Normothermic Kidney Perfusion · ** P

Sarah Hosgood

University of Cambridge

14th November 2015

Normothermic Kidney Perfusion

Refrigeration

Cooling: to Cell Metabolism

• Anaerobic metabolism

• ATP depleted

•

Physiological

Maintains cellular metabolism

• Oxygenation

• Aerobic metabolism

• ATP regenerated

• Pre-transplant assessment

• Pre-transplant therapies

Preservation: Warm or Cold

Static Cold Storage

Hypothermic Machine

Perfusion

Ex-vivo Normothermic

Perfusion (EVNP)

Flow probe

Centrifugal Pump

Pressure line

Oxygenator/heater

Infusion Pumps

Arterial

Venous

Urine collection

Venous

Reservoir

Ex-Vivo Normothermic Perfusion Circuit

Infusion fluids

Urine collection bag

Organ chamber

Venous reservoir

Oxygenator

Centrifugal pump

Isolated Organ Perfusion System

EVNP in Renal Transplantation

Resuscitation to improve early graft function

Pilot series of EVNP (n=36)

Pre-transplant quality and viability assessment

EVNP discarded human kidneys series (n=92)

Pre-transplant therapies and allograft modulation

Noble gases/ nanoparticles

Aim: To determine the feasibility, safety and efficacy of

normothermic perfusion in marginal donor kidney transplantation

vs

n=36 n=47

ECD 23 all ECD

DCD 8

SCD 5

Pilot Clinical Study

Back-table prep

- in ice

Recipient Anaesthetised Dissection Transplant bed

Kidney

cold flushed

EVNP 60 min

EVNP Human Pilot Study Protocol

• 1 unit cross-matched packed red cells + 250 ml crystalloid

• Mean arterial pressure 75 mmHg

• 34-37oC

• TPN (no lipid); Dexamethasone; PGI2

• Co-amoxiclav

• No leucocytes/ little complement

A period of ‘Ideal Perfusion”

EVNP Perfusate

Ex-vivo Normothermic Perfusion

EVNP CS P value

N=36 n=47

First CIT (hr) 10.6 ± 3.9 11.8 ± 3.8 0.240

EVNP (min) 63 ±16 -

Second CIT (min) 26 ± 20 -

Anastomosis (min) 26 ± 6 31 ± 4 0.060

Total ischaemia (hr) 12.9 ± 4.5 12.3 ± 3.7 0.614

Ischaemic Times

• Basiliximab (20mg days 0 & 4)

• Tacrolimus (7-10 ng/ml)

• MMF 0.5g bd

• Prednisolone 20mg 5mg 6/52

Immunosuppression

0

5

10

15

20

25

30

35

40

45

50

EVNP CS

IF

DGF

Results: Early Graft Function

Number of

patients

DGF: 4/36 vs 17/47

(11%) (36%)

P=0.011

Requirement

for dialysis in

first 7 days

0

5

10

15

20

25

30

35

40

45

50

EVNP CS

No AR

AR

Number of

patients

AR: 6/36 vs 9/47

(17%) (19%)

P=0.755

Results: Acute rejection

EVNP CS P value Graft survival 95. 7% 94.3% 0.510 Patient survival 100% 94.3% 1.000

Renal Function

n=36

Arterial thrombosis 0

Venous thrombosis 0

Renal artery stenosis 0

Ureteric stenosis 1

Infection 0

EVNP: Complications

EVNP (n=36 kidneys)

• Feasible and straightforward

• Appears to be safe

• May improve early graft function

in marginal donor kidneys

Summary

0

2

4

6

8

10

12

14

18h CS NP

AT

P:A

DP

ra

tio

ATP : ADP Ratio

**

** P <0.05

Pre CS 18h CS NP

0

5

10

15

20

25

30

35

40

Ex

pre

ssio

n o

f 7

0K

D H

SP

70

vo

lum

e d

ensi

ty (

mm

2)

