal circulation with oxygenated blood in situ
between asystole and procurement This permits dissection without ischemic injury since
oxygen supply to the abdominal organs is guaranteed It also allows assessment of the organs
run tests introduce therapies if necessary NRP relies on an adequate supply of oxygen and
other substrates to fuel processes of cellular homeostasis as well as repair Given that cellular
metabolism is fully restored normothermic perfusion allows a more comprehensive
assessment of organ viability prior to recovery and transplantation1011
The use of NRP to facilitate organ donation was first described in 199712
NRP has been
developed in Spain for uncontrolled DCD donors (Maastricht II DCD II) where it increased
the donor pool The reported experience indicates low rates of PNF and a reduction in DGF
with good 1-year graft survival in kidney transplantation1314
NRP is likely to reduce the rate
of damage caused by warm ischemia as it re-establishes the abdominal circulation allowing a
careful identification of the vascular structures and enables the procedure to be performed
without undue speed compared to traditional DCD organ recovery15
During warm ischemia
ATP degradation leads to the progressive accumulation of xanthine and hypoxanthine
important sources of superoxide radicals at organ reperfusion A period of NRP after warm
ischemia helps to restore cellular energy substrates reduce levels of nucleotide degradation
products and improve the concentrations of endogenous antioxidants16
Maintaining
circulation before retrieval is also thought to condition the organs with the up-regulation of
adenosine receptors which may protect against preservation injury17
The use of NRP in DCD II donors is associated with a lower risk of DGF and with a better
graft function 2 years post-transplantation compared to expanded criteria donor (ECD)
kidneys ECD are defined as donors over 60 years old or aged 50-59 years with at least two
of the following conditions cerebrovascular cause of death serum creatinine over 15 mgdL
or hypertension18
Organs from ECD were associated with a suboptimal post-transplantation
function or shorter graft survival A study by Demiselle et al compared patient survival graft
survival and kidney function between DCD II without NRP ECD and standard criteria
donors The post-transplantation results of DCD II kidneys were comparable to those of ECD
kidneys NRP preservation may improve the results of DCD II transplantation19
Furthermore
the feasibility of NRP in category III DCD donation has also been tested and it is possible to
establish NRP successfully and continue normothermic perfusion for a period of 2 hours In
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situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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blo
od
32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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Ch
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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per
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cir
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Abstractsamenvatting Objectives To decrease waiting time for a kidney transplant donor kidneys of inferior quality
are increasingly being accepted Normothermic regional perfusion (NRP) in donation after
circulatory death restores abdominal circulation with autologous blood and improves organ
quality The use of blood in machine perfusion can cause inflammation injury tissues damage
and cell death Probably it is better to replace the blood in NRP with an artificial preservation
solution To test the different solutions a normothermic machine perfusion model for an
isolated kidney has to be designed The aim of this study is to design a NMP model with
porcine slaughterhouse kidneys to test kidney viability
Methods Porcine kidneys and autologous whole blood were obtained from the
slaughterhouse Kidneys were transported to our lab and reperfused for 4 hours using the
NMP set-up Seven groups were created (n=1-4) Kidneys were transported differently using
cold storage subnormothermic machineperfusion oxygenated- or non-oxygenated
hypothermic machine perfusion Warm and cold ischeamic times differ between groups In
one group mannitol insulin nutrients and dexamethson was added during NMP (NMP+)
Hemodynamics were monitored and perfusate and urine samples were taken regularly
Biopsies were taken to asses renal histology
Results A stable NMP was established There was a significant difference between groups
Kidneys preserved using oxygenated hypothermic machine perfusion and reperfused with
NMP+ performed significantly better These kidneys showed better creatinine clearance
sodium reabsorption and urine production than the control group
Conclusion The model created using oxygenated hypothermic machine perfusion and NMP+
likely is useful for testing different perfusion solutions However more research is required to
optimize this model
Doelstellingen Om wachttijd voor een niertransplantatie te verkorten worden donornieren
van mindere kwaliteit in toenemende mate geaccepteerd Normotherme regionale perfusie
(NRP) na donatie na hartdood hersteld de abdominale circulatie met autoloog bloed en
verbeterd de orgaan kwaliteit Het gebruik van bloed in machine perfusie kan inflammatoire
schade weefselschade en celdood veroorzaken Waarschijnlijk is het beter om bloed
tevervangen door een artificieumlle preservatie vloeistof Om verschillende vloeistoffen te
kunnen testen moet er een normotherm machineperfusie (NMP) model ontworpen worden
Het doel van deze studie is het ontwerpen van een NMP model met varkens nieren uit het
slachthuis om de kwaliteit van de nieren te testen
Methode Varkens nieren en autoloog bloed werden verzameld in het slachthuis De nieren
werden vervoerd naar ons lab en 4 uur op de NMP opstelling geperfundeerd 7 groepen zijn
getest met 1-4 nieren per groep Er zijn verschillende manieren van transport gebruikt cold
storage subnormotherme machineperfusie geoxygeneerde of niet ge-oxygeneerde
hypotherme machineperfusie Warme en koude ischemie tijden verschillen per groep In een
groep is tijdens NMP mannitol insuline voedingsstoffen en dexamethason toegevoegd
(NMP+) De hemodynamica werd gemonitord en perfusaat en urine monsters werden
regelmatig genomen Biopsieeumln werden genomen om renale histologie te beoordelen
Resultaten Een stabiel NMP systeem werd verkregen De groep waarbij geoxygeneerde
hypotherme machineperfusie en NMP+ gebruikt werd presteerde significant beter dan de
controle groep Er was betere creatinine klaring natrium reabsorptie en urine productie
Conclusie Het gecreeumlerde model waarbij gebruik gemaakt wordt van geoxygeneerde
hypotherme machineperfusie en NMP+ is bruibaar om verschillende vloeistoffen voor
perfusiedoeleinden te testen Verder onderzoek is nodig om de nierfunctie tijden de NMP
periode te verbeteren
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Table of contents Abstractsamenvatting 2
Introduction 4
Organ shortage 4
Normothermic regional perfusion 5
Perfusion solutions in NRP 6
Machine perfusion 7
Study objectives 8
Material and methods 9
Experimental design 9
Organ and blood retrieval 9
Kidney transport 10
Perfusion 11
Urine and perfusate analysis 13
Statistical analysis 13
Results 14
Stabilizing the NMP system 14
Renal hemodynamics 16
Renal function 18
Renal Histology 22
Discussion 23
Considerations 23
Study strengths and limitations 24
Recommendations for future research 25
Bibliography 26
Acknowledgements 29
Appendices 30
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Introduction
Organ shortage Kidneys are the most frequently transplanted solid organs Patients suffering from end-stage
renal disease kidney transplantation offers improved quality of life and better life expectancy
when compared to dialysis The persisting organ shortage represents a severe problem in
transplant medicine1 (Table 1) Transplant waiting lists are increasing at a greater rate than the
availability of donors Declining numbers of the classical donation after brain death (DBD)
donors are available due improvements in the management of severe neurologic injuries2
This has triggered interest in marginal donors as an additional organ source Expanded criteria
donors (ECD) and donation after circulatory death (DCD) are examples of such marginal
donors Marginal donors that normally would have been declined in the past are now
considered for transplantation to decrease organ shortage3
Therefore the number of organ
donors has remained more of less stable However on the other hand a decreasing number of
patients become eligible for organ transplants due to obesity excessive alcohol consumption
poorly controlled hypertension and diabetes4 Thereby maintaining the long waiting time for a
kidney transplant Kidney transplantation outcome is negatively affected by this waiting time
with poorer outcome for patients subjected to prolonged dialysis5
A possible solution for organ shortage is the use of donation after circulatory death donors
The different types of DCD may be categorized using the Maastricht criteria6 (Table 2) The
controlled DCD- or Maastricht category III donors are most used in the Netherlands and
are those who have suffered massive brain injury but do not meet the criteria of brain death A
decision to withdraw supportive treatment is made independently of donor status Kidneys
from these patients undergo a period of warm ischemia between asystole and organ retrievel
leading to poorer transplant outcomes with higher incidence of primary non-function (PNF)
and increased complications rates7 In a porcine study an increasing warm ischemic time
(WIT) leads to a proportional impairment of early renal function associated with greater
severity of underlying oxidative tissue injury Kidneys sustaining less warm ischemia
demonstrated better function represented by creatinine clearance urine output renal
hemodynamics and oxygen consumption8
Table 1 Kidney graft shortage in US and Eurotransplant region
United states
(National Kidney
Foundation)
Eurotransplant region
(Eurotransplant
international foundation
statistics report)
Patients on kidney
transplant waiting list
100791 (January 2016) 10282 (March 2016)
Deceased donor kidneys
transplanted
11570 (2014) 1827 (2015)
Median waiting time to
deceased donor kidney
transplant
Up to 5 years Up to 4 years
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Table 2 Maastricht Categories of Donation after Circulatory Death
Category Description
I Dead on arrival at the hospital
II Unsuccessful resuscitation at the hospital
III Withdrawal of supportive treatment
IV Cardiac arrest following establishment of brain death
Patients who have circulatory arrest in relatively uncontrolled situations may also become
cardiac death donors These ldquouncontrolled DCD-rdquo or Maastricht categories I en II donors
experience a longer period of warm ischemia than controlled DCD which results in even
higher incidences of PNF and delayed graft function (DGF)7
Normothermic regional perfusion Organ procurement from DCD donors is associated with a higher rate of organ injury and
discards most likely due to the haste of removing the organs to minimize the WIT9
Normothermic regional perfusion (NRP) is a new and advanced technique which can be
utilized in DCD donors It restores the abdominal circulation with oxygenated blood in situ
between asystole and procurement This permits dissection without ischemic injury since
oxygen supply to the abdominal organs is guaranteed It also allows assessment of the organs
run tests introduce therapies if necessary NRP relies on an adequate supply of oxygen and
other substrates to fuel processes of cellular homeostasis as well as repair Given that cellular
metabolism is fully restored normothermic perfusion allows a more comprehensive
assessment of organ viability prior to recovery and transplantation1011
The use of NRP to facilitate organ donation was first described in 199712
NRP has been
developed in Spain for uncontrolled DCD donors (Maastricht II DCD II) where it increased
the donor pool The reported experience indicates low rates of PNF and a reduction in DGF
with good 1-year graft survival in kidney transplantation1314
NRP is likely to reduce the rate
of damage caused by warm ischemia as it re-establishes the abdominal circulation allowing a
careful identification of the vascular structures and enables the procedure to be performed
without undue speed compared to traditional DCD organ recovery15
During warm ischemia
ATP degradation leads to the progressive accumulation of xanthine and hypoxanthine
important sources of superoxide radicals at organ reperfusion A period of NRP after warm
ischemia helps to restore cellular energy substrates reduce levels of nucleotide degradation
products and improve the concentrations of endogenous antioxidants16
Maintaining
circulation before retrieval is also thought to condition the organs with the up-regulation of
adenosine receptors which may protect against preservation injury17
The use of NRP in DCD II donors is associated with a lower risk of DGF and with a better
graft function 2 years post-transplantation compared to expanded criteria donor (ECD)
kidneys ECD are defined as donors over 60 years old or aged 50-59 years with at least two
of the following conditions cerebrovascular cause of death serum creatinine over 15 mgdL
or hypertension18
Organs from ECD were associated with a suboptimal post-transplantation
function or shorter graft survival A study by Demiselle et al compared patient survival graft
survival and kidney function between DCD II without NRP ECD and standard criteria
donors The post-transplantation results of DCD II kidneys were comparable to those of ECD
kidneys NRP preservation may improve the results of DCD II transplantation19
