A LARGE WATER DIURESIS DURING HYPOXIA: INTERVENTION WITH DDAVP AND FUROSEMIDE

by

Namhee Kim

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Physiology

Cardiovascular Sciences Collaborative Program

University of Toronto

© Copyright by Namhee Kim (2011)

ABSTRACT

Namhee Kim

2011 Master of Science Thesis Project

Department of Physiology

Cardiovascular Sciences Collaborative Program

University of Toronto

A Large Water Diuresis during Hypoxia:

Intervention with dDAVP and Furosemide

Acute kidney injury (AKI) is associated with renal medullary hypoxia. The medullary

thick ascending limb (mTAL) in the renal outer medulla is most susceptible to hypoxic injury,

due to marginal O2 supply and high O2 consumption. The objectives of this study were to

document the earliest effect of hypoxia (8% O2 for 2.5 hrs) on the mTAL function, and to identify

strategies to protect the mTAL from hypoxia. The earliest effect of hypoxia is large water

diuresis, due to a fall in the medullary osmolality and increase in vasopressinase. Desmopressin

acetate (dDAVP), a synthetic vasopressin analogue resistant to vasopressinase that may also

increase O2 delivery, prevented water diuresis. A low dose (0.8mg/kg) of furosemide may

significantly reduce the mTAL work without a large excretion of essential electrolytes. Large

water diuresis may be diagnostically valuable in detecting renal tissue hypoxia, and dDAVP and

furosemide may prevent AKI in the clinical setting.

ii

ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. David Mazer, whose encouragement, guidance and support kept me motivated during my two years in the Master of Science program. His knowledge, generosity and valuable advice have provided me the best possible opportunity to mature as a student. I am grateful to Dr. Mitchell Halperin, for his mentorship, expertise and insight, which inspired me to value the motivation, perseverance, and innovative thought essential for the pursuit of solutions to scientific questions. Without his contribution, this thesis would not have been possible. I would also like to thank Dr. Gregory Hare who, as my committee member, has always conveyed passion and enthusiasm for research and new discoveries in science, which enriched my growth and maturity as a student, a researcher, and a member in the scientific community. It is a great pleasure to show my gratitude to Surinder Cheema-Dhadli, Chee Keong Chong, and Stella Tang for their help in all of the animal experiments for this project, as well as Dr. Daniel Bichet and his laboratory for the radioimmunoassay results. Their support and contributions made the progress of this project possible. I also offer special thanks to Laura Voicu, for her valuable mentorship in the beginning of my Master’s program and providing me with support whenever needed. In addition, I am indebted to my lab members, Albert Tsui and Tina Hu, for their valuable critiques on this project and for helping me to troubleshoot experiments during my Master’s program. I am also grateful to Stephen Chan, for carrying out the Western blot experiments and enzymatic activity assay, which are an invaluable asset of my thesis project. I also thank the Departments of Physiology and Anesthesia for giving me the opportunity to learn and mature as a graduate student and Dr. Andrew Baker and his laboratory, for providing me the wonderful environment in which to work during my Master’s program. I also offer my special thanks to Sharon Klimosco, for her friendly assistance and support. I would like to offer thanks to many individuals who provided me with a very enjoyable and exciting environment to carry out my Master of Science project: Elaine Liu, Eugene Park, Mostafa El Beheiry, Ashley Joseph, and all the summer students who contributed to the lab throughout the two years. Lastly, I would like to thank God, my parents, for their love and guidance, and Hyunhee Kim, who as my sister and my mentor, continues to inspire me with her enthusiasm in research and the wonderful support she provided me during the two years of this project.

iii

TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………. ii ACKNOWLEDGEMENTS……………………………………………………………………. iii TABLE OF CONTENTS………………………………………………………………………. iv LIST OF FIGURES………………………………………………………...…………………... vii ABBREVIATIONS………………………………………………………………………...…... ix CHAPTER 1: INTRODUCTION……………………………………………………………… 1 1.1 Background & Rationale of the Study……………………………………………………… 2

1.2 The Kidney: Anatomy and Function of the Nephron……………………………………..... 5 1.3 O2 Supply to the Renal Outer Medulla …………………………………………………….. 10

1.4 The Function of the Renal Outer Medulla………………………………………………….. 12

1.4.1. Role of the Renal Outer Medulla in the Concentration of Urine………………... 13 1.4.1.1. Maintenance of High Osmolality in the Medullary Interstitial

Compartment…………………………………………………………………………… 13 1.4.1.2. Water reabsorption from the Collecting Duct…………………………. 18

1.5. Role of Vasopressin in the Renal Outer Medullary O2 Balance…………………………… 21 1.5.1. Vasopressin: Regulation of Blood Flow to the Renal Outer Medulla……………. 21 1.5.2. Vasopressin: Urea Reabsorption in the Inner Medulla…………………………... 23 1.6. Maintenance of O2 balance in the Renal Outer Medulla ………………………………….. 26

1.6.1. O2 supply: Desmopressin Acetate (dDAVP)……………………………………. 27 1.6.2. O2 demand: Furosemide…………………………………………………………. 30

1.7. Adaptive Response to Hypoxia……………………………………………………………. 33 1.8. Indices of Renal Tissue Hypoxia………………………………………………………….. 36 1.8.1. Plasma Erythropoietin (EPO)…………………………………………………… 36

1.8.2. Hypoxia inducible factor-1α (HIF-1α)………………………………………….. 38 1.8.3. Nitric Oxide Synthase (NOS)…………………………………………………… 41

1.9. Hypothesis and Specific Aims of the Study………………………………………………. 44 1.10. Synopsis of Thesis Results………………………………………………………………. 45

iv

CHAPTER 2: METHODS………………………. …………………………………………… 48 2.1. Experimental Protocol #1: HYPOXIA………………….. …………………………… 49

2.2. Measured Outcomes from Protocol #1……………………………………………….. 49 2.2.1. Urine and Renal Papillary Osmolality…………………………………………… 51 2.2.2. ELISA for Plasma Erythropoietin ……………………………………………… 52 2.2.3. Renal Medullary Protein Markers for Hypoxia…………………………………. 53 2.2.4. Treatment with dDAVP prior to exposure to Hypoxia………………………….. 54 2.2.5. Detection of Vasopressinase Activity…………………………………………… 55

2.3. Experimental Protocol #2: FUROSEMIDE…………………………………………… 57

2.4. Measured Outcomes: Protocol #2……………………………………………………… 58 2.4.1. Urine and Renal Papillary osmolality…………………………………………… 58

2.5. Statistical Analysis……………………………………………………………………... 58

CHAPTER 3: RESULTS………………………………………………………………………. 59

3.1. Part 1: Hypoxia…………………………………………………………………………. 60 3.1.1. Effect of Hypoxia on Urine Flow Rate…………………………………………. 61 3.1.2. Effect of Hypoxia on Creatinine Clearance and Electrolyte Excretion………… 62 3.1.3. Effect of Hypoxia on the Urine Osmolality…………………………………….. 63 3.1.4. Effect of Hypoxia on Renal Papillary Osmolality ……………………………... 68 3.1.5. Effect of Hypoxia on Plasma Vasopressinase Activity………………………… 70 3.1.6. Effect of dDAVP on Renal Papillary Osmolality………………………………. 73 3.1.7. Effect of dDAVP on Creatinine & Electrolyte Excretions……………………… 77 3.1.8. Signs of Renal Hypoxia and Effect of dDAVP Treatment……………………... 80 3.1.8.1. Blood Lactate Level……………………………………………... 81 3.1.8.2. Plasma EPO……………………………………………………... 81 3.1.8.3. Renal medullary protein markers of hypoxia………………….... 84 PART 1: Summary of Significant Results …………………………………………………… 88

3.2. Part 2: Furosemide………………………………………………………………………. 90 3.2.1. Dose-Effect of Furosemide on Urine Flow Rate………………………………... 91 3.2.2. Dose-Effect of Furosemide on Urine Osmolality……………………………….. 92 3.2.3. Dose-Effect of Furosemide on Papillary Osmolality…………………………… 94

3.2.4. Dose-Effect of Furosemide on Rates of Excretions of Na+, Cl- and K+… 96 3.2.5. Dose-Effect of Furosemide on Excretion of Magnesium……………….. 99 3.2.6. Dose-Effect of Furosemide on Serum magnesium……………………. 100 PART 2: Summary of Significant Results…………………………………………………….. .101

v

CHAPTER 4: DISCUSSION………………………………………………………………… 102

4.1. Summary of Results: HYPOXIA…………………………………………………….. 103

4.1.1. Hypoxia-induced Water Diuresis……………………………………………………... 105 4.1.1.1. Effect of Hypoxia on Urine Flow Rate & Osmolality……………………………. 105 4.1.1.2. Effect of Hypoxia on Renal Papillary Osmolality……………………………… .. 107 4.1.1.3. Effect of Hypoxia on Osmotic Equilibrium in the Collecting Duct…………….… 109

4.1.2. Effect of Hypoxia on Activity of Plasma Vasopressinase…………………………….. 111 4.1.2.1. Vasopressinase: Findings in Literature…………………………………………… 112 4.1.2.2. Hypoxia-induced Release of Vasopressinase: Compensatory Mechanism?........... 114

4.1.3. Hypoxia markers and effect of dDAVP pretreatment……………………………….. . 118 4.1.3.1. Level of Blood Lactate and Plasma Erythropoietin ……………………………... 118 4.1.3.2. Level of Renal Medullary Protein Expression …………………………………… 120 4.1.3.3. Desmopressin acetate (dDAVP): Synthetic Analogue of Vasopressin…………… 123 4.1.3.4. Increasing O2 delivery by dDAVP: Potential mechanism………………………… 126

4.1.4. Clinical Significance of the Hypoxia Study………………………………………….. 128

4.2. Summary of Results: FUROSEMIDE………………………………………………... 131

4.2.1. Dose of Furosmide that ↓ the Function of mTAL: 0.8 mg/kg of Body Weight in Rats...132

4.2.2. Danger of High Doses of Furosemide…………………………………………………..134 4.2.2.1. Depletion of Essential Electrolytes & Fluids……………………….………………135 4.2.2.2. Depletion of Mg2+: Risk of Hypomagnesemia…………………………………...…136

4.2.3. Clinical Significance of Furosemide: ↓ the work of mTAL…………………………... 138

CHAPTER 5: LIMITATIONS OF THE STUDY & FUTURE DIRECTIONS……………… 140

5.1. Limitations of the Study………………………………………………………………. 141

5.2. Future Directions………………………………………………………………………. 143

vi

LIST OF FIGURES

CHAPTER 1: INTRODUCTION

Figure 1-1. Anatomy of the nephron……………………………………………………………... 8 Figure 1-2. The urine concentrating mechanism in the superficial nephron…………………..... 16 Figure 1-3. Reabsorption of Na+ in the medullary thick ascending limb (mTAL)……………… 17 Figure 1-4. Vasopressin-mediated insertion of aquaporin-2 water channels and reabsorption of water from the renal medullary collecting duct…………………………………………………. 20 Figure 1-5. Reabsorption of urea in the inner medulla reduces the need of active reabsorption in the superficial region of renal medulla (mTAL)………………………………………………....25 Figure 1-6. The structures of vasopressin and desmopressin acetate (dDAVP)…………………29 Figure 1-7. Furosemide inhibits Sodium-Potassium-2Chloride (NKCC2) co-transporter in the medullary thick ascending limb (mTAL)……………………………………………………….. 32 Figure 1-8. Hypoxic-inducible factor (HIF) pathway under normoxia and hypoxia…………… 40

CHAPTER 2: METHODS

Figure 2-1. Experimental timeline for Hypoxia experiment……………………………………..50 Figure 2-2. Experimental timeline for Furosemide experiment……………………………….... 57

CHAPTER 3: RESULTS

Figure 3-1. Urine flow rate and osmolality in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2)………………………………………………………………………………… 64 Figure 3-2. Creatinine excretion and clearance in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2)……………………………………………………………………………….... 65 Figure 3-3. Renal excretion of Na+, Cl- and K+ in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2)………………………………………………………………………………… 66 Figure 3-4. Hypoxia-induced water diuresis is prevented with pretreatment with desmopressin acetate (dDAVP) 1 hr prior to hypoxia exposure……………………………………………… 67 Figure 3-5. Urine and renal papillary osmolalities of rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). …………………………………………………………………………69 Figure 3-6. Total change in the absorbance at 380nm detected in the plasma samples of rats exposed to normoxia (21% O2) or hypoxia (8% O2)……………………………………………. 71 Figure 3-7. Activity of plasma vasopressinase, expressed as the rate of production of 1 nmole of p-nitroaniline from S-benzyl-L-cysteine-p-nitroanilide by plasma vasopressinase per minute (mIU/min), in rats exposed to normoxia (21% O2) or hypoxia (8% O2)………………………... 72 Figure 3-8. The fall in the measured renal papillary osmolality is prevented with pretreatment with dDAVP 1 hr prior to hypoxia exposure……………………………………………………. 75 Figure 3-9. The pretreatment with dDAVP prior to exposure to hypoxia prevents the decrease in the urea composition in the renal medullary interstitial compartment………………………….. 76 Figure 3-10. Creatinine excretion and clearance in dDAVP-pretreated rats exposed to normoxia (21% O2) or hypoxia (8% O2)…………………………………………………………………… 78

vii

Figure 3-11. Renal excretion of Na+, Cl- and K+ in dDAVP-pretreated rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2).……………………………………………………….. 79 Figure 3-12. Blood lactate concentration in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2), with or without pretreatment with dDAVP………………………………. 82 Figure 3-13. Plasma erythropoietin (EPO) in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2), with or without pretreatment with dDAVP……………………………….. 83 Figure 3-14. Renal medullary protein expression in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2) with or without pretreatment with dDAVP…………………………………85 Figure 3-15. Renal outer medullary protein expression in untreated and treated with dDAVP rats under normoxia (21% O2)……………………………………………………………………….. 86 Figure 3-16. Urine flow rate in rats injected with different doses of furosemide ………………………………………………....…...…………………………………………….91 Figure 3-17. Urine flow rate and osmolality in rats injected with different doses of furosemide………………………………………………………………………………………..93 Figure 3-18. Urine and renal papillary osmolality in rats injected with different doses of furosemide …………………………………………………………………..…...………………95 Figure 3-19. Excretion of electrolytes (Na+, Cl- and K+) in rats injected with different doses of furosemide………………………………………………………………………………………..97 Figure 3-20. Excretion of electrolytes ([Na+ + K+] and Cl-) in rats injected with different doses of furosemide …………………………………………………………………………………...…..98 Figure 3-21. Excretion of Mg2+ in rats injected with different doses of furosemide …..……….. 99 Figure 3-22. Serum level of Mg2+ in rats injected with different doses of furosemide ..…….... 100 CHAPTER 4: DISCUSSION

Figure 4-1. Summary of Major Findings in this Study………………………………………….130

viii

ABBREVIATIONS

AC AKI ANP

adenyl cyclase acute kidney injury atrial natriuretic peptide

AQP AVP

aquaporin water channel arginine vasopressin

AVR ascending vasa recta BSA bovine serum album Ca2+ calcium cAMP CD

cyclic adenosine monophosphate collecting duct

Cl- chloride dDAVP DCT

desmopressin acetate distal convoluted tubule

DtL descending thin limb of Henle’s loop DVR descending vasa recta EDTA ethylenediaminetetraacetic acid ELISA ENaC

enzyme-linked immunosorbent assay epithelial sodium channel

eNOS endothelial nitric oxide synthase EPO erythropoietin O2 oxygen H+ hydrogen HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIF HO

hypoxia-inducible factor heme oxygenase

iNOS inducible nitric oxide synthase K+ potassium kDa L-NAME

kilodalton N-nitro-L-arginine methyl ester

Mg2+ MgCl2

magnesium magnesium chloride

mRNA messenger ribonucleic acid mTAL medullary thick ascending limb Na+ sodium NaCl NH4+ Na-K-ATPase

saline ammonium sodium-potassium-adenosine triphosphatase pump

NKCC2 sodium - potassium – 2 chloride channel nNOS neuronal nitric oxide synthase NO nitric oxide NOS PCT

nitric oxide synthase proximal convoluted tubule

PHD prolyl hydroxylase PKA protein kinase A ROMK renal outer medullary potassium channel SEM standard error margin

ix

x

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TBS VEGF

Tris-buffered saline vascular endothelial growth factor

CHAPTER 1

INTRODUCTION

1

1.1. Background and Rationale of the Study

Acute kidney injury (AKI) is a significant complication of cardiac and other types of

surgery, and it has serious clinical implications for critically ill patients (12). AKI is

characterized by an abrupt and sustained decreased in glomerular filtration rate, tubular

dysfunction and impaired sodium and water reabsorption (54; 55). The incidence of AKI ranges

from 1-40% (1; 45; 151) following cardiac surgeries, and it is repeatedly associated with high

morbidity and mortality. Although mortality has recently improved marginally, the incidence of

AKI is increasing (13; 15; 95; 120).

The poor clinical outcomes associated with AKI are likely due to the lack of conventional

markers of kidney function for clinical detection of early injury to the kidney (11). Current

diagnostic tools for AKI include an elevation of serum creatinine or a poor urine output (42;

119). However, serum creatinine concentration is a poor marker of early renal dysfunction,

because it is influenced by non-renal factors and does not reflect the real-time changes in kidney

function (27; 42). Recent human studies have introduced several proteins and biochemical

markers as sensitive and specific biomarkers that are capable of early detection of acute tubular

injury (77; 78; 138). These markers include N-acetyl-β-D-glucosaminidase (NAG), neutrophil

gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), cystatin C, and

interleukin-18 (IL-18) (22; 78; 79; 98; 105; 105; 121; 137; 139; 175; 186). However, there are

discrepancies in the perioperative levels of urinary and serum biomarkers and time points of the

peak levels between the studies, thus there is currently a lack of standardization for measuring

these urinary biomarkers (78).

2

One of the major underlying causes of AKI is renal tissue hypoxia. Inadequate supply of

O2 is insufficient compared to demand for O2 to perform renal work. Renal tissue hypoxia is an

important pathophysiological factor in the development of AKI, due to the effects of inadequate

O2 to maintain cytoskeletal structure, membrane physiology, and protein synthesis (24). AKI

may occur in a perioperative setting due to hypoperfusion of the kidney and systemic

redistribution of the decreasing O2 supply in favor of vital organs during periods of hypoxia,

which predisposes the kidneys and thus outer renal medulla, to hypoxic injury. The renal outer

medulla normally functions at very low O2 tension, and therefore is vulnerable to injury, which is

crucial for the development of hypoxic acute necrosis of the renal tubules (111). Under normal

physiological conditions, the O2 supply closely approximates the O2 consumption in the renal

outer medulla (150). This is mostly due to active reabsorption of Na+ and Cl- in the medullary

thick ascending limb (mTAL) in the renal outer medulla, which establishes the concentration

gradient in the interstitium (86; 177). The high osmolality gradient in the renal outer medullary

interstitial is required for the reabsorption of water from the medullary collecting ducts and the

production of concentrated urine. Most of tubular segments have a limited capacity to generate

anaerobic energy and therefore are heavily dependent on O2 to maintain active reabsorption in

the renal outer medulla (57), which renders this region vulnerable to hypoxic injury.

Since renal outer medulla is especially susceptible to hypoxic injury, it is likely that the

earliest physiologic indicator of tissue hypoxia may originate from this region. Thus it was our

goal to examine the effect of hypoxia on the function of the renal outer medulla and to determine

the earliest indicator of hypoxic injury in this region. We expected that the earliest indicator of

hypoxia in the renal outer medulla is the reduced work in the medullary thick ascending limb

(mTAL) in the renal medulla, due to impaired active reabsorption that would lower the

3

osmolality in the medullary interstitial compartment. We expected to find a low urine osmolality

as an early sign of AKI (86; 111), but the exact underlying mechanisms are currently undefined.

Therefore, the purpose of this study is to examine the effect of low supply of O2 as a single

stimulus to impair the function of the mTAL, and to document whether this may be the earliest

physiologic indicator of hypoxic injury in the renal outer medulla.

4

1.2. The Kidney: Anatomy and Function of the Nephron

The kidney is the key organ that is responsible for many processes involved in

homeostasis, such as regulation of Na+, K+, Ca2+ and Mg2+ and water homeostasis, control of

blood pressure, and the maintenance of acid-base balance. Apart from its homeostatic and

excretory functions, the kidney also has endocrine functions. The kidney is the major source of

erythropoietin (EPO) production, which is the main factor for red blood cell formation. Renin,

which acts on the Renin-Angiotensin System (RAS) and mediates arterial vasoconstriction, is

also produced in the kidney and regulates blood pressure.

The basic structural functional unit of the kidney is the nephron, where plasma is filtered,

and excretes the rest of the filtrate as urine. There are two types of nephrons in the kidney: short

superficial nephrons (~85%) that extend down to the renal outer medulla and long

juxtamedullary nephrons (~15%) that extend further deep into the inner medulla (Figure 1-1).

The function of the nephron is regulated by hormones, including vasopressin, aldosterone and

parathyroid hormone, which act on the different segments of the nephron. Each segment of the

nephron is highly specialized in unique function of its own. The segments are glomerulus, the

proximal convoluted tubules (PCT), loop of Henle, and distal convoluted tubules (DCT), which

carry the filtrate to the collecting duct (CD). The nephron has 3 functional units, which are

divided based on water permeability. The first functional unit is the PCT, which is always

permeable to water due to constitutive presence of aquaporin-1 (AQP1) water channels. The PCT

is followed by the middle functional unit, the Loop of Henle (LH), which is always impermeable

to water due to lack of aquaporin water channels in the superficial nephrons. However,

aquaporin-1 water channels are present in the longer LH of juxtamedullary nephrons (75; 187),

5

thus water is permeable in these segments (75). The last functional unit includes the late DCT,

and cortical and medullary CD, and this unit has variable water permeability that is dependent on

the insertion of aquaporin-2 (AQP2) channels via action of vasopressin (76).