HSP 70

**

** P <0.05

Reconditioning

0.0

20.0

40.0

60.0

80.0

100.0

120.0

5 10 15 30 60 90 120 150 180

ml/

min

/10

0g

Reperfusion time (minutes)

Mean Renal Blood Flow

Experimental Evidence

RCT of EVNP in DCD kidney Transplantation

CS Tx 1h NP

3 Centres

Cambridge, Newcastle and Guy’s

Primary Outcome Measure

Delayed graft function – Dialysis in first 7 days

Number of patients

Total sample size = 400 patients across the 3 centres

Tx CS

Degree of injury does not necessarily correlate (inversely) with

viability

What is required is a measure of the potential for recovery from injury

- Restoration of function ex-vivo

EVNP: Viability/Quality Assessment of Discarded Human Kidneys

October 2012 – May 2014

92 Kidneys Declined for Transplantation

Reasons for Decline

Poor Flush 20

Malignancy 18

PMH 11

Donor Age 11

Technical/Anatomical 12

HMP Parameters 8

Histology 6

Prolonged Ischaemia 5

No suitable recipient 1

Demographics (n= 74)

DBD 33

DCD 41

Donor Age (y) 60 ± 15 (31 – 78)

CIT (h) 28.7 ± 13.8 (8.2 – 76.5)

WIT (min) 13.3 ± 3.3 (9 – 20)

Declined Human Kidneys

EVNP for pre-transplant quality assessment

Declined Kidneys from DCD aged 42 years

Right Kidney: Arrival

Left Kidney: Arrival

5 min EVNP

5 min EVNP

60 min EVNP

60 min EVNP

Each kidney assessed after 60 minutes of EVNP

1. Visual assessment

1. Measurement of renal blood flow

2. Total urine output

Quality Assessment Score

Score I: Excellent perfusion (Pink and evenly perfused)

Score II: Moderate perfusion (Pink/purple and patchy appearance)

Score III: Poor perfusion (Global mottled purple appearance)

Score I Score II Score III

n = 27 n = 31 n = 16

Grading: Macroscopic Assessment

Area Sensitivity Specificity Threshold

RBF 0.892 88% (CI 62 - 99%)

72% (CI 59 - 83%)

< 63ml/min/100g

U/O 0.876 94% (CI 68 - 100%)

81% (CI (69 - 90%)

< 50ml

EVNP Assessment Score Macroscopic Score 1, 2, 3 = 1 - 3 RBF <63ml/min/100g = 1 U/O <50ml = 1 Total scores range from 1 to 5

Perfusion Parameter Thresholds

Declined kidneys (n=92)

•18 suspected malignancies (excluded)

•35 kidneys scored (1 - 2)

•25 kidneys scored (3 – 4)

•14 kidneys scored (5)

• 262 Unused kidneys (2013 – 2014)

81% suitable for transplantation

Potential extra transplants last year

= 170

Declined Kidney Series

QUALITY ASSESSMENT SCORE

EVNP Clinical Series

Score 1 – 2 n = 26

Score 3 – 4 n = 10

P value

Donor Age (y) 56.3 ± 11.4 57.3 ± 19.6 0.462

M : F 8 : 18 6 : 4 0.140

ECD 18 5 0.440

DCD 4 4 0.179

SCD 4 1 1.000

Score 1 – 2 Score 3 - 4 P value

Recipient Age (y) 55.9 ± 12.5 61.2 ± 10.4 0.261

M : F 18 : 8 6 : 4 0.700

Pre Dialysis 6 1 0.645

HD 14 4 0.711

PD 6 5 0.224

Demographics

Score 1 – 2 n = 26

Score 3 – 4 n = 10

P value

WIT (min) 13.8 ± 2.8 11.3 ± 2.1 0.196

CIT 1 (h) 10.7 ± 4.9 12.3 ± 3.4 0.332

EVNP (min) 65.2 ± 9.9 51.5 ± 13.9 0.002*

CIT 2 (h) 23.4 ± 17.2 33.3 ± 15.0 0.120

Anastomosis (min)