Furthermore
the feasibility of NRP in category III DCD donation has also been tested and it is possible to
establish NRP successfully and continue normothermic perfusion for a period of 2 hours In
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situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Table of contents Abstractsamenvatting 2
Introduction 4
Organ shortage 4
Normothermic regional perfusion 5
Perfusion solutions in NRP 6
Machine perfusion 7
Study objectives 8
Material and methods 9
Experimental design 9
Organ and blood retrieval 9
Kidney transport 10
Perfusion 11
Urine and perfusate analysis 13
Statistical analysis 13
Results 14
Stabilizing the NMP system 14
Renal hemodynamics 16
Renal function 18
Renal Histology 22
Discussion 23
Considerations 23
Study strengths and limitations 24
Recommendations for future research 25
Bibliography 26
Acknowledgements 29
Appendices 30
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Introduction
Organ shortage Kidneys are the most frequently transplanted solid organs Patients suffering from end-stage
renal disease kidney transplantation offers improved quality of life and better life expectancy
when compared to dialysis The persisting organ shortage represents a severe problem in
transplant medicine1 (Table 1) Transplant waiting lists are increasing at a greater rate than the
availability of donors Declining numbers of the classical donation after brain death (DBD)
donors are available due improvements in the management of severe neurologic injuries2
This has triggered interest in marginal donors as an additional organ source Expanded criteria
donors (ECD) and donation after circulatory death (DCD) are examples of such marginal
donors Marginal donors that normally would have been declined in the past are now
considered for transplantation to decrease organ shortage3
Therefore the number of organ
donors has remained more of less stable However on the other hand a decreasing number of
patients become eligible for organ transplants due to obesity excessive alcohol consumption
poorly controlled hypertension and diabetes4 Thereby maintaining the long waiting time for a
kidney transplant Kidney transplantation outcome is negatively affected by this waiting time
with poorer outcome for patients subjected to prolonged dialysis5
A possible solution for organ shortage is the use of donation after circulatory death donors
The different types of DCD may be categorized using the Maastricht criteria6 (Table 2) The
controlled DCD- or Maastricht category III donors are most used in the Netherlands and
are those who have suffered massive brain injury but do not meet the criteria of brain death A
decision to withdraw supportive treatment is made independently of donor status Kidneys
from these patients undergo a period of warm ischemia between asystole and organ retrievel
leading to poorer transplant outcomes with higher incidence of primary non-function (PNF)
and increased complications rates7 In a porcine study an increasing warm ischemic time
(WIT) leads to a proportional impairment of early renal function associated with greater
severity of underlying oxidative tissue injury Kidneys sustaining less warm ischemia
demonstrated better function represented by creatinine clearance urine output renal
hemodynamics and oxygen consumption8
Table 1 Kidney graft shortage in US and Eurotransplant region
United states
(National Kidney
Foundation)
Eurotransplant region
(Eurotransplant
international foundation
statistics report)
Patients on kidney
transplant waiting list
100791 (January 2016) 10282 (March 2016)
Deceased donor kidneys
transplanted
11570 (2014) 1827 (2015)
Median waiting time to
deceased donor kidney
transplant
Up to 5 years Up to 4 years
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Table 2 Maastricht Categories of Donation after Circulatory Death
Category Description
I Dead on arrival at the hospital
II Unsuccessful resuscitation at the hospital
III Withdrawal of supportive treatment
IV Cardiac arrest following establishment of brain death
Patients who have circulatory arrest in relatively uncontrolled situations may also become
cardiac death donors These ldquouncontrolled DCD-rdquo or Maastricht categories I en II donors
experience a longer period of warm ischemia than controlled DCD which results in even
higher incidences of PNF and delayed graft function (DGF)7
Normothermic regional perfusion Organ procurement from DCD donors is associated with a higher rate of organ injury and
discards most likely due to the haste of removing the organs to minimize the WIT9
Normothermic regional perfusion (NRP) is a new and advanced technique which can be
utilized in DCD donors It restores the abdominal circulation with oxygenated blood in situ
between asystole and procurement This permits dissection without ischemic injury since
oxygen supply to the abdominal organs is guaranteed It also allows assessment of the organs
run tests introduce therapies if necessary NRP relies on an adequate supply of oxygen and
other substrates to fuel processes of cellular homeostasis as well as repair Given that cellular
metabolism is fully restored normothermic perfusion allows a more comprehensive
assessment of organ viability prior to recovery and transplantation1011
The use of NRP to facilitate organ donation was first described in 199712
NRP has been
developed in Spain for uncontrolled DCD donors (Maastricht II DCD II) where it increased
the donor pool The reported experience indicates low rates of PNF and a reduction in DGF
with good 1-year graft survival in kidney transplantation1314
NRP is likely to reduce the rate
of damage caused by warm ischemia as it re-establishes the abdominal circulation allowing a
careful identification of the vascular structures and enables the procedure to be performed
without undue speed compared to traditional DCD organ recovery15
During warm ischemia
ATP degradation leads to the progressive accumulation of xanthine and hypoxanthine
important sources of superoxide radicals at organ reperfusion A period of NRP after warm
ischemia helps to restore cellular energy substrates reduce levels of nucleotide degradation
products and improve the concentrations of endogenous antioxidants16
Maintaining
circulation before retrieval is also thought to condition the organs with the up-regulation of
adenosine receptors which may protect against preservation injury17
The use of NRP in DCD II donors is associated with a lower risk of DGF and with a better
graft function 2 years post-transplantation compared to expanded criteria donor (ECD)
kidneys ECD are defined as donors over 60 years old or aged 50-59 years with at least two
of the following conditions cerebrovascular cause of death serum creatinine over 15 mgdL
or hypertension18
Organs from ECD were associated with a suboptimal post-transplantation
function or shorter graft survival A study by Demiselle et al compared patient survival graft
survival and kidney function between DCD II without NRP ECD and standard criteria
donors The post-transplantation results of DCD II kidneys were comparable to those of ECD
kidneys NRP preservation may improve the results of DCD II transplantation19
Furthermore
the feasibility of NRP in category III DCD donation has also been tested and it is possible to
establish NRP successfully and continue normothermic perfusion for a period of 2 hours In
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situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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18
Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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19
As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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26
Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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k In
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4
Introduction
Organ shortage Kidneys are the most frequently transplanted solid organs Patients suffering from end-stage
renal disease kidney transplantation offers improved quality of life and better life expectancy
when compared to dialysis The persisting organ shortage represents a severe problem in
transplant medicine1 (Table 1) Transplant waiting lists are increasing at a greater rate than the
availability of donors Declining numbers of the classical donation after brain death (DBD)
donors are available due improvements in the management of severe neurologic injuries2
This has triggered interest in marginal donors as an additional organ source Expanded criteria
donors (ECD) and donation after circulatory death (DCD) are examples of such marginal
donors Marginal donors that normally would have been declined in the past are now
considered for transplantation to decrease organ shortage3
Therefore the number of organ
donors has remained more of less stable However on the other hand a decreasing number of
patients become eligible for organ transplants due to obesity excessive alcohol consumption
poorly controlled hypertension and diabetes4 Thereby maintaining the long waiting time for a
kidney transplant Kidney transplantation outcome is negatively affected by this waiting time
with poorer outcome for patients subjected to prolonged dialysis5
A possible solution for organ shortage is the use of donation after circulatory death donors
The different types of DCD may be categorized using the Maastricht criteria6 (Table 2) The
controlled DCD- or Maastricht category III donors are most used in the Netherlands and
are those who have suffered massive brain injury but do not meet the criteria of brain death A
decision to withdraw supportive treatment is made independently of donor status Kidneys
from these patients undergo a period of warm ischemia between asystole and organ retrievel
leading to poorer transplant outcomes with higher incidence of primary non-function (PNF)
and increased complications rates7 In a porcine study an increasing warm ischemic time
(WIT) leads to a proportional impairment of early renal function associated with greater
severity of underlying oxidative tissue injury Kidneys sustaining less warm ischemia
demonstrated better function represented by creatinine clearance urine output renal
hemodynamics and oxygen consumption8
Table 1 Kidney graft shortage in US and Eurotransplant region
United states
(National Kidney
Foundation)
Eurotransplant region
(Eurotransplant
international foundation
statistics report)
Patients on kidney
transplant waiting list
100791 (January 2016) 10282 (March 2016)
Deceased donor kidneys
transplanted
11570 (2014) 1827 (2015)
Median waiting time to
deceased donor kidney
transplant
Up to 5 years Up to 4 years
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Table 2 Maastricht Categories of Donation after Circulatory Death
Category Description
I Dead on arrival at the hospital
II Unsuccessful resuscitation at the hospital
III Withdrawal of supportive treatment
IV Cardiac arrest following establishment of brain death
Patients who have circulatory arrest in relatively uncontrolled situations may also become
cardiac death donors These ldquouncontrolled DCD-rdquo or Maastricht categories I en II donors
experience a longer period of warm ischemia than controlled DCD which results in even
higher incidences of PNF and delayed graft function (DGF)7
Normothermic regional perfusion Organ procurement from DCD donors is associated with a higher rate of organ injury and
discards most likely due to the haste of removing the organs to minimize the WIT9
Normothermic regional perfusion (NRP) is a new and advanced technique which can be
utilized in DCD donors It restores the abdominal circulation with oxygenated blood in situ
between asystole and procurement This permits dissection without ischemic injury since
oxygen supply to the abdominal organs is guaranteed It also allows assessment of the organs
run tests introduce therapies if necessary NRP relies on an adequate supply of oxygen and
other substrates to fuel processes of cellular homeostasis as well as repair Given that cellular
metabolism is fully restored normothermic perfusion allows a more comprehensive
assessment of organ viability prior to recovery and transplantation1011
The use of NRP to facilitate organ donation was first described in 199712
NRP has been
developed in Spain for uncontrolled DCD donors (Maastricht II DCD II) where it increased
the donor pool The reported experience indicates low rates of PNF and a reduction in DGF
with good 1-year graft survival in kidney transplantation1314
NRP is likely to reduce the rate
of damage caused by warm ischemia as it re-establishes the abdominal circulation allowing a
careful identification of the vascular structures and enables the procedure to be performed
without undue speed compared to traditional DCD organ recovery15
During warm ischemia
ATP degradation leads to the progressive accumulation of xanthine and hypoxanthine
important sources of superoxide radicals at organ reperfusion A period of NRP after warm
ischemia helps to restore cellular energy substrates reduce levels of nucleotide degradation
products and improve the concentrations of endogenous antioxidants16
Maintaining
circulation before retrieval is also thought to condition the organs with the up-regulation of
adenosine receptors which may protect against preservation injury17
The use of NRP in DCD II donors is associated with a lower risk of DGF and with a better
graft function 2 years post-transplantation compared to expanded criteria donor (ECD)
kidneys ECD are defined as donors over 60 years old or aged 50-59 years with at least two
of the following conditions cerebrovascular cause of death serum creatinine over 15 mgdL
or hypertension18
Organs from ECD were associated with a suboptimal post-transplantation