Blood entering the kidney is filtered in the glomerulus, and the filtrate moves to the PCT,

where 80% of the filtered Na+ and water are reabsorbed. The filtrate continues to the LH, where

the hairpin tubular structure bends into the renal medulla. The LH consists of descending and

ascending limbs, which have thin and thick membranes that serve distinct functions along the

loop. The main function of this nephron segment is to establish a high osmolality in the

interstitial compartment of the outer medulla which, through reabsorption of Na+ and Cl-, and is

required for the production of concentrated urine. The medullary thick ascending limb (mTAL)

is responsible for the generation of high osmolality in the medullary interstitial compartment, and

the detailed description of the function of mTAL is discussed in Section 1.4. After loop of Henle,

the DCT receives the hypotonic filtrate, and reabsorbs the remaining of the needed Na+ and Cl-

under the influence of hormones such as aldosterone. In this nephron segment, there are flow-

activated epithelial Na+ channels (ENaCs), which reabsorb Na+ and Cl- during periods of large

diuresis to help desalinate the filtrate and prevent loss of Na+ and Cl-. When vasopressin is

present, this nephron segment is permeable to water owing to the insertion of AQP2 and a higher

interstitial than luminal osmolality. Under the influence of parathyroid hormone, the distal

convoluted tubules reabsorb Ca2+, and this process prevents excessive Ca2+ excretion, because

the reabsorption of Ca2+ is not complete in more proximal segment of the nephron, such as the

PCT and the mTAL. After passing through DCT, the filtrate enters the cortical and medullary

collecting ducts, which are the last segment of the nephron, where under the action of

vasopressin, water reabsorption occurs via activated AQP2 channels. The reabsorption of water

6

can achieve osmotic equilibrium between the renal medullary interstitial compartment and the

lumen of the CD, and thereby produce concentrated urine.

7

Figure 1-1. Anatomy of the nephron. The top dashed horizontal line represents the boundary between the renal cortex and the outer medulla, and the bottome dashed line marks the beginning of the region of inner medulla. The loop of Henle of superficial nephrons (~85% of total number of nephrons) extend down to renal outer medulla, whereas the juxtamedullary nephrons (represented by dashed loop) extend further down to the inner medulla. The filtration begins in the glomerulus, followed by the proximal convoluted tubule (PCT), the descending thin (DtL) and ascending limbs (mTAL) of the loop of Henle, the distal convoluted tubule (DCT), and the cortical (CCD) and medullary collecting ducts (MCD).

8

Acute failure of the kidney may occur with complication of surgeries, if kidneys do not

receive adequate blood supply for an extended period of time. In fact, renal tissue hypoxia due to

hypoperfusion of the kidney is highly associated with development of acute kidney injuries (117).

As previously mentioned, the renal outer medulla is most susceptible hypoxic injury and renal

medullary hypoxia is repeatedly associated with development of AKI. In these cases, a mismatch

between O2 supply and O2 consumption occurs within the renal outer medulla, where O2 supply

cannot accommodate the O2 consumption in the medullary thick ascending limb (mTAL), the

area with a high metabolic demand. Therefore, to understand the development of acute kidney

injury associated with renal tissue hypoxia, it is important to focus on the physiology and

underlying mechanisms of the function of the renal outer medulla, and possible defense

mechanisms that may minimize the risk of hypoxic injury in this region. The physiology of O2

balance in the kidney and functions of the renal outer medulla are discussed in the following

sections.

9

1.3. O2 Supply to the Renal Outer Medulla

The O2 supply to the renal outer medulla is regulated by three factors: renal medullary

blood flow, countercurrent exchange mechanism in the vasa recta, and plasma skimming in the

microcirculation perfusing the renal medulla. The three factors govern the oxygen balance in the

renal outer medulla, and provide sufficient O2 delivery for the active reabsorption while limiting

the O2 supply to prevent buildup of oxidative radical species deep in the renal medulla, where

most of the work is passive.

Although the kidneys receive a quarter of cardiac output, approximately 90% of the renal

blood flow remains in the cortex and remaining 10% of the blood flow perfuses the renal

medulla (114). The blood perfusing the renal outer medulla primarily originates from the efferent

arterioles of the juxtamedullary glomeruli, which comprise approximately 10% of the total

number of glomeruli in the kidney (61). These efferent arterioles of juxtamedullary nephrons

have distinct characteristics that allow subtle control over regional perfusion in the renal medulla

separate from the cortex (135). Juxtamedullary efferent arterioles give rise to descending vasa

recta (DVR) in the outer medulla (61; 135). Since medullary circulation arises from small sub-

population of juxtamedullary efferent arterioles, large changes in blood flow in the renal medulla

can occur with relatively minor changes in the total renal blood flow (61). Studies have shown

that smooth muscle cells and pericytes on DVR can modulate blood flow in the renal medulla

(114). The smooth muscle cells on DVR are gradually replaced by pericytes as vasculature

courses towards the outer medulla (61). These contractile cells are under the influence of a

number of factors, including angiotensin II, atrial natriuretic peptide and vasopressin, the last

being the most potent vasoconstricting agent (67; 133; 134; 140). Limiting the blood flow to the

10

renal medulla prevents washout of the concentration gradient established by active reabsorption

(114; 133). As many DVRs turn back towards the cortex, the vessels merge into ascending vasa

recta (AVR) and dense capillary bundles place DVR and AVR in close proximity (61; 133). This

arrangement allows for the countercurrent exchange between the vasa recta that leads to O2

shunting, which limits the O2 supply deep in the outer medulla (135). The countercurrent

exchanger also serves to trap NaCl and urea deposited in the medullary interstitial compartment

by the loops of Henle and collecting ducts (135). This mechanism also serves a physiological

role in limiting O2 supply to the deep parts in the renal medulla, where few nephrons exist and

the bulk of the work occurs via passive reabsorption. The maintenance of low oxygen supply to

the deeper renal medulla prevents accumulation of radical oxygen species in the deep medullary

region (188).

Another factor that limits the O2 supply in deeper areas in the renal outer medulla is a

phenomenon called plasma skimming, which results in a low hematocrit of the blood supply

feeding this region (88). Plasma skimming occurs when a juxtamedullary arteriole branches from

the interlobular arteries in a steep angle. Although this mechanism may improve blood flow to

the renal medulla due to a reduced viscosity, there is lower O2 supply to the deeper areas in the

renal medulla. The O2 balance in the renal outer medulla is discussed in Section 1.5.

11

1.4. Function of the Renal Outer Medulla

Hypoxia is an important pathogenic factor in many renal diseases, and it is also highly

associated with acute kidney injuries induced by ischemia/reperfusion or nephrotoxicity (21).

Under normal physiological conditions, the renal medulla operates at a lower PO2 compared to

the renal cortex. Within the renal medulla, the bulk of supplied O2 is consumed by the outer

medulla and the medullary thick ascending limb (mTAL) in the renal outer medulla functions at

a brink of hypoxia, with marginal O2 supply and high O2 consumption due to active reabsorption

of Na+ (39). In fact, medullary hypoxia is suggested to be one of the major factors involved in

the pathogenesis of acute kidney injury, (149) which is reflected by an early decrease in the urine

concentrating ability of mTAL in the renal outer medulla (38). Therefore, I hypothesized that the

earliest response of the kidney to tissue hypoxia will originate in the renal outer medulla.

12

1.4.1. Role of the Renal Outer Medulla in the Concentration of Urine

The major role of this region is to conserve water during periods of dehydration. As a

result, Na+ and Cl- are excreted in a hypertonic form when there is a low intake of water. There

are three mechanisms that are required for this function. First, as previously mentioned, the

countercurrent exchange mechanism in the vasa recta in the renal outer medulla minimizes the

washout of osmoles from the medullary interstitial compartment. The high osmolality in the

medullary interstitial compartment is generated by active reabsorption of Na+ and Cl- by the

mTAL in the outer medulla without water. However, this step occurs after reabsorption of water

in the collecting duct (CD), which occurs when vasopressin inserts AQP2. Overall, the role of

the renal outer medulla in the urine concentrating mechanism is to maintain the hyperosmolality

in the interstitial and therby, the reabsorption of water from the lumen of the medullary CD.

1.4.1.1. Maintenance of High Osmolality in the Medullary Interstitial Compartment

The current view of the concentrating mechanism in the renal outer medulla requires

modification owing to the fact that there are no aquaporin-1 (AQP1) channels in the majority of

descending thin limbs (DtLs) (187), which implies that this nephron segment is impermeable to

water. This indicates that high luminal osmolality in the bottom of Loop of Henle (LH) is

established by the entry of Na+, rather than removal of water from the lumen of the DtL (Figure

1-2). According to Halperin et al.(75), passive Na+ entry increases the concentration of Na+ in

the lumen of the DtL as it descends deeper into the renal medulla. The filtrate leaving the mTAL

13

is hypotonic, due to the reabsorption of Na+ and Cl- in the mTAL, which raises the osmolality in

the medullary interstitial compartment.

There are two mechanisms by which Na+ is reabsorbed in the mTAL in the outer

medulla, and each mechanism is dependent on the reabsorption of K+ into the lumen by the renal

outer medullary potassium (ROMK) channels (Figure 1-3). Half of Na+ is reabsorbed by the cells

of mTAL via Na-K-Cl2 (NKCC-2) cotransporter, driven by the low intracellular concentration of

Na+ due to the active export of Na+ by the Na-K-ATPase in the basolateral membranes of cells in

the mTAL (76). The low concentration of Na+ in mTAL cells is generated by the Na-K-ATPase

via active reabsorption of Na+. Rest of reabsorption of Na+ occurs via paracellular Na+ channels

driven by the positive voltage in the lumen of mTAL, which results from the build up of K+

established by ROMK channels (76). The lumen-positive voltage also drives the electrogenic

reabsorption of the remaining half of the Na+ that is reabsorbed between cells of the mTAL. The

reabsorption of Na+ and Cl- into the mTAL serves two functions. First, only a small portion of

reabsorbed Na+ is added to medullary interstitial compartment. This component of Na+ maintains

the high concentrations of Na+ that allows reabsorption of water from the water-permeable

nephron segments. The rest of reabsorption occurring in the mTAL is recycled back to the DtL,

which raises the luminal concentration of Na+ in the DtL (75). The recycling of Na+ into DtL

represents the work that conserves the source of Na+ to maintain the hypertonic medullary

interstitial compartment in the mTAL (75).

The traditional view of urine concentrating mechanism considers the reabsorption of Na+

and Cl- as the first step of the process. However, mTAL does not have a mechanism to detect

how much Na+ and Cl- is reabsorbed to the outer medullary interstitial compartment that

provides an osmotic driving force to reabsorb the desired volume of water (75). An excessive

14

reabsorption of Na+ and Cl- may increase the risk of forming kidney stones in the outer

medullary interstitial compartment. Halperin et al (75) suggests that the urine concentrating

mechanism may begin with reabsorption of water from medullary collecting duct. Reabsorption

of water dilutes the medullary interstitial compartment, which decreases the concentration of

unknown signal that inhibits Na+ and Cl- reabsorption. The suggested candidate is Ca2+, because

there is a Ca2+-binding receptor at the basolateral membrane of cells of the mTAL, and binding

of Ca2+ to the receptors generates a signal that inhibits ROMK channels (75; 80; 180). The

inhibition of ROMK channels prevents the reabsorption of Na+ and Cl- in the mTAL via NKCC-

2 and the paracellular route, since both pathways depend on luminal source of K+ in the lumen.

When the concentration of inhibitor falls in the medullary insterstitial compartment, the

reabsorption of K+ by ROMK channels increases, and subsequently the reabsorption of Na+ and

Cl- in the mTAL increases as well, completing the mechanistic loop.

15

Figure 1-2. The urine concentrating mechanism in the superficial nephron. The concentration of urine requires a hypertonic renal interstitial compartment, where concentration gradient increases deeper in the loop of Henle (LH). Since descending thin limbs (DtL) of superficial nephrons lack aquaporin-1 (AQP1) channels, the concentration of Na+ in the bottom of LH increases by the passive entry of Na+ (and not reabsorption of water) from the renal medullary interstitial compartment. The medullary thick ascending limb (mTAL) reabsorbs Na+ via both active and passive processes, and the passive reabsorption of Na+ results from high positive voltage in the lumen of mTAL. Majority of Na+ reabsorbed in the mTAL is recycled back into the lumen of DtL, and the rest of reabsorbed Na+ is used to generate hypertonic renal medullary interstitial compartment. The concentrated urine is produced when reabsorption of water leads to osmotic equilibrium between the medullary interstitial compartment and the medullary collecting duct. The numerical values represent mmol of Na+/day delivered to the nephron segment. Figure adapted from Halperin et al., 2010 (75).

16

Figure 1-3. Reabsorption of Na+ in the medullary thick ascending limb (mTAL). The reabsorption of Na+ and Cl- in the mTAL occurs via Sodium-Potassium-2Chloride (NKCC2) co-transporter, which is controlled by an inhibitory signal, believed to be Ca2+ in the renal medullary interstitial compartment. A high concentration of Ca2+ inhibits the renal outer medullary potassium (ROMK) channel, which also prevents reabsorption of NKCC2, since NKCC2 co-transporter requires high concentration of K+ to function. The driving force of NKCC2 is also a negative voltage in the cells lining the luminal membrane of mTAL, established by the Sodium-Potassium-ATPase (Na-K-ATPase), which pumps 3Na+ out and 2K+ into the mTAL cells. The reabsorption of water from the renal medullary collecting duct lowers the concentration of Ca2+ in the medullary interstitial compartment, removing the inhibitory control on the ROMK channel, which increases the lumen-positive voltage and allows active reabsorption of Na+ and Cl- by NKCC2 co-transporter. The buildup of positive voltage in the mTAL lumen by ROMK also provides driving force for the passive reabsorption of Na+, Ca2+ and Mg2+, which occur via paracellular route. Adapted from Halperin et al., 2010 (76).

17

1.4.1.2. Water Reabsorption from the Collecting Duct

The production of concentrated urine is the result of the reabsorption of water in the

medullary collecting duct (CD). As previously mentioned in the last section, the reabsorption of

water is the first step of the urine concentrating mechanism. This step requires the presence of

AQP2 channels on the CD, which allows osmotic equilibrium between the lumen of CD and the

medullary interstitial compartment.

When there is a need to conserve water, the rise in the plasma concentration of Na+

triggers the release of vasopressin, which is an antidiuretic hormone. The osmoregulatory system

of vasopressin is very sensitive and detects even a small change (1-2%) in the plasma osmolality

(173). The major role of vasopressin in the kidney is to increase water permeability in the

collecting duct, via insertion of AQP2 channels (Figure 1-4). The binding of vasopressin to V2

receptors activates adenyl cyclase via the G-coupled protein pathway, which subsequently

increases intracellular cAMP in the distal nephron segments (29; 82). The increase in cAMP

activates cAMP-dependent protein kinase A (PKA), which leads to phosphorylation of AQP2

(29; 64) and thereby trafficking to AQP2-bearing vesicles to the apical plasma membrane of

collecting duct principal cells (29; 82). The presence of AQP2 on the collecting duct increases

the water permeability, until the collecting duct lumen and medullary interstitial compartment

have equal osmolalities. Vasopressin is required for the permeability of water in the medullary

CD.

Recent reviews on the water permeability of the CD have suggested that there is

‘residual’ water permeability in the inner medullary CD (36). This residual water permeability

plays a significant role during periods of water diuresis, where vasopressin is virtually absent. In

18

the absence of vasopressin, there is no AQP2 in the luminal membrane of upstream nephron

sites, and subsequently, a high volume of water reaches the inner medullary CD. Since there is a

large delivery of Na+ and Cl- in the filtrate, more reabsorption of Na+ and Cl- is required in

upstream nephron segment to avoid large loss of these ions in the urine. The increased

reabsorption of Na+ and Cl- is stimulated by the water reabsorption in the inner medullary CD,

where water without Ca2+ is reabsorbed from the filtrate, diluting the medullary interstitial

compartment. As mentioned in the last section, the dilution of the medullary interstitial

compartment increases Na+ and Cl- reabsorption in the mTAL via the fall in the inhibitory

control possibly by [Ca2+] (Figure 1-3). Therefore, reabsorption of water in the inner medullary

CD serves an important role in desalinating the filtrate reaching the final nephron sites during

periods of water diuresis.

19

Figure 1-4. Vasopressin-mediated insertion of aquaporin-2 water channels and reabsorption of water from the renal medullary collecting duct. The binding of vasopressin to its receptor (V2 receptor), which is coupled to a stimulatory G-protein, activates adenyl cyclase (AC). The activation of AC increases the production of cyclic adenyl monophosphate (cAMP), which phosphorylates and activates protein kinase A (PKA). This leads to increased synthesis and phosphorylation of aquaporin-2 channels (AQP2), and vesicles carrying AQP2 are inserted on the apical membrane. Water is reabsorbed via AQP2 into the renal medullary collecting duct cells, and enters the renal medullary interstitial compartment via aquaporin 3 and 4 channels (not shown in the Figure), which are constitutively present on the basolateral membrane. Adapted from Fenton et al., 2007 (63).

20

1.5. Role of Vasopressin in the Renal Outer Medullary O2 Balance

Vasopressin is a nanopeptide hormone that is synthesized in the neurosecretory cells in

the hypothalamus as preprovasopressin, where it is degraded into provasopressin. Provasopressin

migrates along the neuronal axons to the posterior pituitary gland, to be subsequently released as

vasopressin, neurophysin II and copeptin (41). The neurophysin II acts as a binding protein for

vasopressin, but the exact function of copeptin is yet unclear. The half-life of vasopressin is

approximately 5 to 15 minutes, until it is degraded by circulating vasopressinases (41).

Vasopressin serves two major functions in the kidney: the vasoconstriction of blood vessels

perfusing the renal medulla and the insertion of AQP2 on the medullary CD for reabsorption of

water, via actions on the V1 receptor and the V2 receptor respectively. These two functions of

vasopressin are essential in the urine concentrating mechanism in the kidney.

1.5.1. Vasopressin: Regulation of Blood Flow to the Renal Outer Medulla

As previously mentioned, vasopressin is one of the most potent vasoconstricting agents in

the renal medulla (140). There is a corticomedullary concentration gradient of [Na+ + Cl-] in

deeper regions of the renal outer medulla to levels significantly higher than in plasma (16; 156).

Studies have suggested that the vasoactive action of vasopressin may be more specific to the

renal medulla, with no detectable changes in the total or cortical renal blood flow (61). The

ability of vasopressin to selectively reduce medullary blood flow may be due to vasoconstriction

21

of arterioles of juxtamedullary nephrons, which give rise to vasa recta (VR) perfusing the renal

medulla.

In the renal medulla, the vascular action of vasopressin is primarily mediated by the

activation of V1 receptors (82; 127), which activates PKC and produces inositol triphosphate

(IP3) and diglyceride (DAG), leading to mobilization of intracellular Ca2+ stores. Increase in

intracellular Ca2+ activates receptor-operated Ca2+ channels, and subsequent Ca2+ sensitization of

pericytes leads to vasoconstriction in the VR (82). The paracrine system of vasopressin-mediated

vasoconstriction has been investigated, and the general view suggests that vasopressin does not

influence the whole renal hemodynamics (17). Under most experimental conditions, the acute

vasopressin-mediated vasoconstriction is regulated by the stimulation of V2 receptors and the

subsequent release of counterregulatory endogenous vasodilators such as NO (127). In a paper

by Masuda et al (113), the authors have suggested that vasopressinase may be released in the

renal distal tubules via V2 receptor pathway, as a negative feedback to degrade the excessive

vasopressin in the renal medulla. Other studies have demonstrated that physiological increases in

vasopressin by water deprivation in dogs have decreased renal blood flow (65; 123), which in

turn can be blocked by V2 agonist desmopressin acetate, dDAVP (123). Therefore, the

vasopressin-mediated changes in renal medullary blood flow depend on the intrarenal

localization of V1 and V2 receptor subtypes and on the activities of endogenous vasoactive agents

(127).

22

1.5.2. Vasopressin: Urea Reabsorption in the Inner Medulla

The antidiuretic effect of vasopressin is mediated by the activation of V2 receptors on the

basolateral membranes of CD cells and the subsequent increase in the water permeability in the

CD. Another function of vasopressin in the renal medulla is urea reabsorption in the inner

medullary CD (75). The activation of V2 receptor also leads to insertion of urea transporters

(UTs) on the apical membranes of inner medullary CD cells (Figure 1-5). Urea reabsorption is

required for high concentration of urea in the inner medullary interstitial compartment, which

may be critical in minimizing work in the deep part of the outer medulla. Under normal

physiological conditions, the blood flow to the renal medulla is limited by vasoconstrictory

effects of vasopressin binding on V1 receptors, and consequently there is marginal O2 supply to

meet the demand in the mTAL, where most of work is performed in the renal outer medulla.

Urea recycling plays an essential role in replacing the requirement for active reabsorption of Na+

and Cl- in the mTAL via passive reabsorption of these ions in the inner medulla.

The maintenance of high concentration of urea in the inner medullary interstitial

compartment proceeds as follows: First, under the actions of vasopressin, urea and water are

reabsorbed as an iso-osmotic solution in the inner medulla. The reabsorption of urea and water

lowers the concentration of Na+ and Cl- in the medullary interstitial compartment, and creates a

driving force for the passive reabsorption of these electrolytes in the inner medulla (72). As the

AVR delivers fluid with a lower [Na+ and Cl-] from the inner medulla to the deepest area of the

outer medulla, there is reduced need for more active reabsorption in the mTAL. The work

required to achieve a high urea concentration in the lumen of the inner medullary CD is the high

medullary interstitial osmolality deep in the outer medulla. This work is performed in the renal

23

cortex and renal medulla near the cortex, where there is an abundant supply of O2. The bulk of

active reabsorption of Na+ and Cl- occurs in this area, which is responsible for the driving force

of reabsorption of water in the medullary CD in the outer medulla. Subsequently, there is a rise

of urea concentration in the inner medullary CD lumen, which is sufficient to continue the

intrarenal urea recycling.