29.5 ± 7.4 26.5 ± 6.7 0.266

Demographics

Score 1 -2 n = 26

Score 3 – 4 n = 10

P value

PNF 0 0

DGF 1 3 0.056

SGF <10% Cr fall 5 3 0.149

Acute Rejection 3 4 0.076

0

100

200

300

400

500

600

700

800

Pre 1 2 3 4 5 6 7

Cr

(µ

mo

l/L

)

Post-transplant (Days)

Serum Creatinine Levels

Score 1-2

Score 3-4

P = 0.032

*

Outcome

Score 1 – 2 n = 26

Score 3 – 4 n = 10

P value

Complications 0 0

Patient Survival 100% 100% 1.000

Graft Survival 96.2% 100% 1.000

0

50

100

150

200

250

300

Score 1-2 Score 3-4

Cr

()µ

mo

l/L

)

Serum Creatinine (12 months)

P = 0.018

0

10

20

30

40

50

60

70

80

Score 1-2 Score 3-4

eG

FR

(m

l)

eGFR (12 months)

P = 0.047

Graph Function

Ischaemia Reperfusion Injury Directly administered to the kidney during EVNP

Transplantation Therapies

Nature Reviews Gastroenterology & Hepatology 10, 79-89 (February 2013)

Therapies

Mechanisms of Tissue Protection • TNFα mediated ICAM/VCAM

expression • Inhibition of NFκB activity • HIF1α dependent (EPO/VEGF)*

• Anti-apoptotic signalling

*Ma D et al JASN 2009;20:713 Zhao H et al Am J Transplant 2013;13:2006

Irani Y et al. Nephron Extra. 2011;1:272.

Therapies: Argon and Xenon

17h CS 1h EVNP

10m WIT

1: Argon 70% (n = 5) 2: Control 95%O2 (n = 5) 3: Nitrogen 70% (n = 6)

Reperfusion 3h

Effects of Argon Post-treatment on Renal Ischaemia-reperfusion Injury

0

10

20

30

40

50

60

70

Argon Control NitrogenRB

F (

ml/

min

/10

0g

)

EVNP Mean Renal Blood Flow

P = 0.020

0,0

10,0

20,0

30,0

40,0

50,0

60,0

0 5 10 15 20 25 30 35 40 45 50 55 60

RB

F (

ml/

min

/10

0g

)

Mean Renal Blood Flow - EVNP Argon

Control

Nitrogen

EVNP Results

0

50

100

150

200

250

300

350

400

Argon Control NitrogenRB

F (

ml/

min

/10

0g

)

EVNP Total Urine Output

P = 0.030

0

2

4

6

8

10

Argon Control NitrogenCr

Cl

(ml/

min

/10

0g

) EVNP Creatinine Clearance

P = 0.040

EVNP Results

0

10

20

30

40

50

60

Argon Control NitrogenRB

F (

ml/

min

/10

0g

)

Mean Renal Blood Flow

P = 0.055

0,00

20,00

40,00

60,00

80,00

100,00

0 5 10 15 30 60 90 120 150 180

RB

F (

ml/

min

/10

0g

) Renal Blood Flow

ArgonControlNitrogen

Reperfusion

0

100

200

300

400

500

600

Argon Control Nitrogen

Ur

ine

Ou

tpu

t (m

l)

Total Urine Output

P = 0.008

0

2

4

6

8

10

Argon Control Nitrogen

Cr

CL

(m

l/m

in/1

00

g).

h

AUC Creatinine Clearance

P = 0.016

Reperfusion

Therapeutics and Allograft Modulation during EVNP

CD31 (green) Nanoparticles (red) Overlay

Nanoparticles

Resuscitation

Viability/

Quality

Therapies

Ex-vivo Normothermic

Perfusion

Optimise early graft function

Increased use of

marginal kidneys

Stem cells

Gene therapy

Small molecules

Potential Roles for EVNP

The ice age is ending