function or shorter graft survival A study by Demiselle et al compared patient survival graft
survival and kidney function between DCD II without NRP ECD and standard criteria
donors The post-transplantation results of DCD II kidneys were comparable to those of ECD
kidneys NRP preservation may improve the results of DCD II transplantation19
Furthermore
the feasibility of NRP in category III DCD donation has also been tested and it is possible to
establish NRP successfully and continue normothermic perfusion for a period of 2 hours In
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situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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5
Table 2 Maastricht Categories of Donation after Circulatory Death
Category Description
I Dead on arrival at the hospital
II Unsuccessful resuscitation at the hospital
III Withdrawal of supportive treatment
IV Cardiac arrest following establishment of brain death
Patients who have circulatory arrest in relatively uncontrolled situations may also become
cardiac death donors These ldquouncontrolled DCD-rdquo or Maastricht categories I en II donors
experience a longer period of warm ischemia than controlled DCD which results in even
higher incidences of PNF and delayed graft function (DGF)7
Normothermic regional perfusion Organ procurement from DCD donors is associated with a higher rate of organ injury and
discards most likely due to the haste of removing the organs to minimize the WIT9
Normothermic regional perfusion (NRP) is a new and advanced technique which can be
utilized in DCD donors It restores the abdominal circulation with oxygenated blood in situ
between asystole and procurement This permits dissection without ischemic injury since
oxygen supply to the abdominal organs is guaranteed It also allows assessment of the organs
run tests introduce therapies if necessary NRP relies on an adequate supply of oxygen and
other substrates to fuel processes of cellular homeostasis as well as repair Given that cellular
metabolism is fully restored normothermic perfusion allows a more comprehensive
assessment of organ viability prior to recovery and transplantation1011
The use of NRP to facilitate organ donation was first described in 199712
NRP has been
developed in Spain for uncontrolled DCD donors (Maastricht II DCD II) where it increased
the donor pool The reported experience indicates low rates of PNF and a reduction in DGF
with good 1-year graft survival in kidney transplantation1314
NRP is likely to reduce the rate
of damage caused by warm ischemia as it re-establishes the abdominal circulation allowing a
careful identification of the vascular structures and enables the procedure to be performed
without undue speed compared to traditional DCD organ recovery15
During warm ischemia
ATP degradation leads to the progressive accumulation of xanthine and hypoxanthine
important sources of superoxide radicals at organ reperfusion A period of NRP after warm
ischemia helps to restore cellular energy substrates reduce levels of nucleotide degradation
products and improve the concentrations of endogenous antioxidants16
Maintaining
circulation before retrieval is also thought to condition the organs with the up-regulation of
adenosine receptors which may protect against preservation injury17
The use of NRP in DCD II donors is associated with a lower risk of DGF and with a better
graft function 2 years post-transplantation compared to expanded criteria donor (ECD)
kidneys ECD are defined as donors over 60 years old or aged 50-59 years with at least two
of the following conditions cerebrovascular cause of death serum creatinine over 15 mgdL
or hypertension18
Organs from ECD were associated with a suboptimal post-transplantation
function or shorter graft survival A study by Demiselle et al compared patient survival graft
survival and kidney function between DCD II without NRP ECD and standard criteria
donors The post-transplantation results of DCD II kidneys were comparable to those of ECD
kidneys NRP preservation may improve the results of DCD II transplantation19
Furthermore
the feasibility of NRP in category III DCD donation has also been tested and it is possible to
establish NRP successfully and continue normothermic perfusion for a period of 2 hours In
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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6
situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Ch
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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situ NRP represents a significant advance in DCD organ retrieval and has the potential to
increase the number and quality of the transplanted organs15
Perfusion solutions in NRP Different techniques have been developed to accommodate NRP in DCD II and DCD III
donors The use of NRP in both donation techniques requires a perfusion fluid which
comprises all the components needed during the NRP period The composition of the
perfusion solution is vital to ensure adequate delivery of nutrients and oxygen to maintain
cellular integrity and vascular processes
To ensure that sufficient oxygen for normal metabolic function is provided to the abdominal
organs an oxygen carrier in the perfusion solution is needed All normothermic perfusion
(NP) studies used blood as the perfusion solution with red blood cells as the oxygen
carrier158 NP with oxygenated blood was able to restore depleted ATP levels and reverse
some of the deleterious effects of CS20
Although red blood cells are highly evolved to provide oxygen to tissues there are some
disadvantages to using blood as perfusion fluid Early studies found that leucocytes
haemolysis and platelet activation during perfusion with a blood-based solution caused an
increase in resistance and tissue oedema during prolonged periods of preservation20
Leucocytes play a role in an inflammatory process causing cellular injury Endothelial cell
damage caused by ischemic injury stimulates a pro-inflammatory environment which
activates and stimulates leucocytes Leucocytes migrate and infiltrate into the interstitium
leading to microvascular congestion and the ldquono-reflowrdquo phenomena This increases cytokine
expression production of oxygen free radicals and activates the complement system to sustain
the injury response causing cell death and tissue damage21
Normothermic perfusion using a leukocyte and platelet depleted red cell-based solution limits
infiltration and the inflammatory response to improve circulation and renal function
Apoptosis and inflammatory mediators are also suppressed reducing the likelihood of
injury22
Platelets also have a damaging role in reperfusion injury they mediate
vasoconstriction and inflammatory processes causing injury23
In cardiovasculair surgery the use of cardiopumonary bypass (CBP) is associated with acute
kidney injury (AKI) Haemolysis is a common consequence of cardiopulmonary bypass that is
caused by mechanical stress in the perfusion circuit and results in the release of hemoglobin
from lysed erythrocytes into the plasma Cell-free oxyhemoglobin reacts with nitric oxide to
form methemoglobin and nitrate As nitric oxide is an important vasodilator that has a central
role in blood flow regulation reduction of nitric oxide bioavailability by free hemoglobin may
impair tissue perfusion24
Since the NRP circuit consists of the same technical features as a
cardiopulmonary bypass circuit this problem with AKI seen in CPB could be a serious
detrimental effect that can be the result when using NRP in combination with blood
Currently blood is still the widely used product in perfusion solutions for NRP and other
normothermic machine perfusion (NMP) settings As described above the components of a
blood-based solution causes inflammatory injury tissue damage and cell death Also
mechanical stress in a perfusion circuit contributes to kidney damage Therefore we believe
that the use of blood in machine preservation of organs is not the best solution and that is
would be better to replace the blood with another preservation solution
The artificial perfusion solution has to meet some requirements The perfusion fluid should
deliver enough oxygen to maintain aerobic metabolism Furthermore it needs to consist
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
stu
k B
iblio
grap
hy
27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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ents
29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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sufficient nutrients to prevent depletion of cellular energy substrates With these components
the preservation fluid should minimize injury in organs that have been subjected to warm
ischemia
In an ideal situation the non-blood based NRP solutions are tested in a randomized controlled
trail with NRP in DCD donors However it is unethical to do this in a clinical setting
Therefore the first step is to design an animal model Porcine kidneys are suitable for this
model because the size and geometry of a porcine kidney is comparable to human kidneys
Furthermore various renal functions damage parameters and morphology can easily be
assessed25
Performing NRP in a pig is an expensive procedure large experimental animals
are costly to keep and NRP equipment is expensive as well Also approval for such
experiments is hard to get in the Netherlands Therefore we are aiming to establish a low cost
model using slaughterhouse kidneys instead of laboratory pigs thereby decreasing the cost
and avoiding ethical questions However it is not possible to perform NRP in the
slaughterhouse therefore slaughterhouse kidneys are transported to our lab and tested in and
an isolated perfused porcine kidney system created to simulate warm kidney perfusion
Machine perfusion Preserving the function of the kidney graft during transport is of vital importance for an
effective NRP model In the clinical setting in most countries a kidney is flushed cooled with
a cold preservation solution and cold stored on ice between organ retrieval and
transplantation26
During this preservation period the organ is transported cross matching is
performed and the operating room can be prepared This period of cold ischemia is then
followed by reperfusion More and more research is performed to determine the best
preservation method between organ retrieval and transplantation The goal in these studies is
to decrease the amount of ischemia-reperfusion injury (IRI) caused by tissue ischemia27
IRI is an unavoidable relevant consequence after kidney transplantation and results in a
distinct inflammatory reaction of the graft Clinically IRI is associated with delayed graft
function graft rejection chronic rejection and chronic graft dysfunction28
IRI is principally
caused by blood flow impairment which starts with brain death and is due to severe
hemodynamic disturbances in cadaveric donors Clamping of the renal artery during the
harvesting operation causes a short but severe renal ischemia In addition cold ischemia
during transport causes a further ischemic damage The final and biologically more severe
stage of the injury occurs during the reperfusion as a consequence of the returning blood flow
in the recipient29
Underlying factors of ischemia reperfusion include energy metabolism
cellular changes of the mitochondria and cellular membranes initiation of different forms of
cell death-like apoptosis and necrosis together with a recently discovered mixed form termed
necroptosis Chemokines and cytokines together with other factors promote the inflammatory
response leading to activation of the innate immune system as well as the adaptive immune
system If the inflammatory reaction continues within the graft tissue a progressive interstitial
fibrosis develops that impacts long-term graft outcome30
Machine perfusion could enable active organ conditioning prior to transplantation and
furthermore this technique provides a platform for therapeutic interventions during organ
preservation21
Machine perfusion is preferably done under (sub)physiologic conditions
through (sub)normothermic machine perfusion at or below 37degC31
NMP may be able to
reverse some effects of ischemia by restoring organ metabolism outside the body prior to
transplantation It also allows pre-transplant assessment of organ viability25
Kidneys that
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have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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26
Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
stu
k In
tro
du
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8
have been perfused using NMP have significantly lower rates of DGF than those preserved
cold storage33
Various studies have demonstrated that hypothermic machine perfusion (HMP) is superior to
static cold stored kidney grafts from deceased donors 2634
DCD donors are more likely to
suffer from IRI only cooling the organ during preservation may not be sufficient The
principle of cold preservation is based on temperature reduction to reduce metabolism
Cooling does not completely stop cell metabolism which in turn leads to energy depletion35
HMP however reduces the risk and duration of DGF and leads to improved graft survival26
In response to these convincing data all kidneys recovered from deceased donor kidneys in
The Netherlands are preserved by HMP as of November 2015 Static cold storage has been
largely abandoned in our country for kidney preservation36
However the need for oxygen during HMP persists because the metabolic rate remains at
levels estimated around 10 There has been much debate on whether it is necessary to add
oxygen to support the low level of metabolism under these conditions Evidence suggests that
oxygen is particularly beneficial in restoring cellular levels of adenosine triphosphate after