In conclusion, vasopressin-mediated urea reabsorption plays a critical role in the

integrative physiology of the deep outer medulla, and reduces the work performed in this O2-

poor region of the outer medulla which is most susceptible to hypoxic injury. Therefore, the role

of vasopressin extends beyond the reabsorption of water in the urine concentrating mechanism in

the kidney, since vasopressin-mediated urea recycling also plays a role in reducing the work in

the deep outer medulla, and thereby minimizing the risk of hypoxic injury in this region.

24

Figure 1-5. Reabsorption of urea in the inner medulla reduces the need of active reabsorption in the superficial region of renal medulla (mTAL). When vasopressin is present, urea transporters are inserted on the inner medullary collecting duct of juxtamedullary nephrons (which consists of 15% of total number of nephrons). This allows 1) reabsorption of urea into the inner medullary interstitial compartment. The reabsorption of water and urea decreases the relative concentration of Na+ and Cl- in the renal medullary interstitial compartment, and subsequently increases the passive entry of Na+ and Cl- from the ascending thin limb (AtL) of the loop of Henle. This process reduces the need of active reabsorption of Na+ in the mTAL. Adapted from Gowrishanka et al., 1998 (72).

25

1.6. Maintenance of O2 Balance in the Renal Outer Medulla

It is important to maintain the O2 balance in the renal outer medulla to minimize the

hypoxic injury to this region. AKI often results from hypoperfusion of the kidney during

surgeries requiring CPB, which leads to renal tissue hypoxia. Kuwahira et al. have demonstrated

that exposure to acute hypoxia in rats resulted in the redistribution of blood flow to the essential

organs, such as brain, heart and liver (101). The total blood flow to the kidney remained

unchanged, but the O2 supply to the renal medulla fell significantly. This indicates a serious

danger to the renal medulla, which receives only 10% of the total renal blood flow, and an O2

supply that closely approximates O2 consumption under normal physiological conditions. During

periods of acute hypoxia, the O2 balance could be seriously compromised in this region which is

already vulnerable to hypoxia.

In the next sections, I will discuss the potential interventions to maintain the O2 balance

in the renal medulla during periods of hypoxia. There are two ways to maintain the O2 balance in

this region: increase blood flow and thereby O2 supply or decrease O2 consumption.

26

1.6.1. O2 Supply: Desmopressin Acetate (dDAVP)

It was previously mentioned that vasopressin regulates blood flow in the renal medulla,

by acting as a vasoconstrictive agent that maintains low perfusion to the region. The

vasoconstrictive action of vasopressin depends on the activation of V1 receptors. Under normal

physiological conditions, the V1 receptor-mediated vasoconstriction limits the medullary blood

flow, which prevents the washout of the concentration gradient in the medullary interstitial

compartment (114). Limiting blood flow to the renal medulla may also prevent excessive

perfusion deep in the inner medulla, and therefore prevents buildup of reactive oxygen species

(ROS) in the area where most of the work is passive (155). However, under hypoxic conditions,

limiting the blood supply may have adverse effects, since the blood is low in O2 content.

Desamino 8D-arginine vasopressin, dDAVP, is a synthetic V2 receptor agonist that lacks

the amino group on the N-terminal cysteine, which is cleaved under the action of vasopressinase

(166). The lack of the substrate chain for vasopressinase activity in dDAVP results in the long

lasting activity of dDAVP (Figure 1-6). Although it is a synthetic analogue of vasopressin,

dDAVP is suggested to have no pressor activity, since it has V2 receptor specificity and thus

does not mediate V1 receptor vasoconstriction. The lack of affinity to V1 receptor may be due to

D-conformation of the 8 Arginine, which may hinder dDAVP from interacting with the V1

receptor. In the clinical setting, dDAVP is used to treat patients suffering from bleeding

disorders, such as von Willebrand disease, hemophilia A and several platelet disorders (166).

The effect of dDAVP in these treatments is the increase in the plasma levels of factor VIII, von

Willebrand factor and plasminogen activator activity (90). dDAVP is also used clinically to treat

water diuresis in pregnant women, where an excessive activity of vasopressinase released from

27

the placenta degrades circulating vasopressin (51; 146) and leads to loss of the ability to

concentrate urine. Another clinical use of dDAVP is to treat diabetes insipidus, to mimic the

actions of vasopressin under conditions where there is impaired release of endogenous

vasopressin (51). The specificity of dDAVP to V2 receptor may be of significance in the clinical

settings such as renal tissue hypoxia. dDAVP has been repeatedly associated with V2 receptor-

mediated vasodilatory effect in regulating the renal medullary blood flow via nitric oxide (NO)

production (48; 140). Renal medullary NO was also shown to offset the prolonged

vasoconstrictive effect of vasopressin in rats, suggesting that dDAVP-mediated vasodilatory

effects may improve perfusion to the renal outer medulla during hypoxia. Although there are

numerous studies addressing the role of dDAVP in improving renal medullary perfusion, the

exact underlying mechanism is incompletely understood, and further investigation is required to

understand the potential role of dDAVP in preventing renal medullary hypoxic injury. Therefore

the first part of my study aimed to address the question of whether dDAVP may have clinically

relevant effects in renal tissue hypoxia.

28

Figure 1-6. The structures of vasopressin (top) and desmopressin acetate (dDAVP; bottom). Desmopressin acetate (dDAVP), a synthetic analogue of vasopressin, lacks the amino group on the first cysteine. Vasopressinase attacks the first cysteine and breaks the disulfide bond, which inactivates vasopressin (red arrow). The first group is deaminated in dDAVP, and therefore the disulfide bond is not broken down by vasopressinase and the molecule is resistant to the vasopressinase activity. The degraded fragment of vasopressin (metabolite of vasopressinase) has no effect on either V1 or V2 receptor, suggesting that disulfide bond is critical to the action of vasopressin on these two receptors. In addition, dDAVP, being a V2 receptor agonist, has D-conformation of 8-arginine, which may hinder its interaction with V1 receptor. Therefore, the activity of vasopressin on V2 receptor may only require the disulfide bond, whereas its binding to V1 receptor may require the disulfide bond and the L conformation on 8-arginine, both of which are lost with vasopressinase activity. Adapted from Berg et al (23).

29

1.6.2. O2 Consumption: Furosemide

Furosemide is a loop-diuretic that inhibits active reabsorption by mTAL in the renal outer

medulla, by binding on the Cl- binding region in the NKCC2 channel. It is currently used

clinically to treat patients suffering with chronic congestive heart failure and pulmonary edema,

where rapid diuresis is required. The effectiveness of furosemide is measured by the rise in urine

flow rate. Furosemide is effective in removing excessive extracellular fluid volume, by inhibiting

reabsorption of Na+ and Cl-, and subsequently preventing reabsorption of water in the final

nephron segment.

The actions of furosemide are explained as follows. Furosemide binds on the chloride

binding region of NKCC2, and inhibits the reabsorption of Na+, K+ and Cl- by the mTAL (Figure

1-7). As previously mentioned, the reabsorption of K+ is the source of K+ for the ROMK

channel, which permits K+ and positive voltage to enter the lumen of the mTAL. This provides

the driving force that allows passive reabsorption of Na+, and divalent cations Ca2+ and Mg2+,

which occurs via paracellular pathways. Thus furosemide also diminishes the reabsorption of

Ca2+ and Mg2+ in the mTAL. This explains why prolonged excessive use of furosemide may

result in hypomagnesemia, which is defined by the serum Mg2+ level of <0.7 mmol/L in humans.

Magnesium is a cofactor in more than 300 enzymatic reactions that involve energy metabolism

and protein and nucleic acid synthesis (58). The clinical manifestations of hypomagnesemia

include cardiac arrhythmia, tachycardia and tetanus. Hypomagnesemia is also associated with

high mortality (43). The most common cause of hypomagnesemia is the use of loop-diuretics

such as furosemide (182), which strongly suggests that long-term use of very high doses of

furosemide should be avoided. The risk of hypocalcemia may not be as high, since reabsorption

30

of Ca2+ occurs more distally in the DCT, and thereby may compensate the reduced reabsorption

in the mTAL. Not only is the risk of hypomagnesemia increased with prolonged use of

furosemide, the long-term uses of furosemide are also associated with higher morbidity in

patients undergoing cardiac surgeries. Other adverse side-effects of furosemide include

dehydration and electrolyte imbalance, due to an excessive loss of Na+, K+, Cl- and Mg2+.

Therefore, the use of furosemide should be limited to reducing the active reabsorption of mTAL

in the renal outer medulla, rather than to remove body fluids. The O2 consumption in the renal

outer medulla primarily consists of the active reabsorption in the mTAL. There is evidence that

inhibiting the active reabsorption of mTAL by loop diuretics can increase medullary PO2 from 16

to 35mmHg (31). Therefore, reducing the active reabsorption of Na+ and Cl- in the mTAL may

be helpful in maintaining O2 balance in the renal outer medulla during periods of acute tissue

hypoxia, where O2 supply is low.

However, there is currently a lack of consensus on the dose of furosemide that can

significantly reduce the work within the mTAL in the renal outer medulla without causing an

excessive loss of electrolytes. Hence, the goal of the second part of my study was to address the

question of what dose of furosemide can reduce the work of mTAL in the renal outer medulla,

without causing a large electrolyte excretion that may deplete the body of essential electrolytes.

31

Figure 1-7. Furosemide inhibits Sodium-Potassium-2Chloride (NKCC2) co-transporter in the medullary thick ascending limb (mTAL). Furosemide competes for the Cl- binding region in the NKCC2 co-transporter, and inhibits the active reabsorption of Na+, K+ and Cl- into the mTAL cells. The inhibition of NKCC2 lowers the intracellular K+, which is the source of K+ for the ROMK channel. This also hinders the buildup of positive voltage within the lumen of the mTAL, and therefore the passive reabsorption of Na+, Ca2+ and Mg2+ is also prevented.

32

1.7. Adaptive Response to Hypoxia

Exposure to low PO2 triggers systemic cardiovascular and respiratory adaptations in order

to maintain adequate blood flow and O2 supply to the vital organs, such as the brain, heart and

the liver (104). An early response to exposure to mild hypoxia (10% O2) in rats is a transient fall

in the body temperature, suggesting a decreased metabolism (103). In fact, many cells show

decreased O2 consumption and often switch to alternative anaerobic metabolic pathways (5). One

way to decrease O2 consumption in these cells is to reduce ion permeability to actively maintain

the ion concentration gradient during hypoxia. This hypoxia-induced reduction in the

maintenance of ion gradient is especially significant in the renal outer medulla, where the

primary function is to actively reabsorb Na+ to generate the hyperosmotic gradient in the

medullary interstitial compartment.

A mismatch between O2 supply and O2 consumption in animals can be systemic or region-

specific, and adaptive responses to hypoxia may be different depending on whether the hypoxia

is systemic or restricted to specific tissues. Most often, studying acute response to hypoxia is

focused on systemic hypoxia. At the systemic level, hypoxia causes many adaptive responses

which may increase O2 delivery throughout the body (5). An initial response to hypoxia is an

increase in ventilation, which results in respiratory alkalosis and increases hemoglobin-O2

affinity and O2 loading (20; 154). There is also an increase in the quantity of hemoglobin per litre

of blood volume. The hemoglobin concentration in the blood may be increased through many

processes, such as hypoxia-induced diuresis, release of erythrocytes from storage organs (ie

spleen) and de novo synthesis of red blood cells. Systemic hypoxia can also cause an increase of

synthesis, release and metabolism of atrial natriuretic peptides (ANPs). One study using a rabbit

33

model has shown that the plasma level of ANP increases before the rise in mean arterial pressure

and heart rate (10). One of the major effects of ANP is an increase in the renal excretion of Na+,

which results in a diuresis. Hypoxia-induced diuresis also increases the hemoglobin

concentration in the blood, which can improve pulmonary gas exchange and therefore increase

O2 carrying capacity of the blood.

At the cardiovascular level, the main effect of acute hypoxia is an increase in the heart rate.

The role of the cardiovascular system is to maintain adequate O2 supply throughout the body and

peripheral chemoreceptors which are sensitive to changes in PO2 (fall in PO2) respond to hypoxia

by activating sympathetic nervous system. Cardiac output also increases with hypoxia (145;

174), mainly due to increase in heart rate, which may also be the result of withdrawal of

parasympathetic action on the heart. There is also an increase in myocardial contractility, but the

stroke volume does not show any significant change during hypoxia, possibly due to a fall in the

end-diastolic volume that occurs with higher heart rate (101). In terms of peripheral vasculature

response to hypoxia, there is evidence that release of vasodilators such as nitric oxide synthase

increases local blood flow to maintain an adequate supply of O2 to the tissues (178). The

hypoxia-induced changes in the cardiovascular system also redistribute blood flow to essential

organs, such as the brain, heart and liver (101), whereas renal blood flow does not change

significantly in rats exposed to acute hypoxia (10% O2).

Exposure to acute hypoxia results in many adaptive changes that may maintain the

adequate O2 delivery to the tissues; cells may decrease metabolism to reduce O2 consumption

(136), whereas changes in cardiovascular system may enhance perfusion to the tissues to

maintain adequate O2 supply. Among the changes in the cardiovascular system, peripheral

vasculature releases local vasodilators, which results in a reduced total peripheral resistance and

34

redistributes blood flow to the vital organs (5). Total renal blood flow does not show any

significant changes in response to hypoxia, which may be due to autoregulation. Despite any lack

of significant changes in the blood flow, regions of the kidney may still be vulnerable to hypoxic

injury in clinical settings due to the lack of a uniform distribution in renal blood flow and

different anatomical zones in the kidney with various O2 tensions (148).

35

1.8. Indices of Renal Tissue Hypoxia

Renal tissue hypoxia is one of the major causes of development and pathogenesis of

experimental AKI (32; 129; 148), and hypoxia-induced acute kidney injury is also associated

with various pathophysiological conditions, such as surgeries requiring cardiopulmonary bypass.

In addition to the many adaptive responses to hypoxia previously mentioned, there are also

significant changes at the molecular level. These changes include synthesis of erythropoietin

(EPO), stabilization of hypoxia-inducible factors (HIFs), and the upregulation of nitric oxide

synthases (NOS), all processes which may enhance renal medullary O2 delivery. In this study,

these three indices were used to detect renal tissue hypoxia. The mechanisms by which these

indices are synthesized, stabilized, and upregulated are discussed in the following sections.

1.8.1. Plasma Erythropoietin (EPO)

One of the major responses to hypoxia is the synthesis of erythropoietin (EPO), which is

produced primarily by the fibroblast-like cells in the peritubular space between the renal cortex

and outer medulla. Although EPO is also produced by other organs (68), the major source of

EPO is the kidney, possibly due to the O2 sensors believed to be located in the renal cortex (74).

EPO is a 30.4kD glycoprotein that regulates production of red blood cells, and its levels can

increase up to 1000-fold in cases of severe hypoxia (62; 87). The EPO receptors are expressed

specifically on erythroid progenitor cells, and through its receptors, EPO promotes viability,

proliferation and differentiation of red blood cell precursors.

36

The hypoxia-induced increase in EPO is the result of both increased transcription and

prolonged half-life of EPO mRNA. Studies have demonstrated tissue-specific expression of EPO,

in which different regions on the EPO gene are responsible for its expression in the liver and the

kidney. The expression of EPO in different tissues is also age-specific: EPO is principally

expressed in the liver of mammalian fetus, and liver EPO production is suppressed and kidney

production of EPO becomes predominant as ontogeny continues (161). In the liver of anemic

mice, EPO was shown to be highly expressed in hepatocytes surrounding the central vein,

whereas in better oxygenated areas surrounding the portal triads, EPO production was low (128).

The receptors for EPO have been found in many tissues such as the brain (107; 141; 159),

kidney (168) and human placenta (185). EPO mRNA is also widely expressed in endothelial

cells, cardiomyocytes, and macrophages, which suggests that EPO may serve various roles in

different tissues. The binding of EPO to its receptor involves one protein binding to a dimer of

two EPO receptors. Binding to EPO triggers a conformational change in the receptor dimers, and

this change activates numerous kinases and signaling pathways. EPO receptor activation

phosphorylates many kinases, adaptor proteins and molecules involved in signal transduction,

and the pattern of phosphorylation depends on the cell type. Different cells express receptors for

EPO during periods where red blood cell formation is critical, such as periods of low PO2 or

anemia (96). The net effect of EPO is an increase in the mass of red blood cells, which increases

the O2 carrying capacity of the blood during periods of hypoxia. Other than its role in increasing

the mass of circulating red blood cells, EPO is also suggested to have neuroprotective and

renoprotective effects and therefore may be significant in the clinical settings (70).

37

1.8.2. Hypoxia Inducible Factor-1α (HIF-1α)

Hypoxia increases the transcription of genes involved in adaptive responses which may

enhance blood flow and thereby maintain adequate O2 delivery to the tissues. One of the major

molecular components governing these adaptive responses is hypoxia-inducible transcription

factors (HIF) (149). Increase in HIF expression is an early and lasting response induced by renal

tissue hypoxia which is closely associated with development of acute tubular necrosis and renal

failure(117). HIFs are heterodimers of an O2-regulated α subunit and a constitutive β subunit

(57). More than 100 target genes of HIF-1 have been identified, and these target genes include

EPO, vascular endothelial growth factor (VEGF), glucose transporters and heme oxygenase-1

(HO-1), which suggests crucial adaptive roles of HIF-1 (24; 57; 115). The α-subunit of HIF,

which is sensitive to O2, plays a critical role in cellular responses to hypoxia.

Under normoxic conditions, HIF-1α is rapidly and continuously degraded by the

ubiquitin-proteasome pathway, and therefore it has a short half-life of less than 5 minutes (Figure

1-8) (24; 53). The destabilization of HIF-1α begins with hydroxylation of specific prolyl residues

by prolyl hydroxylation domain proteins (PHD), which require O2 as a co-factor in the process

(24; 33). The hydroxylated prolyl residues on HIF-1α are recognized by pVHL, the product of

the von Hippel Lindau tumor suppressor gene, and a recognition component of the E3 ubiquitin

ligase complex (53). The ubiquitylation of HIF-1α targets the protein for proteasomal

degradation. In addition, the acetylation of lysine residue on HIF-1α also promotes degradation

by proteasomes (53). Under hypoxic conditions, there is a reduced activity of PHD due to the

lack of O2 as a substrate, which results in accumulation of HIF-1α. Subsequently, stabilized HIF-

1α forms dimers with HIF-1β and binds to a DNA motif in the hypoxia response elements,

38

transactivating target genes (24). There is a rapid increase of HIF-1α in rats within 1 hr after the

onset of exposure to hypoxia (10% O2), suggesting that hypoxia may not affect the synthesis of

HIF-1α, but rather its stabilization (35). The HIF system is also shown to be activated by non-

hypoxic stimuli, such as growth factors, cytokines, and vascular hormones, where accumulation

of HIF results from HIF-1α protein translation rather than the stabilization of the protein (53).

Up-regulation of HIF-1α is associated with many pathophysiological conditions, such as

renal ischemia, functional anemia and acute kidney injury (163). In these cases, HIF-1α was

expressed in cells of tubular epithelia and papillary interstitial compartment. There is abundance

of PHD in the deeper areas of renal medulla, suggesting that the role of HIF may be especially

significant in the region where O2 tension is physiologically low (163). There is a cell-specific

induction of HIF in the kidney in response to systemic hypoxia, and therefore up-regulation of

HIF and its target genes may serve a role in the response to regional renal hypoxia (148). Several

studies have addressed the renoprotective role of HIF in preventing hypoxia-induced renal

tubular damage (57; 110). Inhibitors of PHDs inhibit degradation of HIF and mimic the effect of

hypoxia with the presence of O2, which may up-regulate HIF in the kidney and induce

angiogenesis in an experimental model (60; 85). Such stimulation of HIF is shown to confer

protection in an acute kidney model, and there is also evidence that a constitutively active HIF

may serve a protective role in the renal medulla (110; 179).

39

Figure 1-8. Hypoxic-inducible factor (HIF) pathway under normoxia and hypoxia. Under normoxic conditions (right), the presence of O2 leads to hydroxylation of 2 proline residues on HIF-1α by prolyl hydroxylase (PHD). The hydroxylation of proline promotes HIF-1α association with von Hippel-Lindau protein (pVHL) E3 ubiquitin-ligase complex and degradation of HIF-1α via ubiquitin/proteasome pathway. Under hypoxic conditions, the lack of O2 allows HIF-1α to escape the degradation pathway, and HIF-1α dimerizes with HIF-1β to form a heterodimer complex, activating downstream target genes by binding on hypoxia responsive elements (HRE). This process leads to production of proteins associated with hypoxia, such as erythropoietin (EPO) and nitric oxide synthase (NOS).

40

1.8.3. Nitric Oxide Synthase (NOS)

Nitric oxide (NO) is an important signaling and effector molecule with various

physiological roles (69; 94). NO is synthesized from L-arginine by different isoforms of nitric

oxide synthase (NOS) in a reaction which requires the presence of O2, and may be Ca2+

dependent when catalyzed by neuronal or endothelial isoforms of NOS (nNOS and eNOS) (122).