kidneys have been subjected to warm or cold ischemic injury37
The potential benefits of
active oxygenation during HMP have been tested using a pig model Oxygen delivery during
preservation proved to be valuable for improving organ quality Kidney grafts preserved with
oxygenated HMP displayed a lower serum creatinine peak compared to non-oxygenated
HMP Histologic investigation showed a trend towards decreased inflammation in kidneys
preserved with oxygen38
Study objectives The aim of this study is to design a NMP model with porcine slaughterhouse kidneys to test
kidney viability The results of this study serve as a basis for the development of a preclinical
study where different perfusion solutions for NRP will be tested and later verified in a large
animal model
The first priority is to establish a stable perfusion using the IPPK technique for 4 hours The
NMP system should be pressure controlled and maintain a mean pulsatile arterial pressure of
75 mmHg The perfusate in the system must be 37 degC to represent normal physiological body
temperature The oxygenator should be able to deliver enough oxygen to the perfusate to keep
the partial oxygen pressure above 60 kPa
Furthermore the optimal way to preserve the porcine kidneys from the slaughterhouse to the
lab needs to be explored First different WIT will be tested Secondly a different method of
transportation will likely improve kidney quality This is tested by using cold storage
subnormothermic oxygenated machine perfusion hypothermic non-oxygenated machine
perfusion hypothermic oxygenated machine perfusion In the end we will add additives
during reperfusion to support kidney function
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Material and methods
Experimental design
Eight groups were created with 2 kidneys each except for the control group which contains 4
kidneys The WIT way of transport and cold ischemic time (CIT) differs between the groups
as described in table 3 Kidneys were transported differently either using cold storage (CS)
subnormothermic machineperfusion (sNMP) oxygenated hypothermic machine perfusion for
3 hours or non-oxygenated hypothermic machine perfusion for 3 hours(HMP -O2) All
kidneys were reperfused for 4 hours in a normothermic machine perfusion (NMP) set-up at an
arterial pressure of 75 mmHg and temperature of 37degC In the last group the NMP protocol
has changed dexamethson and mannitol was added to the priming solution and insulin
nutrients and bicarbonate were added during perfusion to create a NMP+ group Renal blood
flow and perfusate temperature were recorded every 10 minutes Perfusate and urine samples
were taken every 30 minutes Both were kept on ice before centrifugation and storage at -80C
Blood gas samples were taken and analysed immediately every 30 minutes One needle
biopsy of the cortex was taken prior to perfusion and a surgical biopsy was taken after
perfusion
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Organ and blood retrieval Kidneys were retrieved from two different slaughterhouses in the vicinity of Groningen The
protocol for organ and blood retrieval was the same for both (appendix 1) Pigs were
anaesthetized with a bi-temporal electric shock rapidly followed by exsanguination
following standard slaughterhouse procedures under the supervision of a veterinarian
Approximately 3 liters of autologous blood was collected in a beaker containing 5ml25000
units of heparin The blood was then poured into a jerry can for transport The kidneys were
removed and after a warm ischeamia interval one was flushed with NaCl 09 until the
aspect of the kidney became uniformly pale and clear fluid ran from the vein The kidney was
then stored for transport according to the assigned group Cold ischemic times varied with the
experimental groups
Table 3 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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sys
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Kidney transport After removal and flush of the kidneys they were stored differently using CS HMP or sNMP
For CS after flushing with cold NaCL 09 the kidney was stored in an organ bag containing
NaCL 09 and stored When HMP was applied the kidney was flushed with cold NaCL
09 and connected to a hypothermic machine perfusion pump (Kidney Assist transport
Organ Assist Groningen The Netherlands) seen in figure 3 filled with cold UW-MP
solution (belzers MP Bridge to life Londen United Kingdom) A patch was created using the
aorta and placed in a patch holder and connected to the kidney holder This is shown in figure
1 figure 2 and figure 3 After this the kidney was placed on the machine Hypothermia was
maintained because of crushed ice surrounding the circuit in which the kidney is placed The
oxygen bottle of the device was opened according to the experimental group
Figure 1 Kidney with patch Figure 2 Patch connected to patch holder
Figure 3 Placement in kidney holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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11
For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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k B
iblio
grap
hy
27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
stu
k B
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grap
hy
28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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gem
ents
29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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For sNMP the kidney was also connected to a Kidney Assist transport (KA) (Organ Asisst
Groningen Netherlands) only instead of ice surrounding the circuit the machine is filled with
heat packs and primed with 500 ml autologous whole blood and 500 ml ringerslactate and
perfused at a temperature of 30degC After flushing with warm NaCL 09 excess fat was
removed and the ureter vein and artery were cannulated then placed in a kidney holder and
placed in the KA reservoir
Figure 4 Kidney assist with disposable
Attaining leukocyte depleted autologous whole blood The leukocyte-depleted blood was prepared by filtering the heparinised autologous whole
blood collected at the slaughterhouse First the blood was poured in a catheter bag using a
funnel Then the blood was led through a leukocyte filter (BioR O2 plus Fresenius Kabi
Zeist Netherlands) After filtration the blood was checked for leukocytes with an upper
boundary off 001x10^9
Perfusion The perfusion circuit that was designed contains a KA with a centrifugal pump (Medos
Medizintechnik AG Stolberg Germany) an oxygenator (Hilite 800 LT Medos
Medizintechnik AG Stolberg Germany) and a homemade organ chamber with a cannula
(cannula for organ perfusion ndash 12F INFUSION Warszawa) To keep the perfusate
temperature stable at 37degC an oxygenator with integrated heat exchanger was used A
temperature sensor provided information regarding the temperature Flow was monitored
using an ultrasonic clamp-on flow probe (ME7PXL clamp frac14 inch flow meter Transonic
Systems Inc Ithaca NY) Pressure was measured directly before the cannula using pressure
transducer which was zero-calibrated to the atmosphere (TrueWave disposable pressure
transducer Edwards Lifesciences Irvine CA) All components were attached to each other
using disposable tubing (Rehau Rauclair-E 102 10x14 and 715 7x10 Rehau NV Nijkerk
Netherlands) (appendix 2) The circuit is shown in figure 5
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Figure 5 The perfusion circuit
The set up was primed with 300 ml Ringers Lactate 10 ml voluven 10ml bicarbonate 100microl
sodium nitroprusside (20mgml) and amoxicillin-clavulanate 1000mg200mg (Sandoz BV
Almere Netherlands) Creatinine was added to achieve a concentration of 1 moll After
priming 500 ml leukocyte depleted whole blood was added The perfusate was oxygenated
with 05 Lmin carbogen (95 O2 5 CO2)
Preparation of the kidney was initiated when the perfusate was 37degC Excess fat was removed
and the ureter was cannulated with an 8 Fr nasogastric feeding tube (Nutrisafe 2 gastro-
duodenal feeding tube (Pur) 8Fr Vygon Valkenswaard Netherlands) The artery patch was
removed and the artery was cannulated with an arterial cannula The cannulated kidney is
shown in figure 6 Next the kidney was put in the organ chamber and attached to the
perfusion circuit (figure 7) and perfused in a pulsatile sinusoid fashion at a mean arterial
pressure of 75 mmHg for a total duration of 4 hours
Figure 6 Cannulated Kidney Figure 7 Kidney connected to NMP circuit
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To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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syst
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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ds
13
To create the 30WI+HMPO2+NMP+ group two infusion pumps are added to the circuit
These pumps are connected to the oxygenator using a valve system For this group the
priming solution is altered 6mg mannitol and 6 mg dexamethason is added The infusion
pumps are used to infuse a nutrient solution with added insulin at 20mlhour and glucose
09 at a rate of 7 mlhour
Histology All pre- and postperfusion biopsie were fixed in 4 formalin dehydrated and embedded in
paraffin wax Sections were cut then stained with hematoxylin and eosin (HE) for evaluation
using light microscopy
Urine and perfusate analysis Urine and perfusate were analysed with routine automated test methodology carried out by the
clinical diagnostics laboratory after completing all experiments Creatinine and sodium levels
were determined in every sample both in urine and perfusate Creatinine clearance (=(urine
creatinine concentration x urine flow rate) plasma creatinine concentration) and fractional
excretion (=100 x (Sodium urine concentration x plasma creatinine concentration) (plasma
sodium concentration x urine creatinine concentration)) of sodium were calculated Lactate
dehydrogenase (LDH) was also determined in a number of experiments as marker of
generalized cellular stress (Table 4)
Statistical analysis Values are presented as mean with standard deviations Descriptive statistics were used to
display statistical dispersion of kidney function parameters within each group Continuous
variables such as serum creatinine were plotted as level versus time curves for each kidney
and the mean area under the curve (AUC) was calculated An one-way ANOVA was used to
compare values between groups if the data were normally distributed and had homogeneity of
variances If data failed these assumptions the Kruskal-Wallis H test was used P-values le
005 were assumed to indicate statistical significance Post hoc tests were performed if
necessary
Table 4 Viability assesment
Perfusion
parameters
Renal function Tubulair function Injury markers
Perfusion pressure Serum creatinine
levels
GFR LDH
Flow Creatinine clearance Fractional NA
excretion
Lactate
Oxygen concetration pH
Kidney weight ATP
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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14
Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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21
significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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23
Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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24
25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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26
Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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sys
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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Results
Stabilizing the NMP system The first 4 kidneys that were perfused were used to stabilize the NMP system to our
requirements The results were analysed after perfusion and adjustments were made to the
system or perfusate when necessary The Kidney Assist was able to provide a stable 4 hour
pressure controlled perfusion at 75 mmHg The third kidney was excluded from the analysis
The decision was made to stop the experiment when the oxygenator started to leak vigorously
Perfusate temperatures renal blood flow and diuresis are shown in the table below
The water bath and heat chamber were able to warm-up the perfusate temperature to 37degC
When connecting a cold stored kidney to the perfusion circuit a temperature drop is seen after
which the temperature is increasing to the appropriate level To maintain stable temperatures
sample were taken via a hatch in the surrounding cabinet instead of taking the entire front of
The blood flow values were low in the first two experiments Therefore a vasodilator was
added to the priming solution This resulted in higher blood flows and more diuresis in the
forth experiment (table 6) which was more in line with expectations for a porcine kidney
Table 5 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 6 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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15
After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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per
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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sys
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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After analysing the perfusate a number of improvements were made to create an environment
for the kidneys that was as close to physiological as possible First the partial oxygen pressure
was checked using gas analysis Graph 1 shows that the oxygenator can keep the oxygen level
above 60 kPa
Graph 1 Oxygen pressure in Perfusate
Glucose levels were also monitored in the perfusate during perfusion (graph 2) During the
first experiment glucose levels dropped until 02 mmolL This level is insufficient to support
normal cell metabolism Therefore we added 7 ml 09 glucose hourly in the second
experiment The goal was to achieve a concentration of 8 mmolL The glucose levels during
the second were higher but did not reach the 8 mmolL goal In the fourth experiment we
calculated the amount of glucose 09 needed to be added to increase the concentration up to
8 mmolL at each time point which gave better results In the following experiments we used
the same table and added glucose 09 before starting perfusion to increase the glucose level
at t=0
Graph 2 Glucose concentration in Perfusate
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Ho
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and
blo
od
30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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nal
per
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Renal hemodynamics Kidneys 5 to 22 were used to fill the experimental groups The control group and HMP+O2
group consisted of 4 kidneys the other groups had 2 kidneys each Except for the
30WI+HMP+ O2+NMP+ which has only one kidney The second kidney in this group was
excluded from analysis due to a broken pressure sensor We could not start HMP preservation
until replacing the sensor During the time it took to replace the pressure sensor the kidney
was cold stored instead of HMP preserved When reperfused renal blood flow of this kidney
was much lower than other kidneys which also led to poor ability to re-warm the kidney to
37degC Not meeting the standards set for the experimental group led to exclusion of this
kidney
Renal blood flow increased during the first 30 minutes in all groups After this the flow
remained almost constant until the last two hours in which the flow is gradually decreasing
Mean flow per group with standard deviation is presented in graph 3-9 Each time point is
evaluated using a one-way ANOVA there were no significant differences found P-values of
the statistical analyses are shown in table 7
Graph 3-6 Mean Renal blood flow in mlmin100 gram per experimental group
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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hy
27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Graph 7-9 Mean Renal blood flow in mlmin100 gram per experimental group
Table 7 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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24
25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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26
Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal function Urine production was collected every half hour Graph 4-10 shows mean urine production and
standard deviation per experimental group The high urine production of the
30WI+HMP+O2+NMP+ group suggests a better performance of this kidney Statistical
analysis of all groups using a Kruskal-Wallis H test showed that at t=120 and t=150 a
significant difference is present (table 8) Therefore a post hoc test is performed for both time
points The 30WI+HMP+O2+NMP+ has significantly more urine production compared to the
control group at t=120 and t=150 p=0001 and p=0002
Graph 10 Mean urine production in mlmin per experimental group
Table 8 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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19
As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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24
25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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As a mark for kidney function creatinine clearance and the fractional sodium excretion
(FENa+) were calculated using creatinine and sodium levels in perfusate and urine Mean
creatinine clearance per group is presented in graph 11 Creatinine clearance rates were
analysed using either a Kruskal-Wallis H test or a one-way ANOVA results are shown in
table 9
Graph 11 Mean creatinine clearance per experimental group
At t=15 t=90 t=120 t=180 and t=210 significant results appeared which needed further
evaluating The 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ groups have a significantly
better creatinine clearance level compared to the control group When comparing these last 2
groups there is a significant difference at t=15 and t=90 indicating the
30WI+HMP+O2+NMP+ is even better than the 30WI+HMP+O2 group Post hoc results are
presented in table 10
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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26
Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 10 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
The serum creatinine drop after 4 hour NMP was calculated for each group The
30WI+HMP+O2 and 30WI+HMP+O2+NMP+ group cleared a significantly better percentage
of creatinine then our control group (p=0007 and p=0001) There was no difference when
comparing the 30WI+HMP+O2 with 30WI+HMP+O2+NMP+ (p=0436)
All mean FENa+ are plotted in graph 12 The FENa
+ of the 30WI+CS and 20WI+CS were
high suggesting that kidney function is less than other groups The 40WI+sNMP group
appears to be better than the other groups during the first hour however this can be explained
by the fact that one kidney in this group did not produce any urine for the first hour
Graph 12 Mean fractional excretion of sodium per experimental group
When evaluating these values using a Kruskal-Wallis H test or an one-way ANOVA results
show a significant difference at all time points after t=90 (table 11) Post Hoc testing reveals
that after t=90 the 30WI+HMP+O2 and 30WI+HMP+O2+NMP+ are functioning significantly
better then the control group However comparing the 30WI+HMP+O2 group with the
30WI+HMP+O2+NMP+ group there is no significant difference present as seen in table 12
Since the groups were small the area under the curve (AUC) for FENa+ was calculated and
analysed using an one-way ANOVA This showed a significant difference p=0027 Post hoc
analysis revealed both the 30WI+HMP+O2 and 30WI+HMPO2+NMP+ group were
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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21
significantly better than the control group There was no significant difference between those
two groups
Several other kidney function and tissue injury parameters were evaluated and an overview is
presented in appendix 3 Kidneys were weighed before and after NMP Weight gain is the
highest in the 40WI+sNMP group Lactate and LDH are also analyzed Lactate levels are
increasing during the 4 hours reperfusion except in the 30WI+HMPO2 group were lactate
levels are decreasing Other parameters such as pH pO2 and glucose did not differ between
groups and are also presented in appendix 3 There are no more statistically significant
differences than previously discussed in kidney function and injury markers between groups
as shown in table 13
Table 11 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 12 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
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24
25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Renal Histology Apart from analyzing renal hemodynamics and kidney function parameters we also studied
renal histology of the biopsies A slight difference seemed to occur between groups A t=0
biopsy was taken after preservation before NMP All groups show damaged tubular cells
indicating that acute tubular necrosis (ATN) is present In the CS groups (figure 8) ATN is
more severe than the kidneys preserved with HMP (figure 9) Focal tubular epithelial necrosis
is present and rupture of basement membranes and occlusion of tubular lumens is more severe
in these groups
Figure 8 HE staining at t=0 of CS kidney Figure 9 HE staining at t=0 of HMP kidney
After 4 hours NMP a second biopsy was taken The difference between groups became more
evident evaluating the histology In the CS group most tubules were fully obstructed due to
necrosis of epithelial cells which have detached and sloughed into the tubular lumens Some
tubules appeared relatively normal meaning there was probably some function left Inside
Bowmanrsquos capsule protein deposition was present indicating that the glomeruli were leaking
There is no difference between the histology when WI changes The 40WI+sNMP group did
not differ from the CS group since debris and obstructed tubules are also present The HMP
groups showed open and intact tubules indicating better function as the CS and 40+sNMP
group The oxygenated kidneys had more arearsquos with almost normal tubules then the non-
oxygenated kidneys The best of all is the 30WI+HMPO2+NMP+ which showed more open
tubuli with a larger diameter and Bowmanrsquos space appeared better then all previous described
groups
Figure 8 HE staining at t=240 of CS kidney Figure 9 HE staining at t=240 of HMP kidney
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Discussion We showed that is it possible to develop a stable NMP system by which renal function
parameters can be monitored A pulsatile mean arterial pressure of 75 mmHg is maintained
during 4 hours perfusion at 37 degC The partial oxygen pressure stayed above 60 kPa This
NMP system is suitable to use as a porcine DCD model without using laboratory animals
Further experiments showed that kidneys in the 30WI+HMP+O2+NMP+ group had
significantly better results than our 30WI+CS control group
Considerations In this study we tried to create a NMP system that is stable and useful to test different
perfusion solutions later on When considering renal function a few parameters stood out
First of all we found a relatively low level of creatinine clearance during NMP Other studies
reported a much higher level of creatinine clearance up to 20 mlmin100gr394041
As we
know that warm and cold ischemia are detrimental to the kidney the short warm ischemia
time (6-7 minutes) and relatively short cold ischemia (2 hours) that the kidney were exposed
to in that study could provide a feasible explanation as to why there is such a large difference
in creatinine clearance However in our study we had a similar experimental group with 7
minutes warm ischemia and 2 hours cold storage Creatinine clearance in our group reached
only 5 mlmin100gr This difference could be due to variations in organ retrieval and
reperfusion protocols used in our experiments
Prolonged warm ischemia time is associated with graft failure and mortality after kidney
kidney transplantation7 Also a clear association between increasing warm ischemic time and
more severe IRI and deterioration in renal function has been shown8 However in our results
different WIT did not lead to statistical significant differences This is most likely caused by
the slaughter process The pigs experience a lot of stress during transport and also waiting for
their turn to be exsanguinated Another element is the heat drum used in standard
slaughterhouse procedures for removing hair and softening the skin adding extra warmth
during the ischemic period The small number of kidneys in each experimental group could
also have contributed to the statistical outcome more inclusions could strengthen statistical
tests and reveal significance if present
Also FENa+ values were investigated after NMP These values were spread between
extremely high and close to normal physiological levels The high FENa+ values are most
likely the result of ATN which is also seen in other studies3941
FENa+ is the highest in the
20WI+CS group (FeNA t240 = 8236plusmn471) and lowest in the 30WI+HMP+O2+NMP+ group
(FeNA t240 = 435) Comparing HE staining of both groups support this assumption In the
20WI+CS group there is more tubular damage evident than the 30WI+HMP+O2+NMP+
group
In addition all kidneys showed an increase in weight suggesting oedema formation This is
probably due to ischemia-reperfusion damage leading to intracellular and interstitial swelling
which is also seen in other studies3925
A point of interest from our view was whether adding oxygen to hypothermic machine
perfusion is beneficial during transport A study evaluating oxygenated hypothermic machine
perfusion in a DCD model showed that preservation using oxygenated hypothermic machine
perfusion is efficient in preserving DCD kidneys greatly enhancing the capacity of the graft
to withstand preservation stress and improving outcome38
Re-evaluating results from only the
30WI+HMP+O2 and 30WI+HMP-O2 group revealed a statistical difference in FENa+ after
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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25 hour NMP (t=180 P=0025 t=210 p=0022 t=240 p=0042) However these values we
report are of very early renal function long term results could differ
Interesting are the results of the 30WI+HMPO2+NMP+ group To our belief this is the best
performing kidney Adding mannitol dexamethason to the perfusate and infusing nutrients
and insulin during perfusion is be beneficial to the kidney Mannitol has protective effects
including increasing renal blood flow and decreasing intravascular cellular swelling
Dexamethason is used to reduce the inflammatory response and insulin to stimulate the re-
absorption of glucose Apart from glucose as energy source it is likely that kidneys need
amino acids to build new proteins Other studies have better results using these additives
during experiments 323941
Our analysis shows a difference between the
30WI+HMPO2+NMP+ group and the control group We experienced some difficulties during
one of the experiments due to a broken pressure sensor Excluding this kidney still resulted in
a highly significant findings when evaluating statistics between the 30WI+HMP+O2 and
30WI+HMPO2+NMP+ group Indicating 30WI+HMPO2+NMP+ has better function
compared to 30WI+CS and 30WI+HMP+O2 However to properly evaluate the effects of
these additives more experiments and further investigation is necessary