One of the most apparent roles of NO is relaxation of the vascular smooth muscles and

subsequent vasodilation (69). There is evidence that NO may play a major mediator role in

vasodilation in cerebral (6; 171) and coronary (5, 19) arteries, and to improve perfusion (28; 142;

169) in rats during hypoxia. NO-mediated vasodilation during hypoxia suggests that low O2

tension may trigger production of NO in the vasculature to increase local blood flow (69). Nitric

oxide plays an important role in regulating vascular tone in the kidney, where there is

comparatively lower vascular resistance than in other organs (109). High expression of nNOS in

the macula densa suggests that nNOS may have a role in regulating tubular glomerular feedback

(TGF) response, and this phenomenon was further supported by a micropuncture study that

demonstrated the effect of nNOS on blunting TGF response (183). Studies have demonstrated

the presence of high levels of nNOS in the renal outer medulla and inner medullary thin limb

(164), and this level of nNOS expression may be stimulated by vasopressin (112). The effect of

vasopressin on nNOS activity may be specific to this isoform, since endothelial NOS (eNOS)

does not appear to be influenced by vasopressin (112). These findings suggest that nNOS may be

the isoform with a specific function in regulating Na+ and water handling by the kidney (112;

147). Other isoforms of NOS, such as eNOS and iNOS are also expressed in the kidney, and

eNOS has been shown to be predominantly expressed at high levels throughout the kidney (112).

41

In the kidney, eNOS is expressed at high levels in the renal vascular endothelium including

medullary vasa recta in the mTAL, and is responsible for regulating vascular tone under normal

conditions (112). The role of iNOS or its function in the kidney is not fully understood, but

studies have demonstrated that iNOS expression is associated with pathophysiological conditions,

such as inflammatory response to pathogens (94).

Nitric oxide (NO) production and activity is shown to play a major role in regulating the

blood flow in the renal medulla, and protecting the region from ischemic injury (47). There is a

concentration gradient of NOS in the kidney of Sprague-Dawley rats, with increasing

concentration of NOS in the deeper areas of renal medulla, which is a magnitude higher than in

the renal cortex (48). The role of NO is supported by a study that showed a significant reduction

of medullary blood flow following a corticomedullary infusion of NOS inhibitor, N-G-nitro-L-

arginine methyl ester (L-NAME) (126). Many ex vivo studies have demonstrated that, using

isolated inner stripe of the outer medulla in rats, NO produced in the mTAL diffuses to the

pericytes of surrounding vasa recta capillaries (47). As previously mentioned, NO can

counterregulate the vasoconstrictory effect under the chronic infusion of vasopressin in rats (48).

A study by Cowley et al demonstrated that when renal medullary NO activity was inhibited by

chronic medullary infusion of L-NAME, vasopressin was able to maintain its pressor activity and

led to a sustained elevation of blood pressure (48). The ability of NO to buffer the

vasconstrictory effect of vasopressin is significant in maintaining an adequate blood flow to the

renal medulla, since the O2 delivery to the mTAL in the renal outer medulla depends on this

blood flow. The vasodilatory effect of NO may be specific to the renal medulla, since infusion of

L-NAME significantly reduces renal medullary blood flow, whereas cortical blood flow is

unchanged (114). Altered production and decreased bioavailability of NO are associated with

42

endothelial dysfunction in acute renal failure, which suggests that NO also plays a significant

role in regulating blood flow in pathophysiological settings (44).

43

1.9. Hypothesis and Specific Aims of the Study

Hypothesis

The earliest physiologic response of renal tissue hypoxia originates from the renal outer

medulla, which will lead to the loss of ability to concentrate urine, reflected by a decrease in the

maximum urine and renal medullary osmolality in rats. In addition, the disruption in O2 balance

in the renal outer medulla may be prevented by increasing O2 supply or decreasing O2

consumption, and such mechanisms may be used to prevent hypoxic injury in the renal outer

medulla.

Specific Aims of the Study:

Part 1: Hypoxia

1. To determine the earliest physiologic indicator of hypoxia in the renal outer medulla.

2. To determine the basis or underlying mechanism of renal outer medullary response to

hypoxia.

3. To assess the ability of dDAVP to prevent the hypoxia-induced renal medullary

dysfunction.

Part 2: Furosemide

1. To document the effect of different doses of furosemide (0.4, 0.8, 1.6, 2.4 and 3.2 mg/kg

in rats) on the function of renal outer medulla.

2. To determine the dose of furosemide that may significantly reduce the renal medullary

function while preventing depletion of electrolytes and fluids.

44

1.10. Synopsis of Thesis Results

The aim of the present study was to determine the earliest indicator of renal tissue

hypoxia and I focused on the mTAL as the area of interest of this study, since the innermost

region of the renal outer medulla is most susceptible to hypoxia. Due to its precarious blood

supply and low hematocrit, the outer renal medulla would be the earliest area most likely

affected during periods of reduced supply of O2. Therefore, the objective of this study was to

determine the effect of hypoxia on the concentrating ability of mTAL in the renal outer medulla

and defense mechanisms that may minimize the risk of renal medullary hypoxic injury. To

address the specific aims of this study, I used the rat experimental model of low PO2 (8% O2)

developed in our lab to use hypoxia as a single stimulus to impair the function of mTAL.

The main function of outer renal medulla is to maintain a hyperosmotic medullary

interstitial compartment, which is in essence the renal concentrating mechanism. The

combination of these two processes (high medullary interstitial osmolality and water

reabsorption in the medullary CD) is required to concentrate the urine. Thus I expected that the

earliest response of the hypoxia to the kidney (and specifically, to the renal outer medulla) would

be a reduction in the medullary interstitial osmolality and subsequent fall in the urine osmolality.

As expected, the exposure to 2.5 hrs of hypoxia significantly reduced urine osmolality in rats.

The fall in urine omsolality was accompanied by a significant increase in urine flow rate,

suggesting that rats were experiencing water diuresis. Pretreatment with desmopressin acetate

(dDAVP), a synthetic analogue of vasopressin, prevented the hypoxia-induced water diuresis,

which indicated that there may be a loss of vasopressin during hypoxia. To determine the basis

of water diuresis, renal papillary osmolality was measured. The osmolality in the renal medullary

45

interstitial compartment was significantly higher compared to urine osmolality. The large

difference suggested that water reabsorption in the renal medullary collecting duct was impaired

during hypoxia, possibly due to absence of vasopressin. However, the renal papillary osmolality

was significantly reduced during hypoxia and from these results I concluded that the basis of

hypoxia-induced water diuresis is due to mTAL dysfunction (impaired active reabsorption) and

absence of vasopressin (impaired water reabsorption). To test whether hypoxia releases

vasopressinase that may degrade circulating vasopressin, the activity of plasma vasopressinase

was measured. The results showed that plasma vasopressinase activity increased significantly during

hypoxia, which diminished the antidiuretic action of vasopressin and caused the hypoxia-induced

water diuresis. In addition, the results from my study showed that water diuresis under 2.5 hrs of

hypoxia may be a transient process, since the previous study with 5 hrs of hypoxia exposure did

not show any sign of absence of vasopressin and the difference between renal medullary

osmolality and urine osmolality was not detected.

Although impaired function of mTAL would likely result in decreased active

reabsorption of Na+ and Cl-, a significantly large increase in the excretions of these electrolytes

was not expected due to the previous findings from our lab, which demonstrated that exposure to

5 hrs of hypoxia in rats did not cause a large Na+ excretion (natriuresis) or Cl- excretion

(chloruresis). Indeed, I did not observe any significant increase in the excretions of Na+ or Cl-

after 2.5 hrs of hypoxia exposure in rats.

The second part of my study aimed at determining whether a loop diuretic such as

furosemide can be used to reduce mTAL function and therefore O2 consumption in the renal

outer medulla. The purpose of this part of the study was also to determine the dose of furosemide

that may significantly reduce mTAL function without depleting essential electrolytes or fluids in

rats.

46

From these experiments, I found that with increasing doses of furosemide, a fall in urine

osmolality occurs before urine flow rate increases significantly. Results from this experiment

also showed that 0.8 mg/kg of body weight is the dose of furosemide where the active

reabsorption of mTAL decreases significantly and excretion of electrolytes (Na+, Cl-, K+ and

Mg2+) begins to increase significantly. Therefore the results from this experiment suggested that

doses higher than 0.8 mg/kg may increase electrolyte excretions significantly, to the point that

may lead to depletion of essential electrolytes, as shown by the fall in the level of serum Mg2+ in

rats with higher doses of furosemide. However, it is also important to note that 0.8 mg/kg of

furosemide may still be high if extrapolated into human values (5.6 mg in 70 kg human), and

further studies should be carried out to determine the dose of furosemide for humans that may

mimic the effect of 0.8 mg/kg demonstrated in rats. Furosemide may best be used to reduce the

work by the mTAL and thus to decrease the metabolic demand (O2 consumption) in the renal

outer medulla than to decrease the body fluids when there is a risk of hypoxic injury.

In summary, I demonstrated in this study that acute exposure to hypoxia leads to water

diuresis, as early as 2.5 hrs, in rats. The magnitude of water diuresis is very large and transient,

and the basis of hypoxia-induced water diuresis is reduced active reabsorption of mTAL and the

metabolic clearance of vasopressin that resulted in impaired water reabsorption. As demonstrated

by the second part of my study, I have shown that the dose of furosemide in rats that can

significantly reduce mTAL function without causing a large excretion of essential electrolytes is

0.8 mg/kg of body weight. However, the difference in dose-effect of furosemide between rats

and humans should be considered. The detailed description of results and discussion of my study

are discussed in the following sections.

47

CHAPTER 2

METHODS

48

2.1. Experimental Protocol #1: HYPOXIA

All experiments were approved by the Animal Care and Use Committee at Keenan

Research Center of the Li Ka Shing Knowledge Institute of St. Michael’s Hospital and followed

the guidelines set out by the Canadian Council on Animal Care. Male Sprague-Dawley rats

(Charles River, St. Constant, Quebec, Canada) weighing 400-450g were individually housed in

metabolic cages to allow for the monitoring of food and drink intake overnight.

In the first protocol, the drink consisted of isotonic saline (NaCl; 150mmol/L), and

glucose (5%) was added to ensure the complete consumption of the drink provided. The purpose

of the isotonic saline was to exclude the possibility of diuresis that may result from hypotonic

fluid intake, and to ensure the production of maximally concentrated urine that reflects renal

medullary function.

Eighteen hours following the saline consumption, rats (n = 8/group) were exposed to

either 8% O2 (hypoxia) or 21% O2 (normoxia) for 2.5 hrs. Rats did not have access to food or

drink during this time period. The rats were exposed to hypoxia in tightly-sealed plexiglass

chambers, where 8% O2 gas was continuously flushed at 3L/min (Praxair, Inc. Danbury CT,

USA). Of note, 8% O2 equals 1/3 of the O2 available under normoxia (21%). The partial pressure

of O2 (PO2) at 8% is approximately 53 mmHg, and this level of PO2 represents the level that is

observed in high altitudes, such as Mount Everest, which can lead to high altitude hypoxia.

Previous findings from our lab have shown that the level of O2 (8%) used in this model is

sufficient to induce hypoxia in the kidney without leading to death in rats. The concentration of

O2 in the chamber was measured using a gas analyzer (Datex Ohmeda 5250 RGM, Canada), to

49

ensure that the rats were exposed to 8% O2. Urine samples were collected at every time of

excretion for the calculation of urine flow rate, and stored at -20°C until analysis. After urine

samples were collected, rats were anesthetized using 3% isoflurane, and blood samples from the

descending aorta were obtained at either 1 hr or 2.5 hrs of exposure to hypoxia or normoxia.

Measurements of blood gas, hemoglobin concentration, lactate and electrolyte concentrations

were made immediately after sample collection (Radiometer ABL 725; Radiometer Medical A/S,

Bronshoj, Denmark). The samples were centrifuged at 1,000 rpm for 20 minutes and the isolated

plasma was stored at -80°C until creatinine and EPO analysis, and spectrophotometric analysis

for vasopressinase activity.

Figure 2-1. Experimental timeline of Hypoxia experiment. Rats on a regular diet were given 25mL of isotonic saline overnight (18 hrs) prior to the exposure to hypoxia (8% O2) or normoxia (21% O2). Blood, urine, renal papilla, and renal medullary tissue samples were collected and analyzed as described in Section 2.1. and 2.2.

50

2.2. Measured Outcomes from Protocol #1

2.2.1. Urine and Renal Papillary osmolality

Urine samples were collected at every time of excretion during the exposure to hypoxia

(8% O2) or normoxia (room air). The concentrations of Na+ and K+ were measured using a dual

channel flame photometer and Cl- was measured using eletromimetic titration (Chloride Meter,

CMT 10, London Scientific Ltd, London, Ontario). The concentrations of NH4+, creatinine, and

urea were measured using the same method described in Cheema-Dhadli et al (37). Urine

osmolality was calculated using the sum of cations plus urea: [2(Na+ + K+ + NH4+) + urea]. To

determine the rates of electrolyte excretion, the concentrations were divided by urinary creatinine

concentrations to control for variance within an individual rat, and to allow comparison between

rats. Since a fall in urine osmolality was observed following exposure to low PO2, another group

of rats were pretreated with dDAVP, which prevented the decrease in urine osmolality. Under

normal physiological conditions, urine osmolality usually reflects the renal medullary osmolality,

thus to determine the exact cause of the decrease in urine osmolality, renal papillary osmolality

was measured using the procedures described below.

Untreated and dDAVP-pretreated rats were exposed to 8% or 21% O2 for either 1hr or

2.5 hrs (n = 8 for each group). Rats were anesthetized with 5% isoflurane in 8% O2 (5L/min flow

rate) for 5 min immediately after removal from the hypoxia chamber. Blood was collected from

the descending aorta via a laparotomy using a heparinized syringe and analyzed as previously

described. Both kidneys were quickly removed and cut longitudinally using a surgical blade.

Longitudinally-cut kidney sections were blotted with gauze and papilla was excised with a sharp

51

knife, placed in a pre-weighed 2mL tube and sealed. The tube was weighed again and the weight

of the wet papilla was calculated as the difference in weights. A lithium diluent (1mL) was added

to the tube and the contents were homogenized. Lithium was used as an internal standard to

account for the variations in the measurements of concentration of Na+ and K+.

The dry weight of the papilla was calculated using a coefficient (0.8) based on the water

content from the previous studies. The actual papillary concentrations of Na+, K+, NH4+

, and urea

were calculated using the wet and dry weights of the papilla. The papillary osmolality was

calculated using the same formula used for the urine osmolality: [2(Na+ + K+ + NH4+) + urea].

This calculation was used rather than direct measurement of renal papillary osmolality, since

homogenizing the renal papillary tissue may release molecules from cells that affect the

osmolality.

2.2.2. ELISA for Plasma Erythropoietin

Erythropoietin (EPO) is a 34 – 39 kDa secreted glycoprotein that is responsible for red

blood cell formation. The major source of EPO is the interstitial peritubular fibroblasts in the

kidney and hepatocytes, and its production is up-regulated by hypoxia.

Plasma EPO was measured after exposure to 8% O2 and normoxia, using the quantitative

enzyme linked immunosorbent assay (ELISA) (Category no., MEPOO, R&D systems, Inc., MN,

USA). All samples and reagents were prepared as directed, and added to microplate which was

pre-coated with monoclonal antibody against rat EPO. The incubation and wash procedures were

performed as indicated and the optical density of each well was determined using a microplate

reader set to 450nm, with wavelength correction set to 540nm (SpectraMAX 340, GMI, Inc.,

52

MN, USA). The mean was calculated for the duplicate readings for each standard, control, and

samples, and the average zero standard optical density was subtracted. The standard curve was

generated and used to determine the EPO concentration in each plasma sample.

2.2.3. Renal Medullary Protein Markers for Hypoxia

The following protocol for Western blot of renal tissue hypoxia markers was adapted

from McLaren et al (118). To assess tissue oxygenation after exposure to hypoxia, renal cortical

and outer medullary tissue was harvested as described above and rapidly frozen in liquid

nitrogen (-70°C) after exposure to either hypoxia (8% O2) or normoxia. Harvested renal tissues

were stored in -80°C until further processing for Western blot. Renal cortical and medullary

tissues were mechanically homogenized (PowerGen 125, Fisher Scientific, Ottawa, ON, Canada)

in the cold buffer containing 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.45 M NaCl

supplemented with a mix of general protease inhibitors (leupeptin, PMSF, DTT). The

homogenized lysates were then centrifuged at 14,000rpm at 4°C, and the supernatants were

mixed with the same buffer containing 40% v/v glycerol. The Lowry-High sensitivity protein

assay was carried out using bovine serum albumin (BSA) to generate a standard curve.

Normoxic and hypoxic renal medullary tissue samples (40 µg) were subjected to 7.5 %

SDS-PAGE. The PageRuler Prestained Protein Ladder (cat. no. SM0671, Fermentas, Burlington,

ON, Canada), containing molecular weight markers ranging from 10 to 170 kDa, was used as a

size-standard. Samples were run on the gel at 170V for 1 hr, transferred to a nitrocellulose

membrane (Bio-Rad) at 90V for 1 hr, and blocked with 1% BSA in Tris-buffer overnight. The

membranes were incubated overnight at 4°C with primary antibody for HIF-1α (1:800 dilution of

53

polyclonal goat HIF-1α antibody; cat. no. AF1935, 45 R&D Systems, MN, USA), or eNOS

(1:800 dilution of mouse monoclonal anti-eNOS; cat. no. 610297, BD Biosciences, CA, USA).

Membranes were also probed for α-tubulin (1:1000 dilution of mouse monoclonal anti-α-tubulin;

cat. no. T6074, Sigma Aldrich, Oakville, ON, Canada) to assess if the loading of the sample and

transfer were uniform. On the next day, the membranes were washed for 1 hr, and then incubated

with secondary antibody specific for HIF-1α or eNOS (1:10,000 for HIF-1α, and 1:5000 for

eNOS) for 1 hr. The secondary antibody was conjugated to horseradish peroxidise in non-fat

milk/TBS buffer.

Antibodies were detected by exposing membranes to chemiluminescent reagents for 5

minutes (ECL Western Blotting System, Amersham Pharmacia Biotech, Baie d’Urfe, Quebec,

Canada), followed by exposure to Kodak Bio-max film using a Kodak X-OMat 2000A Processor.

Optical densities of HIF-1α (~120 kDa) and eNOS (~140 kDa) protein bands were quantified

using ImageJ software and were normalized to corresponding α-tubulin band (55 kDa) densities.

2.2.4. Treatment with dDAVP prior to Exposure to Hypoxia

Since a fall in the urine osmolality was observed at 1 hr of exposure to hypoxia, another

group of rats (n = 8) given an intraperitoneal injection (4 µg) of a long lasting synthetic analogue

of vasopressin, desmopressin acetate dDAVP (1-desamino-8-D-Arg vasopressin; Ferring Co.,

Ontario, Canada) 1 hr prior to exposure of rats to hypoxia or normoxia. Treatment of rats with

dDAVP was used to control for unknown endogenous levels of vasopressin and ensure that the

urine osmolality reflects the ability of the kidney to concentrate the urine, and not the level of

circulating vasopressin.

54

2.2.5. Detection of Vasopressinase Activity

Many studies have demonstrated the similar cases of water diuresis in pregnant

women, where water diuresis is observed following dehydration, or levels of vasopressin that

would lead to maximum antidiuresis (153). In these cases, water diuresis despite high measured

levels of vasopressin was attributed to increased activity of vasopressinase, which may increase

local degradation of circulating vasopressin (19; 106; 153). Since observed hypoxia-induced

water diuresis was similar to the state observed in pregnancy (water diuresis treatable with

vasopressinase-resistant desmopressin acetate), an enzymatic assay was used to detect

vasopressinase activity during exposure to hypoxia.

Since there appeared to be an increased activity of plasma vasopressinase during

exposure to hypoxia, the activity of vasopressinase was measured by measuring the rate of

degradation of substrate for vasopressinase. The enzymatic assay used for this experiment was

adapted from Small and Watkins, 1974 (158). The substrate used to measure vasopressinase

activity was S-benzyl-L-cysteine-p-nitroanilide, an enzyme-specific artificial substrate which is a

derivative of p-nitroanilide (30). The method of blood collection was as describe in Section 2.1.

The pKa of the reaction from HPO4- to H2PO4 is 6.8, thus in order to increase the pH of the

phosphate buffer to 7.2, the mixture (0.1 mM of phosphate buffer) was prepared by adding 3

parts of Na2HPO4 and 1 part of NaH2PO4 (log3 = 0.477). The fresh substrate solution (4.25 mM)

was prepared for each experiment. S-benzyl-L-cysteine-p-nitroanilide is only slightly soluble in

aqueous buffers at neutral pH, but remains in solution in water-miscible organic solvents. To

prepare the substrate solution, 14.1 mg of S-benzyl-L-cysteine-p-nitroanilide was dissolved in 10

ml of 2-methoxyethanol, a commonly used solvent specific for this substrate.

55

The phosphate buffer was prewarmed to 37°C and substrate solution (142 µL) was added

into a 10 mm spectrophotometer cuvette. The mixture was left to equilibrate for 5 minutes.

Following equilibration, 40µL of plasma sample was added into the cuvette. The change in

absorbance of the mixture was detected at 380 nm for 10 minutes. The change in absorbance per

minute was determined, and the enzymatic activity was calculated by multiplying the change in

absorbance by a factor of 3600, which was derived from the molar extinction coefficient of p-

nitroaniline, a degraded byproduct of the substrate (158).

56

2.3. Experimental Protocol #2: FUROSEMIDE

In the second protocol, rats were restricted to 15 mL of water overnight prior to the

experiment. Eighteen hours following the saline consumption, rats (n = 6/group) were injected

with different doses of furosemide: 0.4, 0.8, 1.6, 2.4 and 3.2 mg/kg of body weight. The control

group (n = 6) did not receive furosemide. Rats did not have access to food or drink following the

injection. From each rat, two urine samples were collected and stored at -20°C until analysis. The

purpose of the second urine sample was to ensure that the urine reflected the effect of

furosemide, and to exclude the possibility of mixture of previous bladder urine produced before

the injection of furosemide. After second urine sample was collected, rats were anesthetized

using 3% isoflurane, and blood samples from the descending aorta were obtained. The samples

were centrifuged at 1,000 rpm for 20 minutes and the isolated serum was stored at -80°C until

electrolyte measurement and creatinine analysis.