Study strengths and limitations This study has several strengths First of all a major advantage was that this study is
performed using kidneys from commercial slaughterhouses making the use of laboratory
animals unnecessary Porcine kidneys resemble human kidney closely in function and
anatomy Normally a typical model utilizes laboratory animals as organ donors which is
associated with considerable costs Moreover sacrificing a laboratory pig to obtain one or two
kidneys for research may be regarded as inefficient and ethically questionable
We also succeeded in creating a stable model for testing possible improvements for DCD
donation with outstanding results in the 30WI+HMPO2+NMP+ group Although our results
are suboptimal compared to other studies this model is excellent for testing perfusion fluids
There is room for improvement in renal function which could be achieved by one of the
artificial perfusion solutions to be tested
There are also a few limitations of this study one of them being the small groups (n=1 n=2 or
n=4) The small number of kidneys per group makes is difficult to conclude what the effect of
different perfusion techniques are Due to little time and lots of different techniques to
evaluate we were not able to do more experiments per experimental group However most
interventions were also evaluated by other studies and we had to create similar results during
this pilot in order to show our experimental set up is functioning properly
During the last experiments we experienced some technical difficulties leading to delay
during set up or impairment during perfusion A broken pressure sensor caused delay in
machine preservation causing cold ischemia time instead of oxygenated machine perfusion
We also encountered some coagulation during some of the experiments The heater inside the
cabinet caused clotting in the ureter canula or line of the pressure sensor A blocked urethra
can cause congestion inside the kidney and impairment of kidney function When the pressure
sensor line is blocked pressure starts to build inside de pressure sensor and flow will be
regulated down unnecessary We also had some problems regarding oxygenation during some
experiments A leaky oxygenator made a oxygenator replacement required during or a few
minutes prior to reperfusion Fortunately once experienced these difficulties we could
anticipate and take precautions during upcoming experiments
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
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ofd
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k B
iblio
grap
hy
27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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k B
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Recommendations for future research In this study we found that hypothermic oxygenated machine perfusion is superior to cold
storage when evaluating renal function during 4 hour normothermic perfusion in a pig
slaughterhouse model However we do suspect that adding mannitol dexamethason insulin
and more nutrients during reperfusion could improve kidney function More experiments
regarding the last experimental group should be performed to prove our suspicions
Conducting more experiment should make us more familiar with the perfusion techniques
allowing us to better anticipate on technical difficulties Defects in equipment could be solved
more quickly or can be prevented
Conclusions In this study we created a low cost normothermic machine perfusion DCD model with porcine
slaughterhouse kidneys A stable 4 hour pressure controlled perfusion is established with
mean pulsatile arterial pressure of 75 mmHg We were able to keep the perfusate temperature
37 degC and the partial oxygen pressure above 60 kPa After performing several experiments
regarding preservation and perfusion techniques 30 minutes of warm ischemia combined
with hypothermic oxygenated machine perfusion and additives during reperfusion seemed
superior to all other experimental groups However kidney function still remains suboptimal
compared to other studies Due to technical difficulties while performing experiments with
additives during normothermic machine perfusion in the 30WI+HMPO2+NMP+ group the
number of kidneys included in this experimental group is small Further research needs to be
conducted to determine the optimal way of delivering normothermic machine perfusion in the
reperfusion period
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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ofd
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Bibliography 1 Davis AE Mehrotra S McElroy LM et al The extent and predictors of waiting
time geographic disparity in kidney transplantation in the United States Transplantation 2014 May 2797(10)1049-57
2 Gerber LM Chiu Y Carney N et al Marked reduction in mortality in patients
with severe traumatic brain injury J Neurosurg 20131191583ndash1590
3 Maggiore U Cravedi P The marginal kidney donor Current opinion in organ
transplantation 19 (4) 372-380 (2014)
4 Paul KT Avezaat CJ Ijzermans JN et al Organ donation as transition work
Policy discourse and clinical practice in The Netherlands Health (London) 2014
Jul18(4)369-87
5 Perico N Cattaneo D Sayegh MH Remuzzi G Delayed graft function in kidney
transplantation Lancet 364 (9447) 1814-1827 (2004)
6 Kootstra G Daemen JH Oomen AP Categories of nonheartbeating donors
Transplant PRoc 1995272893-4
7 Tennankore KK Kim SJ Alwayn IP Kiberd BA Prolonged warm ischemia time
is associated with graft failure and mortality after kidney transplantation Kidney
Int 2016 89 3 648-658
8 Harper SJF Hosgood SA Nicholson ML et al The Effect of Warm Ischemic
Time on Renal Function and Injury in the Isolated Hemoperfused Kidney
Transplantation 200886 445ndash451
9 Ausania F White SA Pocock P Manas DM Kidney damage during organ
recovery in donation after circulatory death donors Data from UK National
Transplant Database Am J Transplant 2012 12932ndash936
10 Net M Valero R Almenara R et al Hepatic xanthine levels as viability predictor
of livers procured from non-heart-beating donor pigs Transplantation 2001 71
1232
11 Hosgood SA Nicholson ML Normothermic kidney preservation Curr Opin
Organ Transplant 2011 16 169
12 Johnson LB Plotkin JS Howell CD et al Successful emergency transplantation
of a liver allograft from a donor maintained on extracorporal membrane
oxygenation Transplantation 199763910-911
13 Magliocca JG Magee JC Rowe SA et al Extracorporeal support for organ
donation after cardiac death effectively expands the donor pool J Trauma 2005
58 1095-1102
14 Famey AC Singh RP Hines MH et al Experience in renal and extrarenal
transplantation with donation after cardiac death donors with selective use of
extracorporeal support J Am Coll Surg 20082061028-1037
15 Oniscu GC Randle LV Watson CJ et al In situ normothermic regional perfusion
for controlled donation after circulatory death--the United Kingdom experience
Am J Transplant 2014 Dec14(12)2846-54
16 Hessheimer AJ Billault C Barrou B Fondevila C Hypothermic or normothermic
abdominal regional perfusion in high-risk donors with extended warm ischemia
times impact on outcomes Transpl Int 2015 Jun28(6)700-7
17 Valero R Cabrer C Oppenheimer F et al Normothermic recirculation reduces
primary graft dysfunction of kidneys obtained from nonheart-beating donors
Transpl Int 2000 13303-310
18 Port FK Bragg-Gresham JL Metzger RA et al Donor characteristics associated
with reduced graft survival an approach to expanding the pool of kidney donors
Transplantation 2000232263-71
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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ofd
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27
19 Demiselle J Augusto JF Videcoq M et al Transplantation of kidneys from
uncontrolled donation after circulatory determination of death comparison with
brain death donors with or without extended criteria and impact of normothermic
regional perfusionTranspl Int 2016 29 4 432-442
20 St Peter SD Imber CJ Friend PJ Liver and kidney preservation by perfusion
Lancet 2002 359604-613
21 Hosgood SA van Heurn E Nicholson ML Normothermic machine perfusion of
the kidney better conditioning and repair Transpl Int 2015 Jun28657-64
22 Yang B Hosgood SA Harper SJ Nicholson ML leucocyte depletion improves
renal function in porcine kidney hemoreperfusion through reduction of
myeloperoxidase+ cells caspase-3 IL-1 beta and tubular apoptosis J Surg Res
2010164e351
23 Gawaz M Role of platelets in coronary thrombosis and reperfusion of ischemic
myocardium Cardiovasc Res 200461498
24 Vermeulen Windsant IC Snoeijs MG Hanssen SJ et al Hemolysis is associated
with acute kidney injury during major aortic surgery Kidney Int 2010
May77(10)913-20
25 Unger V Grosse-Siestrup C Fehrenberg C et al Reference values and
physiological characterization of a specific isolated pig kidney perfusion model J
Occup Med Toxicol 2007 2 1
26 Moers C Smits JM Maathuis MH Treckmann J van Gelder F Napieralski BP et
al Machine perfusion or cold storage in deceased-donor kidney transplantation N
Engl J Med 2009360(1)7ndash19
27 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
28 Munshi R Hsu C Himmelfarb J Advances in understanding ischemic acute
kidney injury BMC Med 2011911
29 Eltzschig HK Eckle T Ischemia and reperfusion--from mechanism to
translation Nat Med 2011171391ndash1401
30 Salvadori M Rosso G Bertoni E Update on ischemia-reperfusion injury in
kidney transplantation Pathogenesis and treatment World J Transplant 2015 Jun
245(2)52-67
31 Bon D Chatauret N Giraud S Thuillier R Favreau F Hauet T New strategies to
optimize kidney recovery and preservation in transplantation Nat Rev Nephrol
20128(6)339ndash47
32 Hosgood SA Barlow AD Yates PJ Snoeijs MGJ van Heurn ELW Nicholson
ML A pilot study assessing the feasibility of a short period of normothermic
preservation in an experimental model of non heart beating donor kidneys J Surg
Res 2011171(1)283ndash90
33 Nicholson ML Hosgood SA Renal transplantation after ex vivo normothermic
perfusion the first clinical study Am J Transpl 201313(5)1246ndash52
34 Moers C Pirenne J Paul A Ploeg RJ Machine Perfusion or Cold Storage in
Deceased-Donor Kidney Transplantation N Engl J Med 2012366(8)770ndash1
35 Brat A Pol RA Leuvenink HGD Novel preservation methods to increase the
quality of older kidneys Curr Opin Organ Transpl 201520(4)438ndash43
36 Nederlandse Transplanatiestichting 2015 URL
httpwwwtransplantatiestichtingnlnieuwsalle-donornieren-op-de-machine
geraadpleegd (6th July 2016)
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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and
blo
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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28
37 Hosgood SA Nicholson HF Nicholson ML Oxygenated kidney preservation
techniques Tranplantation 201293455
38 Thuillier R Allain G Celhay O Hebrard W Barrou B Badet L Leuvenink H
Hauet T Benefits of active oxygenation during hypothermic machine perfusion of
kidneys in a preclinical model of deceased after cardiac death donors J Surg Res
2013 Oct184(2)1174-81
39 Hosgood S Harper S Kay M Bagul A Waller H Nicholson ML Effects of
arterial pressure in an experimental isolated haemoperfused porcine kidney
preservation system Br J Surg 200693(7)879ndash84
40 Mancina E Kalenski J Paschenda P Beckers C Bleilevens C Boor P et al
Determination of the Preferred Conditions for the Isolated Perfusion of Porcine
Kidneys Eur Surg Res 201554(1-2)44ndash54
41 Bagul A Hosgood S Kaushik M Kay MD Waller HL Nicholson ML
Experimental renal preservation by normothermic resuscitation perfusion with
autologous blood Br J Surg 200895(1)111ndash8
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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and
blo
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30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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syst
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Ho
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29
Acknowledgements This study would not have been possible without the help of all people mentioned below
First of all many thanks to my faculty supervisor prof dr Henri Leuvenink for his
inspirational support and valuable feedback on the design and process of this study
Special thanks to my daily supervisor Leonie Venema who was always available to listen to
my problems and give advice Also for always accompanying me during all our experiments
most of all during our trips to the slaughterhouse at the crack of down I am especially
grateful for all her efforts pushing me to achieve more intellectual milestones then I imagined
reaching at the start of this project
Many thanks to dr Niels lsquot Hart for all his help regarding histological evaluation and most of
all making beautiful pictures of our stainings
Also special thanks to Aukje Brat and Merel Pool for all their help with the experiments from
preparing the kidney to cleaning everything up It would not have been possible without their
support
Last but not least many thanks to Jacco Zwaagstra Wendy Oost employees of the UMCG
surgical research department employees of slaughterhouse Hilbrants and Kroon and to all the
others who were otherwise involved
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Ho
ofd
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ghte
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ki
dn
eys
and
blo
od
30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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Ch
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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Ch
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erm
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nal
per
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on
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cuit
35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Ch
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Ch
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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syst
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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syst
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ho
ofd
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dn
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and
blo
od
30
Appendix 1 Protocol for organ and blood retrieval
Slaughterhouse kidneys and blood
Materials
- Blood collection
o 5L beaker
o Jerrycan
o Funnel
o 5ml25000 IE Heparine
o 5ml syringe with needle
- Kidneys (depending on the manner of transportation)
o General supplies
1L NaCl for flush
Surgical scissors
(sharp) 2x
Surgical forceps 3x
Clamps
Syringe 60 ML with
tip
Catheter (5cm) for
flush
Large gauze
(40x40cm)
Styrofoam box for
inspecting the
kidneys
Gloves
Trash bags
Pen + paper
o Cold storage
Organ bags
NaCL for storage
Transport box with crushed ice
o Hypothermic machine perfusion
Kidney assist +
sensors+ batteries
Oxygen bottle if
needed
KA Disposable
Canularsquos and patch
holder
UW- machine
perfusion solution
Sutures
20 ml syringe
Crushed ice
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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per
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Ch
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Ch
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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the
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
o Subnormothermic machine perfusion
Kidney assist + sensors+ batteries
Oxygen bottle if needed
KA Disposable adapted to fit the canula used for NMP
Oxygen bottle
Canula for artery
Cannula for urether
Sutures to secure cannula
Sutures to repair leakage if necessary
20 ml syringe
Blunt needle
Heat packs (place them in a 37degC incubator the night before)
500 ml Ringerslactate 37degC
Nacl 37degC
500 ml beaker
Scale
Protocol
Blood
- Put the Heparine in the 5L beaker with the syringe
- Catch about 3 liter blood with the beaker
- Poor the blood in a jerry can use a funnel if needed
Kidneys
- Fill the styrofoam box with ice and place the gauze on top Use a syringe and some
NaCL to wet the gauze Place the kidney pair on top with the aorta facing upwards
When the preferred ischemic time is passed Important in case of sNMP donrsquot use ice
or cold fluids
- Cut the aorta lengthwise in half and search the renal arteries Make sure you donrsquot
damage the renal arteries
- Fill the 60 ml syringe with cold NaCl and attach the catheter
- Place the catheter carefully in the renal artery and flush slowly Be careful not to apply
excessive pressure with the syringe Keep flushing until the kidneyrsquos aspect had
become uniformly pale and clear fluid runs from the vena
- Remove the catheter
- Remove the contra lateral kidney
- Store the kidney for transport
o Cold storage
Place the kidney in a organ bag with cold NaCl
Place this bag in a larger bag containing ice
Place the bag in a large transport box filled with ice
o Hypothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using UW-machine perfusion
Fill the Kidney Assist transport box with ice Donrsquot forget to open the
oxygen bottle if needed
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blo
od
32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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per
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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32
Figure 3 Kidney assist with disposable
After flushing the kidney remove excessive fat from the kidney except
near the urether and hilum Connect the aorta patch to the patch holder
Use an artificial cannula if needed Place the patch holder in the kidney
holder check for leakage with a 20ml syringe
Figure 4 Kidney with patch Figure 5 Patch connected to patch holder
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Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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33
Figure 6 Placement in kidney holder
Place the kidney holder inside the kidney assist reservoir and start
perfusion
Take a sample off the perfusate after 15 ml of perfusion and write
perfusion parameters down on the CRF
o Subnormothermic machine perfusion
While one person is taking care of the kidney flush another person has
to assemble and prime the Kidney Assist using 500ml warm ringers
lacate and 500ml whole blood Fill the Kidney Assist transport box
with the heatpacks Donrsquot forget to turn the oxygen bottle open
Once the kidney is flushed weigh the kidney and write it down
Remove all excessive fat from the kidney except near the urether and
hilum
Place the cannula in the renal artery and secure it with a suture Check
for leakage with a syringe
Place a cannula in the urether and secure it with a suture check for
leakage and correct placement with a bolus of warm NaCl by using
syringe and blunt needle
Place the kidney in the reservoir and start perfusion
Take a sample off the perfusate after 15 min of perfusion and write
perfusion parameters down
During the whole procedure note the following time points
- Time of death of the pig start warm ischemia
- Moment of starting flush end warm ischemia
- Moment were transportation starts start cold ischemia
- In case of HMP or sNMP take a sample at 15 minutes of perfusion and the end of
perfusion Also note the hemodynamics
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34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
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syst
em
43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
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ing
the
syst
em
44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
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ing
the
syst
em
45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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syst
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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ing
the
syst
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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b
34
Preparations at the lab
Leukocyte depleted blood
Materials
- Catheter bag
- Funnel with silicone tubing to connect to catheter bag
- Clamps
- Jerrycan filled with blood at the slaughterhouse
- Leukocyte filter (BioR O2 plus BS PF Fresenius Kabi Bad Homburg Germany)
- 2L beaker
Protocol
- Fill the catheter bag with blood using the funnel
- Close the inlet with a clamp
- Attach the leukocyte filter to the outlet off the catheter bag
- Hang the system to a hook an place the beaker underneath
- Open the outlet of the catheter bag and de-air the leukocyte filter place the beaker
underneath NB Make sure you keep an eye on the beaker there is always a risk of
overflow
A blood sample is analysed for Hematocrit and white blood cell count before blood enters the
NMP system
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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per
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Ch
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Ch
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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sys
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Ch
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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syst
em
44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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syst
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Ch
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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per
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cir
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35
Appendix 2 Protocol NMP
Normothermic regional perfusion circuit
Materials Cabinet with heater and thermostat
Kidney Assist (Organ Assist Groningen Netherlands) with heart assist software
Centrifugal pumphead (Deltastream DP3 MEDOS Medizintechnik AG Stolberg Germany)
Pressure sensor (Truewave disposable pressure transducer Edwards lifesciences Irvine
California USA)
Temperature sensor
Clamp-on flow sensor (ME7PXL clamp Transonic Systems Inc Ithaca NY)
Oxygenator with integrated heat exchanger (HILITE 1000 MEDOS Medizintechnik AG
Stolberg Germany)
Orgaan chamber
Arterial canula without patch (cannula for organ perfusion ndash 12F INFUSION Warszawa)
Waterbath
Luer Lock T- connector 14-14
Luer Lock T-connector 316-316
Connector 14-38
14 silicone tubing ndash 40 cm (2x)
14 silicone tubing ndash 15cm
14 PVC tubing ndash 35 cm
14 PVC tubing ndash 5 cm
14 PVC tubing - 60 cm
38 PVC tubing ndash30 cm
ndash 30 cm
Luer Lock three-way valves (2x)
Infusion tubing (2x)
Tie wraps
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36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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Ch
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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sys
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40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
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46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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per
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cir
cuit
36
Assembling the system Connect all the components above
- Connect the outflow of the pump head to the blood inlet of the oxygenator with 30 of
38 frac14 PVC tubing
- The blood outlet of the oxygenator is connected to a luer lock connector ( frac14 - frac14 ) with
10 cm silicon tubing The pressure sensor is connected to the luer lock connector with
the infusion tubing
- The other outlet on the oxygenator is connected to the infusion tube with at the and a
luer lock valve
- Connect 10 cm frac14 PVC tubing from the luer lock connecter with the pressure sensor to
the inlet of the organ chamber
- The outlet of the organ chamber is connected with a frac14 -38 connector A 30 cm 38
PVC tube is then attached and connected to the inlet of the pumphead
- The water bath is connected to the in- and outlet of the water compartment in the
oxygenator with 40 cm frac14 silicone tubing NB pay attention to the water flow the
outflow of the water bath should be connected to the inlet of the oxygenator
- Connect 60 cm frac14 PVC tubing to the oxygen inlet on the oxygenator and attach the
other end to the carbogen supply
- The temperature sensor floats in the organ chamber
- The clamp-on flow sensor is attached to the frac14 silicone tubing leading from the outlet
of the oxygenator to the luer lock connector with the pressure sensor It is optional to
use Vaseline to improve signal transduction
- Make sure every connection is tie wrapped to avoid leakage under pressure
Figure 2 Perfusion circuit
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37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
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Ch
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
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Ch
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
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Ch
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sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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nal
per
fusi
on
cir
cuit
37
Normothermic machine perfusion
Materials
- Assembled perfusion ciruit (See lsquonormothermic machine perfusion circuitrsquo)
- 300ml Ringerslactate
- 10ml Voluven
- 8ml 84 Natrium bicarbonate
- 90 mg Creatinine
- 100mg200mg Augmentin
- 20mgml Sodium nitroprusside (100microl of the dilution)500 ml leukocyte depleted
blood (See lsquoleukocyte depleted bloodrsquo)
Protocol
- Fill the water bath with purified water and set the temperature at 38degC
- Set the temperature off the external thermostat at 37degC this will regulate the
temperature inside the cabinet Place the temperature probe in the cabinet and turn the
heater in the cabinet on
- Prime the system with the priming fluid
o 300ml Ringerslactate
o 10ml Voluven
o 8ml 84 Natrium bicarbonate
o 90 mg Creatinine
o 100mg200mg Augmentin
o 100microl Sodium nitroprusside
- De-air the tubing leading from the organ chamber to the pump head passively Then
attach the pump head to the Kidney Assist pump unit
- Turn on the external flow unit
- Attach the pressure sensor temperature sensor and flow sensor to the pump unit
- Power on the kidney assist and follow the priming menu
o Press lsquopowerrsquo button
o lsquoSelftest OKrsquo press push-dial button
o ldquodisposable connectedrdquo press push-dial button
o ldquoPerfusate level OKrdquo press push-dial button
o In priming mode remove air from oxygenator bubble trap by opening the
valve on top of oxygenator Close valve once air is removed
o Remove air from infusion lines
o Turn valve on pressure sensor in direction of the perfusion circuit remove caps
on the pressure sensor Pull bleu snap tap and use a syringe to aspirate the
perfusate until a few drops drip out
o Press push-dial button to calibrate the pressure sensor
o Replace the caps on the pressure sensor and turn the valve in direction of the
side port
o press push-dial button and set pressure on 75 mmHg
o Stop when ldquoconnect heartrdquo shows on the display
- Open the carbogen source and set the flow regulator at 05 mlmin
- Add 500 ml leukocyte depleted blood
- Wait until the priming solution reaches 37degC before connecting the kidney
- Meanwhile prepare the kidney for perfusion