Figure 2-2. Experimental Timeline for Furosemide experiment. Rats were restricted to 15mL of water overnight (18 hrs) prior to injection of different doses of furosemide (0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). The control group did not receive furosemide. Blood, urine, renal papillary samples were collected and analyzed as described in Section 2.3. and 2.4.

57

2.4. Measured Outcomes: Protocol #2

2.4.1. Urine and Renal Papillary Osmolality

The second urine sample from each rat was analyzed to ensure that the urine reflected the

effect of furosemide. The method of measurement of urinary Na+, Cl-, K+, NH4+, urea, and

creatinine concentrations were as described in Section 2.2.1. As in the previous protocol, urine

osmolality was calculated using the sum of cations plus urea: [2(Na+ + K+ + NH4+) + urea]. The

excretions of electrolytes and urea were calculated by dividing the concentrations of Na+, Cl-, K+,

NH4+, Mg2+, and urea in the urine to creatinine concentrations to control for variance in

creatinine excretion within individual rat. The method of collection of renal papilla was carried

out as described in the previous protocol.

2.5. Statistical Analysis

All values are expressed as means + standard error margin (SEM). Statistical analysis

was carried out using SigmaPlot software (Sigma Plot Version 11.0; Systat Software Inc.,

Chicago, IL, USA) and mean values between different groups were compared using a one-way

analysis of variance (ANOVA), repeated ANOVA, or two-way ANOVA. When significant

interact effect was shown, a Tukey’s test was used to perform a post hoc pair-wise analysis.

Comparisons in urine and papillary osmolality between normoxia and hypoxia in the same rat

were performed using a paired t-test. Differences were considered statistically significant when

p<0.05.

58

CHAPTER 3

RESULTS

59

3.1. Part 1: Hypoxia

The renal outer medulla is susceptible to hypoxic injury due to marginal blood flow

despite high O2 consumption by active reabsorption of Na+. This study examined an early effect

of low PO2 on the function of the renal outer medulla, and defense mechanisms that may

minimize the risk of hypoxic injury in this region. My results have shown that the earliest

physiologic indicator of a low PO2 in arterial blood is a water diuresis, which may be a

compensatory response to compromised delivery of O2 in the renal outer medulla, the region

susceptible to injury. Results also have demonstrated that dDAVP may prevent the effects of

hypoxia in the deepest region of the renal outer medulla.

60

3.1.1. Effect of Hypoxia on Urine Flow Rate

To examine the effect of exposure to hypoxia on the major function of the renal outer

medulla, rats were exposed to 8% O2 (hypoxia) for 2.5 hrs. The purpose of inhaled 8% O2 was to

observe the effect of hypoxia as a single stimulus on otherwise, healthy animals and minimize

the confounding variables that could be involved with use of anesthesia (i.e., release of

vasopressin).

The first sign of change was a significant increase in the urine flow rate (60 + 7 µl/min)

at 1 hr of exposure to hypoxia relative to normoxia (6 + 1 µl/min; p<0.05; Figure 3-1; Left). An

increase in the urine flow rate may be an osmotic diuresis, where the major factor that influences

the urine flow rate is the number of effective osmoles that are excreted. To test whether the

increase in the urine flow rate during hypoxia was due to an osmotic induced diuresis, the rates

of excretions of electrolytes (Na+, Cl-, NH4+, and K+), glucose, and urea were measured.

61

3.1.2. Effect of Hypoxia on Creatinine Clearance and Electrolyte Excretion

There was no significant change in creatinine excretion or the calculated creatinine

clearance (Figure 3-2), suggesting that the glomerular filtration rate stayed constant throughout

the exposure to hypoxia. It was expected that the exposure to hypoxia may impair the function of

mTAL, which would significantly increase the excretions of Na+ and Cl-. The rates of electrolyte

excretions were expressed by dividing the concentration of Na+ and Cl- by the concentration of

creatinine excretion in the urine. Since the excretion of creatinine is relatively constant over the

collection period (176), ratios of the electrolytes to creatinine excretion provide a measure of

excretion that accounts for the incomplete bladder emptying within the individual rat, which may

result in mixing of old urine in the bladder and ureters with freshly produced urine.

The excretions of Na+ did not change (10 + 2 vs. 12 + 3 / mM creatinine; Figure 3-3) and

there was a significant fall in the rate of Cl- excretion (12 + 2 vs. 5 + 2 / mM creatinine) after 1hr

of exposure to low PO2 relative to normoxia (p<0.05). There was a small increase in the K+

excretion (12 + 2 vs. 15 + 1 / mM creatinine) during the same time period during hypoxic

exposure. Since there was no significant change in the excretions of Na+ and K+, while the Cl-

excretion was significantly decreased, this suggests that there was an increase in the excretion of

unmeasured anions after 1 hr of hypoxic exposure. It was unexpected that there was no increase

in the rate of excretion of electrolytes, given the significant rise in urine flow rate that was

observed (6 + 1 vs. 60 + 7 µl/min; p<0.05; Figure 3-1; Left). To study the basis of the increase in

urine flow rate, the urine osmolality was measured.

62

3.1.3. Effect of Hypoxia on the Urine Osmolality

Results showed that the urine flow rate increased significantly in about half of the rats

exposed to hypoxia. To determine whether the increase in the urine flow rate in this group of rats

was a water diuresis, the osmolality in the urine was measured after 1hr of exposure to low PO2,

using the formula [2(Na+ + K+ + NH4+) + urea]. Immediately after 2.5 hrs of hypoxia exposure,

the urine osmolality was significantly decreased to 333 + 42 mOsm/kg H2O compared to 1455 +

109 mOsm/kg H2O in the control normoxic rats (p<0.05; Figure 3-1; Right). This suggested that

these hypoxic rats had a water diuresis following exposure to hypoxia.

During water diuresis, most of the aquaporin-2 (AQP2) channels must be absent from the

luminal membrane in the late distal nephron, which means that there must be no activity of

vasopressin. To test whether the hypoxia-induced water diuresis was due to a near-absence of

vasopressin, a second group of rats were pretreated with dDAVP 1hr prior to exposure to

hypoxia. In this group, the urine flow rate did not rise (3 + 0.4 µL/min; Figure 3-4) and the

hypoxia-induced decrease in the urine osmolality was not detected (2193 + 162 mOsm/kg H2O)

during exposure to hypoxia relative to normoxia. Since dDAVP prevented the hypoxia-induced

water diuresis, this suggests that hypoxia may release a vasopressinase, a peptidase that degrades

vasopressin, thereby impairing water reabsorption in the kidney.

63

Figure 3-1. Urine flow rate and osmolality in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). Urine flow rate increased significantly following exposure to hypoxia for 2.5 hrs. The rise in urine flow rate was accompanied by a significant decrease in urine osmolality, suggesting that rats were experiencing water diuresis with low urine osmolality under hypoxia exposure. N = 8; Mean + SEM; *p<0.05 vs. Normoxia.

64

Figure 3-2. Creatinine excretion and clearance in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). There was no significant change in creatinine excretion or clearance during exposure to hypoxia relative to normoxia, suggesting a constant glomerular filtrate rate throughout hypoxia exposure. N = 8; Mean + SEM; *p<0.05 vs. Normoxia.

65

Figure 3-3. Renal excretion of Na+, Cl- and K+ in rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). Excretions were calculated by normalizing electrolyte concentrations to that of creatinine. Excretion of Na+ and K+ did not change after exposure to 2.5 hrs of hypoxia (black bars) relative to normoxia (white bars). There was a significant decrease in the Cl-

excretion, which suggests that hypoxia may increase excretion of unmeasured anions. N = 8; Mean + SEM; *p<0.05 vs. Normoxia.

66

#

Figure 3-4. Hypoxia-induced water diuresis is prevented with pretreatment with desmopressin acetate (dDAVP) 1 hr prior to hypoxia exposure. When rats were pretreated with dDAVP before exposure to hypoxia, water diuresis did not occur (no significant decrease in urine flow rate or osmolality). Since pretreatment with a synthetic analogue of vasopressin prevents a hypoxia-induced water diuresis, this suggests that hypoxia increases metabolic clearance of vasopressin that may be mediated by a release of plasma vasopressinase. N = 8; Mean + SEM; *p<0.05 vs. Normoxia; #p<0.05 vs. Hypoxia (-dDAVP).

67

3.1.4. Effect of Hypoxia on Renal Papillary Osmolality

To test whether the hypoxia-induced water diuresis was due to vasopressinase-mediated

degradation of vasopressin, the renal papilla was collected and its osmolality was measured. If

the reabsorption of water in the collecting duct was impaired due to loss of vasopressin, there

would be a significantly higher osmolality in the renal papillary compared to the urine osmolality.

Since the papillary osmolality was significantly higher than the urine osmolality at the end of the

hypoxia exposure (869 + 57 vs. 333 + 42 mOsm/kg H2O; p<0.05; Figure 3-5), this suggests that

water reabsorption was impaired during hypoxia, which is consistent with the hypothesis that

hypoxia may trigger release of vasopressinase and thereby diminish the actions of vasopressin.

There was a significant fall in the papillary osmolality (869 + 57 mOsm/kg H2O) relative

to normoxia (p<0.05), which suggests that the active reabsorption in the mTAL was impaired

during exposure to hypoxia. Although there was a significant fall in the papillary osmolality

during hypoxic exposure, the active reabsorption in the mTAL was not severe enough to cause a

rise in the Na+ excretion (natriuresis) or Cl- excretion (chloriuresis).

68

Figure 3-5. Urine and renal papillary osmolalities of rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). Previously, it was shown that dDAVP pretreatment prevented hypoxia-induced water diuresis, suggesting that hypoxia may increase plasma vasopressinase and increase metabolic clearance of vasopressin. An absence of vasopressin would limit number of aquaporin-2 water channels on the medullary collecting duct, resulting in a failure of osmotic equilibrium between renal medullary interstitium and collecting duct. The measured renal papillary osmolality (black bar) was significantly higher compared to urine osmolality (white bar) during hypoxia, indicating that vasopressin was absent during hypoxia and thus osmotic equilibrium did not occur. These results suggest that hypoxia may increase the level of vasopressinase, which leads to increased metabolic clearance of vasopressin. The results also show that the measured renal papillary osmolality (black bar) significantly decreased after exposure to hypoxia, indicating that the function of mTAL was significantly compromised with low PO2. N = 8; Mean + SEM; *p<0.05 vs. Normoxia; #p<0.05 vs. Renal papilla.

69

3.1.5. Effect of Hypoxia on Plasma Vasopressinase Activity

The activity of plasma vasopressinase was determined by measuring the rate of

degradation of S-benzyl-L-cysteine-p-nitroaniline. This compound is a synthetic substrate for

this analogue of vasopressin that liberates a chromogenic compound, p-nitroaniline when

vasopressinase cleaves the amide bond between the N-terminal cysteine and adjacent tyrosine

bond. The activity of vasopressinase is expressed as milli International Units (mIU), which

defines the quantity of vasopressinase that will release 1 nmole of p-nitroaniline/min from S-

benzyl-L-cysteine-p-nitroanilide.

As previously mentioned, the assay measured the change in absorbance that resulted from

the liberation of p-nitroaniline from the substrate under vaopressinase activity. The increase in

the absorbance at 380 nm (optimal wavelength for p-nitroaniline) reflected the activity of

vasopressinase. There was an increase in the absorbance at 380 nm over 10 minutes, in both

normoxic and hypoxic rats (Figure 3–6). The activity of plasma vasopressinase in rats exposed to

normoxia was 14.5 + 3.0 mIU/min (Figure 3-7). Immediately after exposure of rats to 2.5 hrs of

hypoxia, the vasopressinase activity increased significantly to 32.4 + 4.9 mIU/min (p<0.05). The

increases in the rate of degradation of S-benzyl-L-cysteine-p-nitroanilide in the samples support

the finding that the activity of vasopressinase is increased during exposure to hypoxia, which

explains the loss of vasopressin effect and the water diuresis that was observed during hypoxia

exposure in rats.

70

Figure 3-6. Total change in the absorbance at 380nm detected in the plasma samples of rats exposed to normoxia (21% O2) or hypoxia (8% O2). In order to measure the activity of plasma vasopressinase, enzymatic assay was used to detect the change in absorbance at 380nm that resulted from the liberation of p-nitroaniline from the substrate under vaopressinase activity. The increase in absorbance at 380 nm was greater in the hypoxia group, suggesting that the rate of production of p-nitroaniline from S-benzyl-L-cysteine-p-nitroanilide was higher in rats exposed to hypoxia relative to normoxic group. This indicates that hypoxia increases vasopressinase activity, either due to release or increased stability of vasopressinase. N = 8; Mean + SEM; *p<0.001 vs. Normoxia.

71

Figure 3-7. Activity of plasma vasopressinase, expressed as the rate of production of 1 nmole of p-nitroaniline from S-benzyl-L-cysteine-p-nitroanilide by plasma vasopressinase per minute (mIU/min), in rats exposed to normoxia (21% O2) or hypoxia (8% O2). There was a significant increase in the rate of degradation of the substrate into p-nitroaniline during exposure to hypoxia. This indicates that the exposure to hypoxia in rats increases the activity of plasma vasopressinase, leading to a loss of vasopressin in the circulation and subsequent water diuresis observed in rats during hypoxia. N = 8; Mean + SEM; *p<0.001 vs. Normoxia.

72

3.1.6. Effect of dDAVP on Renal Papillary Osmolality and Papillary Urea Composition

As previously shown, when rats were pretreated with dDAVP 1hr prior to hypoxia

exposure, the urine osmolality remained high relative to the untreated group. Since dDAVP is a

synthetic analogue of vasopressin, it was hypothesized that the hypoxia-induced fall in the urine

osmolality was absence of vasopressin, and therefore dDAVP may have mimicked this action of

vasopressin. This hypothesis was supported by the measurement of renal papillary osmolality,

which was significantly higher compared to the urine osmolality (869 + 57 vs. 333 + 42

mOsm/kg H2O; p<0.05). Since the urine osmolality in the dDAVP treated group was much

higher compared to the untreated group during exposure to hypoxia, renal papillary osmolality

was also measured in this group to explain the basis of high urine osmolality. Renal papillary

osmolality in the dDAVP pretreated group remained high throughout the hypoxia exposure

(2024 + 117 mOsm/kg H2O; Figure 3-8). Thus this result suggests that the pretreatment with

dDAVP prevented hypoxia-induced water diuresis by preventing the fall in the osmolality in the

medullary interstitial compartment. dDAVP also allowed osmotic equilibrium, because the renal

papillary osmolality was equal to the urine osmolality. The baseline renal papillary osmolality

during normoxia was higher in dDAVP-treated group when compared to the group that was

untreated.

As previously mentioned in Section 1.5.2., reabsorption of urea in the inner medullary

interstitial compartment reduces the need of active reabsorption deep in the renal outer medulla,

by replacing the active reabsorption of Na+ and Cl- in the mTAL with passive reabsorption in the

inner medulla. Therefore high concentration of urea in the inner medullary interstitial

compartment is important for the high composition of urea in the inner medulla that allows

73

passive reabsorption of Na+ and Cl- in the outer medulla. After 2.5 hrs of exposure to hypoxia,

there was a decrease in the papillary concentration of urea relative to normoxia (472 + 35 to 224

+ 29 mOsm/kg H2O; Figure 3-9). The pretreatment with dDAVP prevented the hypoxia-induced

fall in the urea concentration in the medullary compartment (1229 + 72 mOsm/kg H2O; Figure

3-9), which suggests that the reabsorption of urea in the inner medullary interstitial compartment

was occurring under hypoxia, generating the high urea concentration.

74

Figure 3-8. The fall in the measured renal papillary osmolality is prevented with pretreatment with dDAVP 1 hr prior to hypoxia exposure. Hypoxia-induced water diuresis was prevented with dDAVP pretreatment. In addition, the graph shows that the renal papillary osmolality (black bars) did not decrease when rats were pretreated with dDAVP before hypoxia exposure. The pretreatment maintained the high renal papillary osmolality, suggesting that both active reabsorption in the mTAL of outer medulla and water reabsorption occurred. These results suggest that dDAVP may maintain adequate O2 delivery to the renal outer medulla during hypoxia. N = 8; Mean + SEM; *p<0.05 vs. Normoxia; #p<0.05 vs. Renal papilla.

75

Figure 3-9. The pretreatment with dDAVP prior to exposure to hypoxia prevents the decrease in the urea composition in the renal medullary interstitial compartment. The fall in the urea concentration (black bars) in the renal medullary interstitial compartment occurs after 2.5 hrs of exposure to hypoxia. The pretreatment with dDAVP prevented this hypoxia-induced fall in renal medullary urea composition, suggesting that reabsorption of urea was occurring during hypoxia, generating high urea concentration that is required for decreasing the need of active reabsorption in the mTAL in the renal outer medulla (discussed in Section 1.5.2.). N = 8; Mean only.

76

3.1.7. Effect of dDAVP on Creatnine & Electrolyte Excretions

The pretreatment of dDAVP 1 hr prior to hypoxia exposure prevented the fall in the renal

papillary osmolality. In the dDAVP pretreated group, the rate of excretion of creatinine did not

change during exposure to hypoxia (Figure 3-10). There was no significant change in the

excretions of Na+, Cl- and K+ relative to normoxia. As mentioned in the previous section, the

renal papillary osmolality did not decrease during hypoxia. Moreover, since the excretions of

Na+, Cl- and K+ did not change throughout exposure to hypoxia (Figure 3-11), the results show

that active reabsorption in the mTAL in the renal outer medulla was maintained, which

prevented the fall in the osmolality in the renal medullary interstitial compartment. These results

suggest that dDAVP may improve O2 delivery that provides the source of O2 for metabolic

demand in the mTAL of renal outer medulla during exposure to hypoxia. To examine whether

dDAVP increases delivery of oxygen during exposure to hypoxia, indices of tissue hypoxia, such

as plasma erythropoietin and lactate, were measured. The results demonstrating the signs of

tissue hypoxia are discussed in the following section, which also includes the effect of dDAVP

pretreatment on these signs during exposure to hypoxia.

77

Figure 3-10. Creatinine excretion and clearance in dDAVP-pretreated rats exposed to normoxia (21% O2) or hypoxia (8% O2). There was no significant change in creatinine excretion or clearance during exposure to hypoxia relative to normoxia, suggesting a constant glomerular filtrate rate throughout hypoxia exposure. N = 8; Mean + SEM; *p<0.05 vs. Normoxia.

78

Figure 3-11. Renal excretion of Na+, Cl- and K+ in dDAVP-pretreated rats exposed to 2.5 hrs of normoxia (21% O2) or hypoxia (8% O2). Excretions were calculated by normalizing electrolyte concentrations to creatinine. Excretion of Na+, Cl- and K+ did not change after exposure to 2.5 hrs of hypoxia (gray bars) relative to normoxia (white bars). N = 8; Mean + SEM; *p<0.05 vs. Normoxia.

79

3.1.8. Signs of Renal Hypoxia and Effect of dDAVP Treatment

Previous unpublished findings in the lab have demonstrated that the degree of hypoxia

(8% O2) used in this experiment is not sufficiently severe to cause death in rats. The findings

from the previous studies suggested that exposure up to 5 hrs exposure to this degree of hypoxia

causes physiological changes that are reversible with recovery under normoxia. The results from

the current study suggested that the degree of hypoxia caused a large response within the kidney,

demonstrated as a large water diuresis, within as early as 1 hr of exposure. This water diuresis

during hypoxia was prevented with pretreatment with dDAVP. It is important to determine

whether the water diuresis observed was indeed due to hypoxia, and whether dDAVP prevents

water diuresis during exposure to hypoxia by increased O2 delivery. To document the tissue

response to hypoxia, plasma erythropoietin and blood lactate were measured in rats exposed to

hypoxia, with or without pretreatment of dDAVP.

80

3.1.8.1. Blood Lactate Level

Under normoxia, the blood lactate concentration was approximately 1.5 mM in the

control group. After 2.5 hrs of exposure to hypoxia, blood lactate levels increased slightly to 2

mM (Figure 3-12). Previously findings from our lab have demonstrated that blood lactate levels

increased significantly immediately after 5h of exposure to hypoxia, but decreased to control

levels within 6h post-hypoxia exposure. Thus the model of systemic hypoxia (8% O2) used for

this study appears to be of modest degree, and not detrimental to cause irreversible renal damage.

In rats pretreated with dDAVP 1h prior to hypoxia exposure, there was no significant change in

the blood lactate concentration.

3.1.8.2. Plasma EPO

The baseline concentration of plasma EPO was 33.5 + 1.8 pg/ml. At 1hr of hypoxia, the

plasma EPO concentration increased significantly to 68.6 + 4.2 pg/ml (p<0.05; Figure 3-13).

When exposure to hypoxia was increased up to 2.5h, the EPO level increased even further to

235.6 + 84.8 pg/ml. According to previous findings in the lab, the elevation in EPO levels was

reversible, since it returned to control values 6 hrs after re-exposure to normoxia. In the dDAVP

pretreated group, the baseline concentration of plasma EPO was 68.5 pg/ml, which was higher

relative to the untreated group (33.5 + 1.8 pg/ml; p<0.05). There was no change in the plasma

EPO levels throughout exposure to hypoxia in the dDAVP pretreated group.

81

*

#

Figure 3-12. Blood lactate concentration in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2), with or without pretreatment with dDAVP. The concentration of blood lactate (white bars) showed a slight increase throughout exposure to 2.5 hrs of hypoxia. When rats were pretreated with dDAVP, the increase in blood lactate concentration did not change throughout the hypoxia exposure (gray bars). N = 6; Mean + SEM; *p<0.05 vs. Normoxia; #p<0.05 vs. No dDAVP.

82

Figure 3-13. Plasma erythropoietin (EPO) in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2), with or without pretreatment with dDAVP. Plasma EPO concentration (white bars) increased significantly throughout exposure to hypoxia. The significant increase in plasma EPO under hypoxia did not occur when rats were pretreated with dDAVP prior to hypoxia exposure. N = 8; Mean + SEM; *p<0.05 vs. Normoxial #p<0.05 vs. No dDAVP.

*

*

*#

# # #

83

3.1.8.3. Renal Medullary Protein Markers of Hypoxia

Western blot analysis was used to detect the renal medullary expression of HIF-1α and

eNOS, with molecular weights of 120 and 140 kD, respectively (Figure 3-15). The protein band

densities were normalized to α-tubulin loading controls in the protein samples. Immediately

after 2.5 hrs of exposure to hypoxia, there was a significant increase in the renal outer medullary

expression of HIF-1α (~4 fold; p<0.05). Although non-significant, there was a slight increase in

the density of renal outer medullary eNOS (0.41 + 0.08) protein band relative to normoxia (0.32

+ 0.01). In the dDAVP pretreated group, the renal outer medullary expression of HIF-1α did not

change significantly (2-fold). There was a non-significant, slight increase in the expression of

eNOS in the renal outer medulla in the dDAVP-pretreated group after 2.5 hrs of exposure to

hypoxia (Figure 3-14). There was a significant increase in the renal outer medullary expression

of eNOS with dDAVP pretreatment under normoxia, relative to untreated normoxic rats (Figure

3-15).

84

HIF‐1 α

eNOS

α ‐tubulin

Normoxia Hypoxia

A) Renal outer medullary expression of proteins after 2.5 hrs of hypoxia

HIF‐1 α

eNOS

α ‐tubulin

Normoxia Hypoxia

B) Renal outer medullary expression of proteins after 2.5 hrs of hypoxia in dDAVP pretreated rats

Figure 3-14. Renal medullary protein expression in rats exposed to 2.5 hrs of hypoxia (8% O2) or normoxia (21% O2) with or without pretreatment with dDAVP. Renal medullary hypoxia-inducible factor 1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) protein expressions were measured using western blotting technique in rats exposed to normoxia (white bars) or hypoxia (black bars). There was a significant increase in HIF-1α (~5-fold) and statistically non-significant increase eNOS protein expressions in the renal outer medulla after exposure to 2.5 hrs of hypoxia. The dDAVP pretreatment group did not show any significant changes in the renal outer medullary expression of these proteins. N = 3; Mean + SEM; *p<0.05 vs. Normoxia.

85

HIF‐1 α

eNOS

α ‐tubulin

‐dDAVP +dDAVP

Renal outer medullary expression of proteins in untreated and treated dDAVP rats under normoxia

Figure 3-15. Renal outer medullary protein expression in untreated and treated with dDAVP rats under normoxia (21% O2). Renal outer medullary hypoxia-inducible factor 1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) protein expressions were measured using western blotting technique in rats under normoxia without (white bars) or with (gray bars) treatment with dDAVP. There was no change in the expression of HIF-1α and a significant increase in eNOS protein expression in the renal outer medulla with dDAVP treatment. N = 3; Mean + SEM; *p<0.05 vs. No dDAVP.

86

In summary, the exposure to hypoxia resulted in significant increases in the levels of

blood lactate and plasma EPO and a slight increase in the renal medullary expression of eNOS.

The significant increases in lactate and EPO were not detected in hypoxic rats pretreated with

dDAVP, suggesting that dDAVP treatment may lead to improved global O2 delivery. The renal

outer medullary expression of eNOS was significantly higher in dDAVP pretreated rats, which

indicates that the signs of improved O2 delivery with dDAVP treatment may be regulated by

NO-mediated vasodilation.

87

PART 1: Summary of Significant Results

1) An early physiological response to exposure to hypoxia in rats is a large water diuresis,

which is indicated by a significant increase in the urine flow rate accompanied by a

significant decrease in the urine osmolality.

2) The excretions of Na+ did not change significantly after exposure to hypoxia and

excretion of Cl- decreased significantly, suggesting that the degree of hypoxia used in this

study did not severely impair the active reabsorption by mTAL in the renal outer medulla.

3) The pretreatment with dDAVP prior to exposure to hypoxia prevented water diuresis,

suggesting that hypoxia may increase the level of vasopressinases, which increases the

metabolic clearance of vasopressin required to insert sufficient number of AQP-2

channels and allow reabsorption of water in the renal collecting duct.

4) There was a significant fall in the renal papillary osmolality after exposure to hypoxia,

suggesting that the active reabsorption by mTAL in the renal outer medulla was

compromised by a lower PO2. Renal papillary osmolality was significantly higher

compared to the urine osmolality, and the activity of plasma vasopressinase increased

during hypoxia, which indicated that hypoxia may increase the metabolic clearance of

vasopressin, causing the large water diuresis that was observed.

88

5) The pretreatment with dDAVP prevented the fall in the renal papillary osmolality,

suggesting that active reabsorption of Na+ by the mTAL occurred and sufficient O2

delivery was present to meet the metabolic demand in the renal outer medulla during

exposure to 8% O2. The pretreatment with dDAVP prevented a significant increase in

blood lactate and plasma EPO, suggesting that dDAVP improved global delivery of O2.

In normoxic rats pretreated with dDAVP, there was a significant increase in the renal

outer medullary expression of eNOS, which indicates that dDAVP may improve O2

delivery by NOS-mediated vasodilation.

89

3.2. Part 2: Furosemide

The objective of the second part of this study was to determine an optimal dose of

furosemide, a common loop-diuretic used to treat hypertension, edema and heart failure, that can

reduce the work in the renal outer medulla without depleting the body of electrolytes, such as

Na+, Cl-, K+, and Mg2+. This study examined the effect of different doses of furosemide (0, 0.4,

0.8, 1.6, 2.4 and 3.2mg/kg of body weight) on the function of the renal outer medulla. 0.8mg of

furosemide/kg of body weight is the dose that caused a significant increase in the rate of

excretion of electrolytes; there were much higher excretions with higher doses, which suggest

that doses of furosemide higher than 0.8mg/kg may deplete the body of electrolytes.

90

3.2.1. Dose-Effect of Furosemide on Urine Flow Rate

Since the effect of furosemide is often determined by the urine flow rate in the clinical

setting (91), rats were injected with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4 and

3.2mg/kg of body weight) and the urine flow rates were measured. The urine flow rate increased

significantly at 1.6mg/kg of furosemide (Figure 3-16), with increasing trend with higher doses.

Figure 3-16. Urine flow rate in rats intraperitoneally injected with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). Urine flow rate increased with higher doses of furosemide, with significant increase beginning at 1.6 mg/kg. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg.

91

3.2.2. Dose-Effect of Furosemide on Urine Osmolality

To avoid any confounding variables with mixtures of residual urine due to incomplete

emptying of bladder, two urine samples were collected for analysis. Since the urine flow rate

increased significantly at 1.6 mg of furosemide/kg, it was expected that the urine osmolality

would show a significant fall at this dose of furosemide, reflecting the reduced function in the

renal outer medulla. However, the urine osmolality decreased significantly at 0.8mg/kg of

furosemide, which indicates that fall in the urine osmolality occurs before the urine flow rate

increases (Figure 3-17).

92

Figure 3-17. Urine flow rate and osmolality in rats injected intraperitoneally with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). Urine osmolality (dashed line) decreased significantly with increasing doses of furosemide. Beginning at 0.8 mg/kg urine osmolality started to significantly decrease, which occurred before a significant rise in urine flow rate (solid line) at 1.6 mg/kg of furosemide. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg (Urine Flow Rate); #p<0.05 vs. 0 mg/kg (Urine Osmolality).

93

3.2.3. Dose-Effect of Furosemide on Papillary Osmolality

To test whether the fall in the urine osmolality reflected the independent action of

furosemide (without any external factors, such as impaired water reabsorption in the distal

collecting ducts), the renal papilla was collected and its osmolality was measured. The renal

papillary osmolality paralleled the urine osmolality (Figure 3-18), which suggests that the fall in

the osmolality was only due to reduced active reabsorption in the renal outer medulla. There was

a significant decrease starting at 0.4 mg/kg of furosemide, and the decrease continued with

higher doses of furosemide until 1.6 mg/kg, where both urine and papillary osmolality showed

no further decrease.

94

Figure 3-18. Urine and renal papillary osmolality in rats injected intraperitoneally with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). Renal papillary osmolality (dashed line), which reflected urine osmolality (solid line), decreased with higher doses of furosemide, with significant decrease beginning at 0.4 mg/kg. However, renal papillary osmolality was still high at 0.4 mg/kg (~2000 mOsm/kg H2O) and a large decrease in the osmolality occurred at 0.8 mg/kg of furosemide, where it decreased to ~1100 mOsm/kg H2O. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg.

95

3.2.4. Dose-Effect of Furosemide on Rates of Excretions of Na+, Cl- and K+

As expected, the excretions of Na+, Cl-, and K+ increased significantly with higher doses

of furosemide (Figure 3-19). The excretions of Na+ and K+ increased up to 8.6-fold and 2.7-fold

respectively. There was a detectable fall in Na+ excretion at 0.4 mg/kg of furosemide, which may

reflect the increased Na+ reabsorption in the distal nephron segment by ENaC, due to increased

Na+ in the filtrate reaching the distal convoluted tubules. There was a significant increase in the

rate of Cl- excretion with increasing dose of furosemide, and Cl- excretion approximately equaled

the sum of excretions of Na+ + K+ (Figure 3-20), which was expected since the inhibition of

NKCC2 cotransporter by furosemide will impair reabsorption of Na+, K+ with 2Cl-.

96

Figure 3-19. Excretion of electrolytes (Na+, Cl- and K+) in rats injected with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight by the intraperitoneal route). Rates of excretions of Na+ (filled diamond; solid), Cl- (white square; dashed) and K+ (white triangle; dashed) increased with higher doses of furosemide, with significant increase beginning at 0.8 mg/kg. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg.

97

Figure 3-20. Excretion of electrolytes ([Na+ + K+] and Cl-) in rats injected with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight by the intraperitoneal route). Sum of excretions of the Na+ plus K+ (white triangle; dashed) and the excretion of Cl- (black diamond; solid black) increased with higher doses of furosemide, with significant increase beginning at 0.8 mg/kg. The rate of Cl- excretion rate was approximately equal to the sum of rates of excretion of Na+ and K+, which reflects the impaired reabsorption of Na+ and Cl- by NKCC2 cotransporter, which is inhibited by furosemide. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg.

98

3.2.5. Dose-Effect of Furosemide on Excretion of Magnesium

The excretion of magnesium (Mg2+) increased with increasing doses of furosemide

(Figure 3-21). Large increases in the rate of excretion of Mg2+ with higher doses of furosemide

suggest that very large doses of furosemide leads to renal Mg2+ wasting and increase the risk of

decrease in plasma Mg2+ levels.

Figure 3-21. Excretion of Mg2+ in rats injected with different doses of furosemide by the intraperitoneal route (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). Excretion of Mg2+ increased with higher doses of furosemide, with significant values beginning at 0.8 mg/kg. N = 6; Mean + SEM; *p<0.05 vs. 0 mg/kg.

99

3.2.6. Dose-Effect of Furosemide on Serum Magnesium

As expected, the concentration of serum magnesium decreased with increasing doses of

furosemide (Figure 3-22). However, there was a rise in the serum magnesium in the highest dose

group at 3.2 mg/kg, which suggests that rats may have different mechanisms to handle or

reabsorb Mg2+ during periods when the rate of excretion is very high, to compensate for the loss

of Mg2+.

Figure 3-22. Plasma level of Mg2+ in rats intraperitoneally injected with different doses of furosemide (0, 0.4, 0.8, 1.6, 2.4, and 3.2 mg/kg of body weight). Plasma level of Mg2+ decreased with higher doses of furosemide, with significant decrease beginning at 1.6 mg/kg. N = 6; Mean + SEM; *p<0.05 vs. 0.4 mg/kg.

100

PART 2: Summary of Significant Results

1) A significant fall in the urine osmolality occurs before a significant rise in the urine flow

rate, and this should be used to indicate the effectiveness of furosemide.

2) 0.8mg/kg (of body weight) is the dose of furosemide that significantly reduces the

medullary osmolality and therefore is the dose that may significantly reduce the function

and thereby metabolic demand of mTAL in the renal outer medulla.

3) The rates of excretions of electrolytes (Na+, Cl-, K+ and Mg2+) increase significantly

beginning at 0.8mg/kg of furosemide, and higher rates of excretions may lead to

depletion of these electrolytes.

101

CHAPTER 4

DISCUSSION

102

4.1. Summary of Results: HYPOXIA

The purpose of the first part of this study was to determine the earliest indicator of

hypoxia in the renal outer medulla, the area in the kidney most susceptible to hypoxic injury, and

to examine the underlying mechanisms that may minimize the risk of injury in this region. The

results of the present study showed the following. Overall at the renal level, the creatinine

clearance did not change during hypoxia, suggesting that the degree of hypoxia (8% O2) was not

severe enough to significantly change the glomerular filtration rate (GFR).

With exposure to hypoxia, there were signs of changes in the function of mTAL in the

renal outer medulla. The urine flow rate increased significantly (~10 fold) in rats exposed to

hypoxia. The significant increase in the urine flow rate suggested a fall in the renal medullary

osmolality that may have resulted from impaired active reabsorption of mTAL in the renal outer

medulla. However, the excretions of Na+ and K+ did not change while the excretion of Cl-

decreased significantly, which suggested that hypoxia was not severe enough to compromise the

function of mTAL significantly. There was a significant fall in the urine osmolality during

hypoxia, which suggested that rats were experiencing a large water diuresis. In addition, the

significantly lower osmolality in the urine compared to the renal papilla suggested that water

reabsorption in the medullary CD was impaired, and the vasopressin failed to act during hypoxia.

The level of vasopressinase activity was increased during hypoxia, which explained the hypoxia-

induced loss of antidiuretic action of vasopressin. The origin of vasopressinase may be the liver,

since the liver is also susceptible to hypoxia (56).

Of note, the hypoxia-induced large water diuresis did not occur in every rat exposed

hypoxia. Lower degree of water diuresis was observed, which may have been due to a higher

103

level of endogenous vasopressin or a lower magnitude of activity of vasopressinase in these rats

during hypoxia. The lower magnitude of vasopressinase may not have been strong to diminish all

of the action of vasopressin in the circulation.

In addition, pretreatment with dDAVP prevented the hypoxia-induced water diuresis and

the increase in the hypoxic markers, such as blood lactate and plasma EPO. Lower levels of

hypoxic markers with dDAVP pretreatment indicate that dDAVP may improve O2 delivery,

likely by increasing perfusion. The results from this study are discussed in the following

sections.

104

4.1.1. Hypoxia-Induced Water Diuresis

4.1.1.1. Effect of Hypoxia on Urine Flow Rate & Osmolality

The earliest physiological response to hypoxia in rats observed in this study was a

significant rise in the urine flow rate from 6 + 1 to 60 + 7 µL/min (Figure 3-1; Left). To

determine whether the rise in the urine flow rate was an osmotic-induced diuresis, concentration

of electrolytes such as Na+, Cl- and K+ in the urine were measured. In the case of osmotic-

induced diuresis, the excretion of effective osmoles determines the urine flow rate together with

the maximum medullary interstitial osmolality. It was previously expected that acute exposure to

hypoxia would result in increasing rate of excretion of Na+, Cl- and K+, which would reflect a

diminished mTAL function. The large diuresis was not an osmotic-induced diuresis, since there

was no increase in the excretions of Na+, Cl- or K+, which would occur if active reabsorption of

the mTAL is impaired by hypoxia. The higher excretions of Na+ and K+ than Cl- suggests that

there may have been a significant increase in the excretion of unmeasured anions during

hypoxia. To further test the effect of hypoxia on mTAL function, urine osmolality was measured

to examine the ability of the mTAL to concentrate the urine.

There was a significant fall in the urine osmolality from 1455 + 109 to 333 + 42

mOsm/kg H2O following exposure to hypoxia, which suggested that rats were experiencing

water diuresis during hypoxia. During water diuresis, urine flow rate is determined by the

volume of filtrate reaching the distal nephron, excluding the volume reabsorbed through residual

water permeability in the inner medullary collecting duct (CD) (17). A significant increase in the

105

urine flow rate with low urine osmolality suggests that there was a failure of osmotic equilibrium

during hypoxia. Previous studies have shown that hypoxia leads to diuresis in rats, dogs and

humans (132), and diuresis has been demonstrated to occur under hypoxic conditions between 10

to 16% of O2 (81; 83; 93). The authors of these studies suggested that diuretic response is an

early adaptive response to hypoxia that increases the blood O2 capacity before the long-term

adaptive responses come into effect. Although it had previously been proposed that the hypoxic

diuretic response may be the result of hypoxic ventilatory response, this view has not been

confirmed by many studies (81). Hypoxia-induced water diuresis in rats which was observed at

this stage of my study suggested two possible hypotheses of effect of hypoxia on the loss of

antidiuretic ability of the kidney.

Under normal physiological conditions, the concentrated urine originates from

hyperosmotic renal medullary interstitial gradient generated by the mTAL, which with

reabsorption of water from the CD produces concentrated urine. Therefore, I measured

osmolality in the renal papilla, which reflects the renal medullary osmolality, to test whether the

fall in the urine osmolality resulted from the decrease in renal medullary osmolality.

On the other hand, since excretions of Na+, Cl- and K+ did not change during hypoxia, I

hypothesized that the hypoxia-induced fall in the urine osmolality results from release of

vasopressinases, enzymes that degrade endogenous vasopressin and prevent water reabsorption

in the medullary CD. To test whether hypoxia causes release of vasopressinase, I pretreated

another group of rats with dDAVP prior to exposure to hypoxia. Since dDAVP is not hydrolyzed

by vasopressinases, I expected that dDAVP will retain the ability to insert AQP2 water channels

in the medullary CD and result in an osmotic equilibrium, producing concentrated urine during

hypoxia. As expected, when rats were pretreated with dDAVP before hypoxia exposure, the

106

urine osmolality did not change significantly following hypoxia (2193 + 162 mOsm/kg H2O;

Figure 3-4). This finding supported the hypothesis that hypoxia leads to a loss of vasopressin. If

there is absence of vasopressin during hypoxia, there will be a failure of osmotic equilibrium in

the medullary CD. Therefore to confirm this theory, I compared the osmolality between the urine

and the renal papilla during hypoxia. A higher renal papillary osmolality than the urine

osmolality would confirm the absence of vasopressin.

4.1.1.2. Effect of Hypoxia on Renal Papillary Osmolality

To test my first hypothesis that hypoxia-induced fall in the urine osmolality results from

reduced osmolality in the renal medullary interstitial compartment, renal papillary osmolality

was measured. Papillary osmolality is a direct measurement of the mTAL function, since the

osmolality in the renal medullary interstitial compartment is generated by the active reabsorption

of Na+ in the mTAL. As expected, the papillary osmolality decreased significantly during

hypoxia (1343 + 53 to 869 + 57 mOsm/kg H2O; Figure 3-5), which indicated that hypoxia

reduced the active reabsorption of mTAL in the renal outer medulla. Of note, there was no

increase in the excretions of Na+, or Cl-. This is consistent with the revised description of the

urine concentrating mechanism proposed by Halperin et al (75), which suggests that of the total

Na+ reabsorbed in the mTAL, approximately only ¼ is used to generate hyperosmotic medullary

interstitial compartment and the remainder recycles back in the DtL of Henle’s loop (discussed in

1.4.1.1.). Therefore, hypoxia may compromise the active reabsorption of mTAL which impairs

the generation of hyperosmotic renal medullary interstitial compartment but recyling of Na+ into

DtL may prevent a large natriuresis or chloriuresis during hypoxia.

107

The hypoxia-induced fall in the osmolality in the urine was significantly greater than in

the renal medullary interstitial compartment, which suggests that there was a failure in the

osmotic equilibrium in the distal CD. When rats were given dDAVP prior to hypoxia exposure,

water diuresis did not occur (Figure 3-4) and there was no difference between the papillary and

urine osmolalities (Figure 3-8). These findings indicate that the basis of hypoxia-induced water

diuresis is the impaired function of mTAL in the renal outer medulla and the failure of osmotic

equilibrium due to an absence of vasopressin. Hence it was of interest to determine the

underlying mechanism of hypoxia-induced water diuresis and identify defense mechanism that

may minimize the risk of hypoxic renal damage. The possible basis of hypoxia-induced water

diuresis is discussed in the following sections.

108

4.1.1.3. Effect of Hypoxia on Osmotic Equilibrium in the Collecting Duct

To test whether hypoxia leads to a loss of vasopressin, I compared the osmolalities

between the urine and the renal papilla. The renal papillary osmolality was significantly greater

than the urine osmolality (869 + 57 vs. 333 + 42 mOsm/kg H2O; Figure 3-5), suggesting that an

osmotic equilibrium failed to occur. There are two possible reasons for the difference between

urine and papillary osmolalities: an absence of vasopressin, or impaired actions of vasopressin

which is present.

Each milliosmole (mOsm) exerts an osmotic pressure of 19.3 mm Hg; a difference of

~500 mOsm/kg H2O suggests a driving force of almost 10,000 mm Hg. The osmotic equilibrium

failed to occur under the enormous driving force between the lumen of the collecting duct and

the medullary interstitial compartment. The pretreatment with dDAVP restored the osmotic

equilibrium during exposure to hypoxia. The response to dDAVP during hypoxia suggests that

water reabsorption failed to occur due to the absence of vasopressin, which did not allow the

insertion of the functional AQP2 water channels on the luminal membrane in the final nephron

segment. The absence of vasopressin during hypoxia suggests that hypoxia may influence its

release or metabolic clearance from the circulation. Studies have demonstrated that acute

exposure to hypoxia attenuates the renal effect of vasopressin (25), but does not directly affect

the release of vasopressin or the relationship between the plasma concentration of vasopressin

and plasma osmolality (162). Therefore, the absence of vasopressin effect observed in this study

may be the result of increased metabolic clearance of vasopressin by vasopressinases.