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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sio
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39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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ing
the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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syst
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42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
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44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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n
38
Preparing the kidney
Materials
- Icebox with crushed ice
- Large gauze (40x40)
- Syringe 60 ml and 20 ml
- Blunt needle
- Artery cannula
- Urether cannula
- Surgical instruments
- Biopsy gun
- 4 Formalin + biopsy holder and gauze
- SONOP
- Liquid nitrogen
- scale
Protocol
- Place kidney on wet gauze with crushed ice
underneath
- Remove all excessive fat from the kidney except near
the urether and hilum
- Place a cannula inside the urether and tie 2-0 braided
suture around distal end of urether to make sure it
remains in the same place Check for leakage and
correct placement with a bolus of NaCl by using a
syringe and blunt needle
- Place a cannula inside the renal artery secure it with a
suture and check for leakage using a syringe
- Weigh the kidney and write it down
- Take a biopsy using the biopsy gun Store one half in
formalin store the other half in SONOP in liquid
nitrogen
Perfusion
To start perfusion
- Place the prepared kidney in the organ chamber
- Check if the system is still free of air bubbles If not remove them
- Connect the artery cannula to the perfusion circuit make sure to keep the system air
free
- Press push-dial button to start perfusion
- Close the cabinet
During perfusion
Materials
- 1ml syringes
- 5 ml syringes
- 10 ml syringes
Figure 7 Cannulated kidney
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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sio
n
39
- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
sys
tem
40
Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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41
Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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- Infusion caps
- Beaker
- Crushed ice in a styrofoam box
- CRF
- Warm ringers lactate (place within the heat cabinet)
- 5 Glucose
- 5ml tubes
Protocol
- Place a beaker under the cannula of the ureter Make sure that the distal tip of this
cannula is below the level of the renal pyelum
- Write down the start time and hemodynamics on the CRF
- Take samples on given time points 05 ml from sample line and 05 ml from the vena
and analyse immediately using the ABL800 bloodgas analyser Store 5ml perfusate
drawn from the sample line on ice Before taking the sample draw some perfusate
from the sample line to remove death volume
- Replace the beaker underneath the cannula of the ureter at the correct time points
Store urine on ice
- Replace the sample and urine volume using the sample line 6ml for the samples + the
amount of urine collected
- Check the glucose concentration on the bloodgas results If the number drops below 8
mmolL add glucose according to the scheme
Figure 8 Kidney connected to NMP circuit
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Cleaning the system
Materials
- 4 formalin + biopsy holder
- Liquid nitrogen
- Filtration paper
- petridish
- Biotex
- Purified water
- Blade (mesje)
- ethanol
Protocol
- Shut down the Kidney Assist using the power button
- Disconnect the kidney and weigh it
- Take 2 large biopsies store 1 in formalin Place the other on a filtration paper and
place it on a petridish Then drop de petridish on the liquid nitrogen with the biopsy on
the upper side
- Discard the kidney following regulations
- Close the carbogen and disconnect tubing leading to the oxygenator
- Disconnect al sensors carefully
- Turn off heater inside cabinet (and external thermostat)
- Turn off the water bath and disconnect tubing leading to the oxygenator
- Remove the pump from the Kidney Assist
- Remove the perfusion circuit from the cabinet and disconnect Rinse tubing with
plenty of purified water until the tubing appears clean Then rinse it with more purified
water Rinse the oxygenator with plenty of purified water
- Dry the system and oxygenator using carbogen
- Clean cabinet with ethanol and close it
- Check if the area surrounding the experimental set up is clean
- Store all samples in -80 degC after 10 minutes centrifugation at 4degC 1000g Except for
the formalin biopsies they must be embedded in paraffin wax immediately
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
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Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
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Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
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Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
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Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
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Appendix 3 Results overview
Table 1 experimental groups
Group N= WIT (min) Transport CIT(hour) Reperfusion
1 (control) 4 30 CS 3 NMP
2 2 78 CS 2 NMP
3 2 20 CS 3 NMP
4 2 40 sNMP 2 NMP
5 4 30 HMP + O2 2-3 NMP
6 2 30 HMP - O2 3 NMP
7 2 30 HMP + O2 3 NMP +
No cold ischemia in this group because preservation of the kidney was with oxygenated
subnormothermic machine perfusion
Table 2 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of first 2 kidneys
Kidney 1 Kidney 2
Time Temperature Flow Diuresis Temperature Flow Diuresis
t=0 351 53 341 40
t=15 36 51 354 35 4
t=30 384 87 369 120 5
t=60 37 215 6 369 145 10
t=90 362 230 37 130 105
t=120 363 194 6 366 94 55
t=150 371 175 353 38 6
t=180 369 173 5 361 48 4
t=210 375 180 36 74 65
t=240 37 194 11 364 85 105
Total 28 Total 62
Table 3 Perfusate Temperature (degC) Renal bloodflow (mlmin) and diuresis (ml) of kidney 4
Time Temperature Flow Diuresis
t=0 323 25
t=15 339 156 35
t=30 367 196 9
t=60 373 204 17
t=90 372 205 115
t=120 371 205 15
t=150 374 204 16
t=180 36 190 12
t=210 371 184 105
t=240 373 183 8
Total 1025
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Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
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Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
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Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
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Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
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the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
42
Graph 4 Oxygen pressure in Perfusate
Graph 5 Glucose concentration in Perfusate
Graph 6amp4 Mean Renal blood flow in mlmin100 gram per experimental group
40
45
50
55
60
65
70
75
80
-10 15 30 60 90 120 150 180 210 240
kPa
Time
pO2
Kidney 1
Kidney 2
Kidney 4
0
2
4
6
8
10
12
-10 15 30 60 90 120 150 180 210 240
mm
ol
L
Time
Glucose
kidney 1
kidney 2
kidney 4
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+CS
0
50
100
150
200
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
7WI+CS
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
43
Graph 5amp6 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 7amp8 Mean Renal blood flow in mlmin100 gram per experimental group
Graph 9 Mean Renal blood flow in mlmin100 gram per experimental group
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
20WI+CS
0
20
40
60
80
100
120
140
160
0
15
3
0
50
7
0
90
1
10
1
30
1
50
1
70
1
90
2
10
2
30
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
40WI+sNMP
0
20
40
60
80
100
120
140
160
0 20 50 80 110 140 170 200 230
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2
0
20
40
60
80
100
120
140
160 0
15
3
0
50
70
9
0
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP-O2
0
20
40
60
80
100
120
140
160
0
15
30
50
70
90
11
0
13
0
15
0
17
0
19
0
21
0
23
0
flo
w (
ml
min
10
0gr
)
Time (min)
Flow
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
44
Table 4 statistical analysis of renal blood flow per time point using one-way ANOVA
Time point (min) Significance difference (p-value)
Time point (min) Significance difference (p-value)
0 0063 120 0833
10 0135 130 0954
15 0372 140 0971
20 0586 150 0986
30 0743 160 0997
40 0743 170 0998
50 0777 180 0998
60 0759 190 0998
70 0798 200 0997
80 0723 210 0991
90 0714 220 0979
100 0852 230 0968
110 0846 240 0930
` Graph 10 Mean urine production in mlmin per experimental group
Table 5 statistical analysis of urine production per time point in all groups using a Kruskal-Wallis H test
Time point (min) Significance difference (p-value)
15 0066
30 0163
60 0071
90 0065
120 0039
150 0050
180 0110
210 0078
240 0079
0
1
2
3
4
5
6
7
15 30 60 90 120 150 180 210 240
uri
ne
pro
du
ctio
n (
ml
min
10
0gr
)
Time point (min)
Urine production
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
45
Table 6 Post Hoc analysis of urine production per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
120 0062 0001 0008
150 0065 0002 0025
Graph 11 Mean creatinine clearance per experimental group
0
1
2
3
4
5
6
7
8
15 30 60 90 120 150 180 210 240
cle
aran
ce r
ate
(m
lm
in1
00
gr)
Time point (min)
Creatinine clearance
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 8 Post Hoc analysis of creatinine clearance per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
15 0645 0004 0001
90 0028 0000 0034
120 0043 0000 0157
180 0074 0000 0137
210 0052 0000 0165
Table 7 statistical analysis of creatinine clearance per time point
Time point (min) Significance difference (p-value) Test used
15 0000 One-way ANOVA
30 0252 Kruskal-Wallis H
60 0072 Kruskal-Wallis H
90 0000 One-way ANOVA
120 0050 Kruskal-Wallis H
150 0080 Kruskal-Wallis H
180 0046 One-way ANOVA
210 0030 One-way ANOVA
240 0066 Kruskal-Wallis H
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
46
Graph 12 Mean fractional excretion of sodium per experimental group
0
20
40
60
80
100
120
140
30 60 90 120 150 180 210 240
Frac
tio
nal
exr
eti
on
of
sod
ium
(
)
Time point (min)
Fractional exrection of sodium
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
Table 9 statistical analysis of FeNa+ per time point
Time point (min) Significance difference (p-value) Test used
30 0654 One-way ANOVA
60 0087 One-way ANOVA
90 0038 Kruskal-Wallis H
120 0027 Kruskal-Wallis H
150 0025 Kruskal-Wallis H
180 0040 Kruskal-Wallis H
210 0024 Kruskal-Wallis H
240 0031 Kruskal-Wallis H
Table 10 Post Hoc analysis of FeNa+ per time point
Timepoint (min) 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
90 0001 0029 0518
120 0001 0033 0189
150 0000 0012 0653
180 0000 0015 0876
210 0001 0019 0657
240 0000 0020 0548
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
47
Table 11 Mean values and standard deviation of kidney function and tissue injury parameters
Group Creatinine clearance (mlmin100gr) AUC
Edema FeNa+ () AUC
30WI+CS 24 (plusmn2794) 12 (plusmn 14) 16317 (plusmn186)
7WI+CS 578 (plusmn21953) 30 (plusmn01) 7309 (plusmn889)
20WI+CS 11 (plusmn51) 95 (plusmn1202) 17050 (plusmn2568)
40W+sNMP 160 (plusmn19814) 27 (plusmn14) 3896 (plusmn54)
30WI+HMP+O2 383 (plusmn25962) 1367 (plusmn 305) 7346 (plusmn6240)
30WI+HMP-O2 354 (plusmn285) 13 (plusmn848) 8685 (plusmn2356
30WI+HMP+O2+NMP+ 1250 15 3134
Table 12 Mean values and standard deviation of kidney function and tissue injury parameters
Group LDH concentration (UL) AUC
Serum Creatinine drop ()
Urine production accumulated (ml)
30WI+CS 172250 (plusmn 132080) 15 (plusmn 54) 61 (plusmn39)
7WI+CS 107190 (plusmn 6639) 75 (plusmn 42) 616 (plusmn83)
20WI+CS 212280 (plusmn 69176) 175 (plusmn 07) 34 (plusmn23)
40W+sNMP 111120 (plusmn 5441) 12 (plusmn 14) 33 (plusmn6)
30WI+HMP+O2 261603 (plusmn 203398) 67 (plusmn 249) 211 (plusmn112)
30WI+HMP-O2 143748 (plusmn 1118) 645 (plusmn 2333) 493 (plusmn447)
30WI+HMP+O2+NMP+ 348465 92 812
Table 13 statistical analysis of kidney function and tissue injury parameters
Variable Significance difference (p-value)
Creatinine clearance (mlmin100gr) AUC 0054
Edema 0279
FeNa+ () AUC 0027
LDH concentration (UL) AUC 0648
Serum creatinine drop () 0001
Urine production accumulated (ml) 0076
Table 14 Post Hoc analysis of kidney function parameters
Parameter 30WI+CS vs 30WI+HMP+O2
30WI+CS vs 30WI+HMPO2+NMP+
30WI+HMP+O2 vs 30WI+HMPO2+NMP+
FeNa+ () AUC 0032 0006 0589
Serum creatinine drop ()
0007 0001 0436
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
48
Table 15 statistical analysis of pH per time point
Time point (min) Significance difference (p-value)
-10 0836
15 0993
30 0953
60 0690
90 0349
120 0382
150 0237
180 0273
210 0168
240 0187
7
71
72
73
74
75
76
77
78
79
8
-10 15 30 60 90 120 150 180 210 240
pH
Time point (min)
pH 30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
0
500
1000
1500
2000
2500
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μL
)
Time point (min)
Lactate dehydrogenase
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2 30WI+HMP-O2
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+
A porcine slaughterhouse model for normothermic regional perfusion of kidneys
Ch
apte
r C
lean
ing
the
syst
em
49
Table 16 statistical analysis of LDH per time point
Time point (min) Significance difference (p-value)
15 0066
30 0061
60 0070
90 0098
120 0074
150 0079
180 0076
210 0070
240 0075
Table 17 statistical analysis of lactate per time point
Time point (min) Significance difference (p-value)
15 0438
30 0664
60 0647
90 0524
120 0596
150 0571
180 0501
210 0306
240 0737
10
12
14
16
18
20
22
24
26
15 30 60 90 120 150 180 210 240
con
cen
trat
ion
(μ
L)
Time point (min)
Lactate
30WI+CS
7WI+CS
20WI+CS
40WI+sNMP
30WI+HMP+O2
30WI+HMP-O2
30WI+HMP+O2+NMP+