The large difference in the osmolalities between the medullary interstitial compartment

and the urine may also occur due to many reasons. Even in the absence of AQP2 in the last

109

functional nephron segment, the DCT and the CD, the reabsorption of water can occur in the

inner medulla since the inner medullary CD is somewhat permeable to water (36; 76), although

the magnitude of water reabsorption is small relative to the permeability in the earlier nephron

segment in the presence of AQP2. In addition, the large difference between the medullary

interstitial osmolality and the urine osmolality can be explained by the contraction of the renal

pelvis, which is an important feature that affects urine flow rate during periods of water diuresis.

Each time the renal pelvis contracts, a small volume of luminal fluid travels in a retrograde

direction up in the inner medullary CD, which allows more contact time for water reabsorption to

occur via diffusion (76). Overall, the enormous difference in the osmolalities may be due to

aforementioned processes, but the major cause of greater osmolality in the renal medullary

interstitial compartment compared to urine osmolality may be the lack of water permeability in

the last functional nephron segment due to absence of AQP2.

110

4.1.2. Effect of Hypoxia on Activity of Plasma Vasopressinase

Results from this study have shown that the basis of hypoxia-induced water diuresis is

not only due to a fall in the osmolality in the renal medullary interstitial compartment. There was

also a failure of osmotic equilibrium during hypoxia, which resulted from increased metabolic

clearance of vasopressin by vasopressinase, as suggested the enzymatic assay that showed an

increase in the activity of vasopressinase (Figure 3-8).

The hypoxia-induced increase of vasopressinase activity is a novel and interesting

finding, which has not been reported before. As previously mentioned, the hypoxia-induced large

water diuresis did not occur in every hypoxic rat in the hypoxia model used in this study. Results

also showed that hypoxia does not impair the release of endogenous vasopressin. These two

findings suggest that the lower degree of water diuresis in some rats may be due to a lower

magnitude of release/activity of vasopressinase during hypoxia, which may not have been

sufficiently strong to diminish most of the circulating vasopressin.

The metabolic clearance of vasopressin is found in many pathophysiological states, such

as the symptom of transient central diabetes insipidus during pregnancy and liver diseases, where

the activity of vasopressinase increases significantly. Literature findings on vasopressinase and

its possible physiological role in hypoxia are discussed in the following sections.

111

4.1.2.1. Vasopressinase: Findings in Literature

Vasopressinase is an α-aminopeptide amino acid hydrolase that degrades vasopressin and

oxytocin, by cleaving the amide bond between the N-terminal hemicysteine and adjacent

tyrosine, without prior reductive cleavage of the disulfide bridge (71; 71; 158). This cleavage

breaks the ring structure of vasopressin, which is further degraded by the release of tyrosine,

phenylalanine, glutamine and asparagine (71). Vasopressinase is a type II integral membrane

protein which is homologous to aminopeptidase A, aminopeptidase N, and other zinc-dependent

aminopeptidases in the gluzincins family (84; 144). Gluzincin aminopeptidases contain a zinc-

binding active site, (84) which makes their activity sensitive to metal ion chelators.

Vasopressinase is widely distributed in animal tissues and found in many subcellular organelles

(92; 146; 160). Vasopressinase has been identified in fat and muscle tissues as a major

component of glucose transporter isoform 4 (GLUT4) vesicles (144). Vasopressinase is

suggested to be responsible for the intracellular distribution of vesicles containing GLUT4

transporters and the retention of GLUT4, inhibiting its exocytosis to the plasma membrane (89;

181). In the presence of insulin, the vesicles containing both vasopressinase and GLUT4

translocate to the surface, increasing glucose uptake into cells for glycogen synthesis or

glycolysis (89; 144; 181). This response of vasopressinase to insulin gives the aminopeptidase its

other name, insulin-responsive aminopeptidase (IRAP) (181). For the relevance to this study, it is

important to note that insulin and hypoxia (low O2 supply) can induce a common set of target

genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), and also

GLUT4, enhancing glucose transport (8; 184; 189). Acute hypoxia is suggested to increase

sensitivity to insulin (108), suggesting that these two signals overlap in activating the target

112

genes essential for physiological compensatory response to hypoxia. Furthermore, the results

from this study have shown that hypoxia may increase the level or activity of vasopressinases,

which is also seen under the influence of insulin in normoxic conditions (89). These two signals,

insulin and hypoxia, manifest opposite effects in terms of glucose uptake into cells: insulin

increases glucose uptake for lipogenesis and adipogenesis, whereas hypoxia can suppress these

effects in a HIF-1α-dependent manner (184). Therefore, hypoxia may enhance vasopressinase

expression onto cell membranes and hence its activity and increase glucose transport via GLUT4

for glycolysis via increased vasopressinase trafficking, while inhibiting pathways for glucose

storage through HIF-1α pathway.

In the kidney, vasopressinase is expressed in distal and collecting tubules but not in

proximal tubules. The exact role of vasopressinase in the renal tubules is currently undefined but

studies have shown that the release of vasopressinase is closely related to the circulating

vasopressin (113; 125). These studies have demonstrated that vasopressin-mediated cAMP

pathway stimulates the trafficking of vasopressinase carrying vesicles to the plasma membrane

of renal medullary CD tubule cells. The close relationship between vasopressin and

vasopressinase in the renal tubular cells may serve as a regulatory role in maintaining the level of

vasopressin in the kidney. The results from this study showed that the level of plasma

vasopressin is significantly increased during hypoxia, which suggests that the increased level of

vasopressinase may be a compensatory response to prevent an excessive level of vasopressin in

the renal medulla.

Studies have shown that vasopressinase is significantly elevated in pregnant women (51).

In the case of pregnancy, the vasopressin secretion cannot compensate the increased metabolic

clearance of vasopressinase, leading to water diuresis (52), a state which was also observed in

113

rats under exposure to hypoxia in this study. The polyuric state in pregnancy can be treated by

dDAVP, which is not degradable by vasopressinase. The pretreatment with dDAVP also

prevented the water diuresis seen during exposure to hypoxia, which strongly suggests that the

underlying mechanism of hypoxia-induced water diuresis is increased metabolic clearance of

vasopressin by vasopressinase. The origin of hypoxia-induced release of vasopressinase is not

yet certain; it is likely that vasopressinase is released by the liver or the kidney, or by both

organs.

4.1.2.2. Hypoxia-Induced Release of Vasopressinase: Compensatory Mechanism?

Masuda et al (113) has demonstrated that vasopressinase is expressed in the renal distal

tubules and the CD and its release is regulated by V2R-mediated activation of cAMP pathway.

The stimulation of cAMP pathway translocates vasopressinase to plasma membrane of renal CD

cells, which may facilitate the degradation of excessive vasopressin, as a regulatory mechanism

(113). Under normal conditions, degradation of vasopressin regulates the vasopressin actions on

water reabsorption in the renal medullary CD, but this mechanism may be also significant under

pathophysiological conditions, such as periods of renal tissue hypoxia. Vasopressin is a potent

vasoconstricting agent, which limits the blood supply in the deep in the renal outer medulla (66;

67). Although limiting renal medullary blood supply prevents the washout of the interstitial

concentration gradient essential for concentration of urine, limiting blood flow may be adverse

during periods of hypoxia where there is already inadequate supply of O2 in the renal medulla.

There is evidence that the metabolite of vasopressinase (degraded byproduct of vasopressin) does

not have vasoconstrictor properties, and vasopressinase can cleave many vasoactive peptides,

114

such as oxytocin and angiotensin II (71). Therefore it is possible that vasopressinase may play a

role in increasing blood flow by degrading vasoactive agents. In the case of renal outer medulla

where vasopressin is most abundant vasoconstricting agent (140), the degradation of vasopressin

during hypoxia may be a compensatory mechanism to increase renal medullary blood supply.

There is evidence that the oxygenation levels improve in the renal medulla following a water

diuresis that was induced with water loading in humans (167). The improvement in the renal

medullary oxygenation level may be the result of a reduced level of vasopressin following the

water loading, which would eliminate the vasoconstrictory effect of vasopressin and increase

renal medullary blood flow. Microcirculation measurements have shown that during hypoxia,

the blood flow in the superficial renal cortex decreases by 20% while the total blood flow in the

kidney remains almost constant, suggesting that there may be a redistribution to the benefit of the

renal medulla (157). In fact, Sinagowitz et al (157) has demonstrated that the average PO2 falls

considerably less in the renal medulla than in the cortex under the same hypoxic conditions.

Other studies have shown that purified vasopressinase-mediated degradation of vasopressin may

limit the significant effect of vasopressin on the tonicity and blood flow in pregnancy. During

pregnancy, vasopressinase is released by the placenta (18), and its release continues until just

before the onset of labour. It has also been shown that pregnancy-related polyuria (eg. water

diuresis) is also associated with acute liver dysfunction (18). It was suggested that acute liver

dysfunction impairs degradation of vasopressinase, thereby extending its half-life activity and

increasing the metabolic clearance of vasopressin (18). Similarly, hypoxia-induced water diuresis

resulting from the loss of vasopressin activity may be due to increased release or increased half-

life of vasopressinase.

115

Under normal conditions, the level of vasopressin required for V1-receptor-mediated

vasoconstriction is much greater than the concentration required for V2-receptor-mediated

antidiuretic effect. During hypoxic conditions, the degradation of vasopressin may eliminate

vasoconstriction to the renal outer medulla, the area in the kidney most susceptible to hypoxic

injury. However, as shown by this study, the hypoxia-induced increase of vasopressinase activity

may be too great for the body to compensate with the release of endogenous vasopressin. The

metabolic clearance of vasopressin may diminish its antidiuretic effect on V2 receptors, which

leads to failure of osmotic equilibrium in the final nephron segment and subsequent water

diuresis. Therefore there is a price to pay for increasing the renal medullary blood supply during

periods of hypoxia. Duration of hypoxia-induced water diuresis is only transient, as the

preliminary results from this study have shown that water diuresis stops when the hypoxic

exposure is extended up to 5 hrs in rats. Since the water diuresis is large and transient, it may

provide the trigger for the release of endogenous vasopressin, which increases in response to the

rise in plasma Na+. The release of endogenous vasopressin is also triggered by the significant

change in the blood volume, and changes greater than 10% of blood volume may stimulate

vasopressin release (173). The results from this study indicated that the magnitude of water

diuresis was very large to the extent that could decrease the blood volume in rats, which suggests

that there is a strong trigger to release endogenous vasopressin. Therefore the metabolic

clearance of vasopressin may improve blood flow during acute hypoxia exposure; it may be

dangerous in the long-term, where endogenous vasopressin may be secreted, with significant

vasoconstrictory effects in the renal outer medulla. It is important that renal medullary blood

flow is increased while the antidiuretic effect of vasopressin is maintained. Results from this

study have shown that the pretreatment with dDAVP prevented hypoxia-induced water diuresis.

116

The effect of dDAVP during hypoxia is significant; dDAVP is a V2 receptor agonist that does

not have the V1 mediated vasoconstrictory effect of vasopressin. Furthermore, dDAVP is not

degradable by vasopressinases, and subsequently retains antidiuretic effect while vasopressin

itself is inactive under rapid action of vasopressinases. The underlying mechanism of action and

significance of dDAVP is explained more in detail in the section 4.1.3.

117

4.1.3. Hypoxia Markers and Effect of dDAVP Pretreatment

This study has demonstrated that dDAVP prevented the rise in the urine flow rate and the

fall in the urine osmolality in rats exposed to hypoxia. The renal papillary osmolality remained

high, which suggested that active reabsorption still occurred under hypoxia. The signs of tissue

hypoxia, such as blood lactate and plasma EPO, stayed at low levels in dDAVP-pretreated rats

throughout hypoxia exposure. The results from this study also showed that the pretreatment with

dDAVP prevented a significant rise in renal outer medullary expression of HIF-1α during

exposure to 2.5 hrs of hypoxia. The treatment with dDAVP alone significantly increased the

expression of eNOS in the renal outer medulla in rats. In addition, there is evidence that dDAVP

up-regulates the local expression of eNOS in the skeletal muscle vasculature via activation of V2

receptors (90), leading to vasodilatory effects. Therefore, results from this study suggest that

dDAVP may improve O2 delivery through a NO-dependent mechanism, and thus may prevent

hypoxic injury in the areas susceptible to hypoxia, such as renal outer medulla.

4.1.3.1. Level of Blood Lactate and Plasma Erythropoietin

This study demonstrated that exposure to hypoxia in rats resulted in significantly elevated

levels of plasma erythropoietin. There was also a small but significant rise in blood lactate level

during hypoxia, but the increase was relatively small (~1 mmol/L), suggesting that the degree of

hypoxia (8% O2 for 2.5 hrs) used in this study was not severe enough to induce a very low

supply of O2, which may limit the regeneration of ATP by mitochondria. Plasma erythropoietin

118

and blood lactate are markers of local and a low PO2 in mitochondria in some organs in the body,

respectively.

Erythropoietin (EPO) is a glycoprotein that is primarily produced by the fibroblast-like

interstitial cells in the renal cortex, and although it is also produced in the liver, EPO is mainly

the marker of renal cortical tissue hypoxia (9; 97; 99; 102; 116). EPO is responsible for the

production of red blood cells, and also replace those cells continuously removed from the

circulation by the spleen, liver and bone marrow (73). Aside from erythropoiesis, EPO also acts

on non-hematopoietic cells as a proliferation factor, and also a tissue-protective factor that

protects against ischemic injuries in neuronal, cardiovascular and renal tissues (40; 70; 131).

Under normal conditions, the concentration of EPO in the plasma ranges from 30 pg/mL to 80

pg/mL, but can increase up by 1000-fold in hypoxic conditions (73). The synthesis of EPO is

dependent on the O2-sensitive transcription factor HIF, and under hypoxic conditions, it takes up

to 2 hours for the concentration of EPO to increase substantially and distribute uniformly in the

circulation (73). In this study, EPO concentration rose significantly after 2.5 hrs of exposure to

hypoxia, and previous findings in our lab have demonstrated that the concentration of EPO

continues to rise when hypoxia exposure is extended up to 5 hrs, and this increase is sustained

until 6 hrs post-hypoxia. This study also showed that when rats were pretreated with dDAVP, the

concentration of EPO did not show any increase throughout the exposure to hypoxia. However,

under normoxia the EPO concentration was significantly higher (2-fold) in the dDAVP-

pretreated group relative to the group that did not get treated with dDAVP. Although it has been

shown that dDAVP selective activation of V2 receptors does not affect synthesis of EPO (59), the

results from this study suggests that dDAVP may indirectly influence its synthesis or half-

life/stability, as suggested by the plasma concentration of EPO under normoxia. The underlying

119

mechanism that may explain how dDAVP treatment resulted in the significantly higher EPO

concentration during normoxia is yet unknown. Overall, this study showed that dDAVP

prevented a significant rise in the plasma concentration of EPO, and this suggests that dDAVP

may have improved the renal cortical O2 balance. In addition, the concentration of blood lactate

stayed lower in dDAVP-pretreated group relative to rats that did not receive dDAVP, and this

therefore indicates that dDAVP may also improve O2 delivery at the global systemic level as

well. The possible mechanisms of improved O2 delivery with dDAVP are explained in Section

4.1.3.2.

4.1.3.2. Level of Renal Medullary Protein Expression

In this study, the renal medullary expressions of two proteins that may be associated with

tissue hypoxia were measured. There is experimental evidence that HIF-1α is activated even

under normoxic conditions (35), especially in areas with relatively lower O2 tensions, such as the

renal outer medulla (57). Other studies have demonstrated that under normoxic conditions, HIF-

1α is exclusively expressed in the deeper areas in the renal medulla. The selective expression of

HIF-1α in the renal medulla under normoxic conditions suggests that a low PO2 may be

specifically maintained in this region.

There was a significant rise in the renal outer medullary expression of HIF-1α

immediately after exposure to 2.5 hrs of hypoxia, which was prevented when rats were treated

with dDAVP 1 hr prior to exposure to hypoxia. The lower level of HIF-1α in dDAVP-pretreated

rats during hypoxia suggests that dDAVP may have improved renal medullary O2 delivery. We

also showed that dDAVP pretreatment also prevented the hypoxia-induced fall in the renal

120

medullary osmolality, which indicates that dDAVP maintained adequate supply of O2 for active

reabsorption of mTAL in the renal outer medulla. The precise underlying mechanism by which

dDAVP improves renal medullary O2 delivery is yet undefined, but the possible mechanism may

be an improvement of renal medullary blood flow which may be regulated by NO-mediated

vasodilation.

There were non-significant increases in the renal outer medullary expression of eNOS

after exposure to 2.5 hrs of hypoxia. Nitric oxide synthase is involved in the catalysis of NO

production. Among many factors that can influence renal medullary blood flow, the NO

production plays the major role in regulating renal medullary blood flow and protects this region

from ischemic injury (48). There is evidence that exposure to hypoxia (9% O2) significantly

upregulates eNOS in the renal tubules in rats (69). In this study, exposure to 2.5 hrs of hypoxia

did not significantly increase the renal medullary expression of eNOS, which suggests that the

duration of hypoxia exposure may not have been sufficient to significantly upregulate these

proteins.

There was a significant increase in the renal medullary expression of eNOS with dDAVP

treatment alone in normoxic rats. Endothelial NOS is the NOS isoform which is responsible for

vasodilation by generating NO in blood vessels. The increase in eNOS expression with treatment

with dDAVP suggests that a mechanism by which dDAVP enhances O2 delivery may be eNOS-

mediated vasodilation. Cowley et al (46) suggested that V2 receptors may be involved in the NO-

mediated regulation of blood flow to the renal medulla. This study demonstrated that significant

reduction of renal medullary blood flow induced by continuous infusion of vasopressin was

diminished with progressive upregulation of V2 receptors, whereas in the absence of V2 receptor

stimulation, chronic infusion of a specific V1 receptor agonist in rats led to a sustained reduction

121

of renal medullary blood flow (49). These findings indicate that dDAVP action on V2 receptors

may enhance renal medullary blood flow, and upregulation of eNOS may be involved in this

process. Improved renal medullary blood flow with dDAVP may have maintained adequate O2

supply to the renal outer medulla, allowing active reabsorption of mTAL and generation of high

renal medullary osmolality. Further details on the possible underlying mechanism by which

dDAVP may increase renal medullary blood is discussed in Section 4.1.3.4.

122

4.1.3.3. Desmopressin Acetate (dDAVP): Synthetic Analogue of Vasopressin

The present study showed that dDAVP was able to prevent water diuresis in rats exposed

to hypoxia, by substituting the action of vasopressin under the hypoxia-induced release of

vasopressinase. Therefore, many actions of dDAVP in these settings suggest its role in various

clinical settings.

The specificity of dDAVP to V2-receptor may be of significance in the clinical settings

such as renal tissue hypoxia. Hypoxia-induced water diuresis is the result of degradation of

circulating vasopressin, which obliterates its antidiuretic effect via V2 receptors. Previously, it

was mentioned that the loss of vasopressin effect may be a compensatory mechanism to prevent

its vasconstrictory effect and increase blood flow to the areas susceptible to hypoxic injury, such

as the renal outer medulla. Since dDAVP only acts on V2 receptors, it still retains the antidiuretic

effect of vasopressin under the action of vasopressinase. Furthermore, dDAVP has been

repeatedly associated with V2-mediated vasodilatory effect in regulating the renal medullary

blood flow via nitric oxide (NO) production (48; 140). Renal medullary NO was also shown to

buffer the prolonged vasoconstrictory effect of vasopressin in rats. The underlying mechanism

by which dDAVP may increase the renal medullary blood flow and maintain the O2 balance in

the renal outer medulla during hypoxia is discussed in the following section.

Another possibility that dDAVP prevented the fall in the renal medullary osmolality

during hypoxia may be by preventing the washout of the concentration gradient in the medullary

interstitial compartment. As previously mentioned, during a typical water diuresis, there is a high

volume of filtrate reaching the distal part of the end of nephron segment. The residual water

permeability in the inner medullary collecting duct may help to minimize the loss of Na+ in the

123

urine (36). In this study, the urine osmolality was very low and paralleled the value of plasma

(~300mOsm/kg H2O) during exposure to hypoxia. The urine flow rate was also high, suggesting

that there was very large volume of filtrate reaching the last nephron segment. The presence of

residual water permeability in the inner medullary collecting duct may have resulted in the

reabsorption of large volume of water into the inner medullary interstitial compartment, causing

washout of the concentration gradient. The treatment with dDAVP prevented hypoxia-induced

water diuresis, which reduced the volume of filtrate entering the last nephron segment and

thereby prevented the washout of the concentration gradient. The prevention of washout of this

concentration gradient may have contributed in maintaining the high osmolality in the inner

medullary interstitial compartment relative to the low renal medullary osmolality in untreated

group during exposure to hypoxia.

In addition, the renal medullary composition of urea in dDAVP-pretreated rats was

relatively high during hypoxia (Figure 3-9). The insertion of AQP2 channels on the last

functional nephron unit (DCT and the CD) reduces the volume of filtrate entering the inner

medullary CD. Due to the reabsorption of water that occurred in the previous nephron segments,

the volume of the filtrate is small and therefore the concentration of urea in the filtrate is

relatively high. DDAVP also inserts urea transporters in the inner medulla, which results in

reabsorption of urea into the inner medullary interstitial compartment, generating a high inner

medullary urea composition. The high concentration of urea in the inner medullary interstitial

compartment is an important feature in the integrative physiology of the renal medulla. As

previously mentioned in Section 1.5.2., the generation of high urea composition in the inner

medulla reduces the relative concentration of Na+ and Cl- in more superficial outer medulla. The

decrease in the concentrations of Na+ and Cl- drives the passive reabsorption of these electrolytes

124

from the mTAL lumen, which subsequently decreases the need of active reabsorption by the

mTAL.

In summary, the maintenance of high renal medullary interstitial osmolality during

hypoxia in dDAVP-pretreated rats may have resulted from many processes. First, the active

reabsorption by the mTAL may have been maintained due to improved perfusion via V2

receptor-mediated vasodilation, and the reabsorption of urea in the inner medulla generated high

inner medullary urea concentration which may have reduced the need of active reabsorption of

Na+ and Cl- by the mTAL. In addition, the reabsorption of water due to insertion of AQP2 on the

final functional nephron segment led to osmotic equilibrium between the lumen of the inner

medullary CD and the medullary interstitial compartment, which resulted in the urine osmolality

that paralleled the renal medullary osmolality.

125

4.1.3.4. Increasing O2 Delivery by dDAVP: Potential Mechanism

Many studies have demonstrated that dDAVP has vasodilator properties, as shown by an

increase in heart rate, and decrease in systolic and diastolic blood pressure by decreasing the

activity of ionized Ca2+ in the interstitial compartment and thereby increase the active

reabsorption of Na+ in the mTAL (90). The vasodilator action of dDAVP may be mediated by V2

receptors, since these signs of vasodilation are not observed in the case of nephrogenic diabetes

insipidus (NDI), where AQP2 is dysfunctional and therefore cannot respond to circulating

vasopressin (26; 90). Park et al (140) showed that infusing medullary interstitium locally with

dDAVP enhanced the medullary blood flow, which could not be repeated when selective V1R

agonist was used. The mechanism of dDAVP-mediated vasodilation was therefore suggested to

be due to the activation of V2 receptor pathway, which leads to an increased production of nitric

oxide (NO) in the medullary interstitium. In a dog experimental model, vasopressin increased

renal blood flow when treated with V1 receptor antagonist, suggesting that vasopressin leads to

V2 receptor-mediated vasodilation (123). The V2 receptor-mediated NO production by

vasopressin may depend on the mobilization of intracellular Ca2+ in rat inner medullary

collecting ducts (130). Vasopressin binds to V2 receptor, which activates adenyl cyclase (AC)

and phospholipase C (PLC), where the latter hydrolyzes inositol triphosphate (IP3) and

stimulates the release of Ca2+ from IP3-sensitive sarcoplasmic reticulum stores (48). The increase

in intracellular concentration of Ca2+ activates NO production via calmodulin pathway (152).

The specificity of dDAVP for V2 receptors suggests that dDAVP may selectively activate NO-

mediated vasodilation via increase in Ca2+ from intracellular stores, while not affecting the V1

receptor pathway. More importantly, this study has demonstrated that the selective action of

126

dDAVP is especially significant during hypoxia, where dDAVP may retain its function on V2

receptors and subsequently activate NO-mediated vasodilation, possibly providing adequate

blood supply to the renal medulla while vasopressin is degraded by the increased activity of

vasopressinases, diminishing the vasoconstrictory effect of vasopressin and further increasing the

blood flow to the renal medulla.

127

4.1.4. Clinical Significance of the Hypoxia Study

It is critical to be able to detect an early disruption of renal function for a quick

intervention to prevent the development of AKI. The detection of AKI must be also standardized

to avoid discrepancies between the measurements. Currently, AKI is still a serious clinical

problem with high mortality and morbidity, largely due to the lack of effective preventive

strategies and intervention. Many diagnostic markers of AKI have been proposed in recent

studies, and among these biomarkers, NGAL is suggested as the early and reliable indicator of

perioperative AKI. There is evidence that urinary NGAL levels begin to increase within 1 hr and

remain significantly high 3, 18, and 24 hrs after cardiac surgery requiring cardiopulmonary

bypass in patients who develop perioperative AKI (78; 175).

The findings from the present study are clinically significant for many reasons. The large,

hypoxia-induced water diuresis began at 1 hr of exposure to hypoxia, which was the earliest

physiologic sign that indicated the renal medullary response to inadequate O2 supply. As

expected, the renal medullary response to hypoxia occurred under a modest degree of hypoxia in

which there was no significant change in the function of renal cortex. The change in renal

medullary function indicates that the sign of reduced function in the renal medulla is a reliable

tool that is sensitive to detect small changes in the renal O2 balance. Water diuresis is easy to

detect, as its diagnosis only requires the analysis of urine flow rate and urine osmolality. In this

study, urine osmolality was calculated from the ‘effective’ electrolytes and urea. The method of

measuring urine osmolality is quick (a few minutes), and relatively simpler compared to the

method used to measure urinary proteins, which may be delayed in time to show a significant

change after a hypoxic insult to the kidney. Therefore, measurement of urine flow rate combined

128

with urine osmolality may be clinically significant in detecting an early stage of renal hypoxic

injury that may develop into AKI.

This study also demonstrated the potential use of dDAVP in enhancing the renal

medullary perfusion during periods of renal tissue hypoxia, which indicates clinical significance

of dDAVP in protecting the renal outer medulla. In addition with detection of water diuresis

which may be used as a quick diagnosis of renal hypoxia, dDAVP may serve a prophylactic role

in preventing the development of hypoxic injury in the kidney.

NOTES:

The proposed underlying mechanism of dDAVP was improved blood flow, which may

increase the medullary O2 supply and maintain O2 balance in the renal outer medulla during

hypoxia. The potential role of dDAVP in maintaining the renal medullary O2 balance by

increasing O2 supply suggests that renal outer medulla can also maintain this balance by

decreasing O2 consumption (Figure 4-1). As previously mentioned, a possible agent to maintain

the renal medullary O2 balance by reducing the O2 consumption is furosemide, the loop diuretic

that reduces the active reabsorption of mTAL in the renal outer medulla. The results from this

study are discussed in the following sections.

129

Figure 4-1. Summary of Major Findings in this Study. The O2 balance in the renal outer medulla (bottom) depends on O2 supply, which consists of O2 content in the blood and renal medullary perfusion, and O2 consumption, which is almost entirely due to the active reabsorption of Na+ by the mTAL. Due to low perfusion and high demand of O2 by the mTAL, the renal outer medulla is vulnerable to injury during renal hypoxia. Hypoxia may release the vasopressinase as a compensatory mechanism to diminish the vasoconstrictive effect via V1 receptor and increase blood flow, but may degrade vasopressin to the extent that takes away all of its functions, leading to water diuresis, which was the earliest physiologic indicator of renal hypoxia that was detected with this experimental model. The treatment with dDAVP prevents this effect of hypoxia since it is resistant to the activity of vasopressinase, and may increase O2 delivery via improved perfusion. Non-natriuretic dose of furosemide may decrease O2 consumption by reducing the active reabsorption in the mTAL, and these two mechanisms may be significant in maintaining the O2 balance in the renal outer medulla and prevent hypoxic injury in this setting.

130

4.2. Summary of Results: FUROSEMIDE

The first part of the study aimed to determine the earliest renal response to hypoxia,

which was, as previously expected, a significant fall in the mTAL function in the renal outer

medulla by inhibiting active reabsorption (91). It was shown that dDAVP prevents the fall in the

function of mTAL during hypoxia, possibly by improving O2 delivery via adequate blood supply

to meet the metabolic demand of the mTAL in the renal outer medulla. It was of interest whether

the O2 balance in the renal outer medulla could also be maintained by decreasing the work of

mTAL. Since furosemide targets the cotransporter (NKCC2) primarily responsible for the active

reabsorption of mTAL, the second part of this study aimed at determining the dose that would

significantly reduce the active work in the mTAL. Prolonged use of large doses of furosemide is

associated with increased risks of hypomagnesemia, which results from increased excretion of

Mg2+ (182). Therefore the purpose of this part of the study was to document the effect of

different doses of furosemide on the function of the mTAL, and to determine a dose of

furosemide that significantly reduces the metabolic demand of mTAL and therefore minimizes

the need of O2 consumption in the renal outer medulla without causing a depletion of essential

electrolytes and fluids in rats.

In summary, the second part of my study demonstrated that doses of furosemide that

substantially reduce the active reabsorption of mTAL in the renal outer medulla and significantly

increase the where rates of excretion of electrolytes is 0.8 mg/kg in rats. The concentration of

plasma Mg2+ decreased with higher doses of furosemide, suggesting that very high doses of

furosemide may increase the risk of hypomagnesemia. The results from the furosemide study are

discussed more in detail in the following sections.

131

4.2.1. Dose of Furosmide that ↓ the Function of mTAL: 0.8 mg/kg of Body Weight in Rats

The result showed that urine flow rate increased significantly at 1.6 mg/kg of body

weight of furosemide (10 + 1 vs. 57 + 16 µL/min; Figure 3-16). The urine osmolality showed a

significant fall beginning at 0.8 mg/kg (2374 + 114 vs. 1164 + 101 mOsm/kg H2O; Figure 3-17),

suggesting that the fall in the urine osmolality occurs at a lower dose relative to urine flow rate.

The finding that urine osmolality decreases before a rise in urine flow is significant, since the

effectiveness of furosemide in the clinical settings is generally measured by the rise in the urine

flow rate. In all dose groups, the renal papillary osmolality paralleled urine osmolality,

suggesting that the fall in the osmolalities was primarily due to a reduced active reabsorption in

the mTAL, rather than distal factors, such as impaired water reabsorption. The excretion of

electrolytes such as Na+ (14 + 2 vs. 38 + 10; Figure 3-18), Cl- (20 + 2 vs. 67 + 13) and K+ (20 +

2 vs. 45 + 6) increased significantly starting at 0.8 mg/kg and continued to increase with higher

doses of furosemide. The rate of Mg2+ excretion showed a similar trend, with significant rise

beginning at 0.8 mg/kg of furosemide. The significant increase in Mg2+ excretion suggests that

inhibition of NKCC2 may substantially affect the reabsorption of Mg2+ via paracellular pathway.

The magnitude of increase in Mg2+ excretion was also reflected by the significant fall in the

plasma Mg2+ concentration beginning at doses higher than 0.8 mg/kg of furosemide, which

suggests that high doses of furosemide may increase the risk of hypomagnesemia.

However, the doses of furosemide required for the diuretic effect observed in rats may be

different from humans. The dose of furosemide in rats, which significantly reduces the function

of mTAL (0.8 mg/kg of body weight), could be considered as very high if applied intravenously

in humans (~60mg/day for 70kg human). The doses of furosemide in clinical settings range from

132

10 to 20 mg per day, thus 0.8 mg/kg would be more than double the doses used in clinical

settings. Regular rat diet contains higher concentrations of NaCl, and hence rats generally have a

higher intake of Na+ and Cl- compared to humans. Higher intake of these electrolytes increases

the content of Na+ and Cl- in the filtrate entering the lumen of mTAL. As previously mentioned,

furosemide competes for the Cl- binding region on NKCC2. Thus higher doses of furosemide

may be required in rats to compete against high luminal content of Cl- for the binding site on

NKCC2, since there is higher luminal content of Cl- entering the DtL in rats. Therefore, it is

important to determine the doses for humans that mimic the effect of 0.8 mg/kg of furosemide in

rats, which significantly decreases the work of mTAL without causing large excretions of

electrolytes.

133

4.2.2. Danger of High Doses of Furosemide

Furosemide is used to treat hypervolemia, edema and heart failure, to reduce body fluids

in a short period of time. Clesnick et al. demonstrated that the effect of furosemide does not

begin until 10 minutes after the furosemide administration (34; 50), and there is a 20-min to 60-

min delay between the time of administration of furosemide and the peak diuretic effect (50).

Other studies have shown that the extrarenal effects of furosemide occur prior to the onset of

diuretic effects of the drug, as evidenced by decreases in volume of extravascular water in

patients with pulmonary edema (143). The findings in these studies suggest that furosemide

affects blood volume by redistribution before the onset of its diuretic effects that significantly

change the distribution of water in the body. There is evidence that the diuretic action of

furosemide is significantly decreased in patients suffering from AKI (91), and hence in the

setting of AKI, a significantly high dose of furosemide is required for a significant increase in

urine flow rate. By contrast, the effect of furosemide on excretion of Na+ and K+ is enhanced in

AKI patients, due to seriously compromised concentrating ability in the latter part of the nephron.

Based on the findings from this and other studies (172), high doses of furosemide required to

obtain a large diuretic effects may potentially be dangerous, since the large osmotic diuresis

induced by high doses of furosemide may result in significant loss of fluids and electrolytes.

134

4.2.2.1. Depletion of Essential Electrolytes & Fluids

The results from this study demonstrated that high doses of furosemide (> 0.8 mg/kg of

body weight) significantly increase excretion of electrolytes such as Na+, Cl-, K+ and Mg2+.

Although the increase in the rate of excretion of these electrolytes is expected with furosemide,

the study showed that this effect is accompanied by the concurrent decrease in the serum

electrolytes, such as plasma concentration of Mg2+. This therefore suggests that high doses of

furosemide may be harmful if the excretions of electrolytes stay at very high rates as observed in

this study. In addition, the increases in excretions of these electrolytes subsequently lead to a

large diuresis, which may be dangerous in patients at risk of hypovolemia.

There is evidence that low doses of furosemide may attenuate or reduce the severity of

early onset of AKI (14). These low doses (ranging from 0.03 mg/kg/hr to 0.4 mg/kg), which are

insufficient for a diuretic effect, significantly reduced the ischemia/reperfusion induced apoptosis

and prevented the suppression of angiogenesis-related genes (3). Renal blood flow was also

shown to be significantly increased with low doses of furosemide, which may serve a preventive

role in the development of hypoxia-associated AKI (4). By contrast, studies demonstrated that

higher doses of furosemide (0.5 mg/kg/hr), which result in significant increases in the urine flow

rate and the rate of Na+ excretion (>2-fold increase, similarly observed in this study; Figure 3-19),

failed to prevent the onset of renal failure (170). Significant reductions in extracellular and

intracellular volumes are also associated with high doses of furosemide (2; 124).

The potential for low doses of furosemide to attenuate the progression of early onset of

AKI indicates that there is a possible underlying mechanism by which furosemide may enhance

or maintain renal function in the setting of AKI. The action of furosemide is to inhibit active

135

reabsorption of Na+ in the mTAL, therefore it is possible that non-natriuretic doses of furosemide

may reduce metabolic demand and create an imbalance for O2 in the kidney. In addition, higher

doses of furosemide (> 0.8mg/kg) increase excretion of electrolytes (Figure 3-19; 3-21) and

water to the extent of depletion, and therefore may increase the risks of electrolyte imbalance and

hypovolemia.

4.2.2.2. Depletion of Mg2+: Risk of Hypomagnesemia

This study showed that the rate of excretion of Mg2+ increased significantly with

increasing doses of furosemide (Figure 3-21). The increase in the urinary Mg2+ excretion was

accompanied by a significant decrease in the plasma Mg2+ to less than 1 mmol/L. The normal

range of Mg2+ is between 0.74 mmol/L to 0.94 mmol/L in humans, and thus suggests that rats

may have a different mechanism of Mg2+ reabsorption that results in higher plasma Mg2+ levels

(7). The major site of Mg2+ reabsorption in the renal medulla is mTAL, where filtered Mg2+ is

reabsorbed via paracellular route. Inhibition of NKCC2 in the mTAL prevents the reabsorption

and subsequently leads to a high concentration of Na+, Cl-, and K+ in the lumen of mTAL. High

luminal positive voltage hinders the influx of K+ into the mTAL lumen by ROMK, which

provides the driving force for paracellular reabsorption of Ca2+ and Mg2+. Therefore, large

excretion of Mg2+ resulting from high doses of furosemide may seriously deplete Mg2+ stores in

the body. In fact, loop diuretics are the most common causes of hypomagnesemia (182), and the

long-term use of furosemide is often associated with this condition. In addition, in the setting of

congestive heart failure, furosemide may enhance Mg2+ loss and produce Mg2+ deficiency.

136

Although the adverse side-effects of furosemide also include hypokalemia and

hypocalcemia, the risks of these states are relatively lower since there is reabsorption of K+ and

Ca2+ more distally in the nephron that could compensate the reduced reabsorption in the mTAL.

Unlike the reabsorption of K+ or Ca2+, the reabsorption of Mg2+ in the distal convoluted tubules

only makes up 5% of the filtered Mg2+ (7). Hypomagnesemia and the depletion of intracellular

Mg2+ stores resulting from very high doses of furosemide (>80 mg/day) are highly associated

with variety of cardiovascular abnormalities, such as atrial fibrillations, impairment of cardiac

contractility and vasoconstriction (43). These findings suggest that the use of furosemide should

be limited to lower doses that do not result in a very high excretion of Mg2+.

137

4.2.3. Clinical Significance of Furosemide: ↓ the work of mTAL

The potential role of furosemide in decreasing the O2 consumption in the renal outer

medulla has been discussed in other literatures. Loop diuretics such as furosemide has been

shown to alleviate renal medullary hypoxia (31; 32; 165), and it also has been demonstrated that

furosemide selectively increases PO2 in the renal medulla within 20 minutes of administration,

but not in the cortex (31; 100). These findings strongly suggest that furosemide may be helpful in

reducing the metabolic demand in the renal outer medulla and prevent hypoxic injury in patients

at risk of AKI. As previously mentioned, AKI patients have reduced diuretic effects of

furosemide, and therefore require high doses of the drug for a significant rise in the urine flow

rate. However, the findings from this study suggest that, in rats, doses higher than 0.8 mg/kg

significantly increases the excretion of electrolytes and the rise in the urine flow rate occurs after

urine osmolality has significantly decreased. Urine flow rate was accompanied by the significant

increase in the rate of excretion of electrolytes, suggesting that higher doses of furosemide that

result in significant increase in the urine flow rate may compromise the body of essential

electrolytes, such as Na+, Cl-, K+ and Mg2+. Urine flow rate should not be the only sign to detect

effect of furosemide, since the rise in urine flow rate occurs after the work of mTAL has

significantly decreased. The function of the mTAL in the renal outer medulla should be defined

by the changes in the urine osmolality, which reflects the active reabsorption in the mTAL.

138

NOTES:

In summary, this study demonstrated that in rats, 0.8 mg/kg of furosemide is a dose

where there is a significant fall in the renal medullary osmolality (which reflect the active

reabsorption of mTAL), while the excretion of electrolytes begin to increase significantly. Very

high doses of furosemide (>1.6 mg/kg of body weight in rats) may lead to depletion of fluids and

electrolytes, which may cause hypovolemia or hypomagnesemia. Therefore, the purpose of the

use of furosemide should be to decrease the work of mTAL in the renal outer medulla and

therefore the need of O2 consumption in the periods of renal medullary hypoxia. Reducing the

metabolic demand in the renal outer medulla may be significant in maintaining the O2 balance in

the renal outer medulla and prevent the risk of mTAL dysfunction that may lead to AKI (Figure

4-1).

139

CHAPTER 5

LIMITATIONS OF THE STUDY & FUTURE DIRECTIONS

140

5.1. Limitations of the Study

The underlying mechanisms of causes leading to perioperative AKI involve many factors,

such as hypovolemia, sepsis, and ischemic injury. This study aimed to study the effects of

hypoxia in isolation from other confounding variables such as anesthesia and surgery. Thus one

limitation of this study is that only hypoxia was singled out from many other factors involved in

the development of AKI to simulate the model of AKI in rats, and therefore may not accurately

reflect the real settings of AKI.

Another limitation of this study is the dose of dDAVP that was administered to rats. The

dose of dDAVP (4µg) used in this study is a very large dose for rats and therefore may not

accurately depict efficacy in the clinical settings. In addition, the study showed that dDAVP may

prevent the hypoxia-induced renal medullary dysfunction by increasing the O2 supply. The exact

underlying mechanism is not yet certain whether the increase in O2 supply was the result of

improved medullary perfusion. The signs of increased O2 supply were plasma EPO and lactate,

and hypoxia markers in the renal medullary protein, which were an indirect measurement of

renal medullary O2 supply during hypoxia. Future experiments should directly measure the renal

medullary blood flow following administration of dDAVP with techniques such as Laser-

Doppler flowmetry, to determine whether dDAVP enhances the renal medullary perfusion.

The degree of hypoxia used in this study was 8% O2 and results showed that this degree

of hypoxia did not cause severe renal injury in rats, as demonstrated by a significant change that

was limited to renal medullary function. Although acute exposure to 8% O2 did not disrupt renal

functions beyond that of the renal outer medulla, this degree of hypoxia may be considered

relatively severe in terms of clinical settings such as AKI. Further experiments should be carried

141

out with exposure to different levels of O2 in rats, to examine the time-course effect of different

degrees of hypoxia on renal medullary dysfunction.

Lastly, the goal of this study was to document the early markers and examine underlying

mechanisms of development of renal tissue hypoxia that may lead to AKI and prevent permanent

renal damage. However, the study did not examine histology following exposure to hypoxia, or

long-term effects of acute exposure to hypoxia and therefore does not confirm the lack of

permanent injury with the hypoxia model.

The second part of this study showed that 0.8 mg/kg of furosemide is the dose where the

mTAL function significantly falls and therefore reduces the metabolic demand in the renal outer

medulla. This dose of furosemide may be the optimum dose in rats that reduces the O2

consumption in the renal outer medulla while preventing large excretions of essential electrolytes.

The dose of the furosemide used in rats in this study may not accurately reflect the doses used in

clinical settings. In fact, rats may require higher doses of the drug for the same diuretic effect

required for humans. It is important to determine the dose of furosemide in humans that mimics

the diuretic effect of 0.8 mg/kg in rats.

142

5.2. Future Directions

This study evaluated the potential use of dDAVP and furosemide in improving the O2

balance in the renal outer medulla, the area in the kidney most susceptible to hypoxic injury. As

previously mentioned, the study examined the effect of only one dose (4µg) of dDAVP in

preventing the hypoxia-induced renal medullary dysfunction. Therefore, it is of interest whether

there is an optimum dose of dDAVP to prevent effects of hypoxia on function of the renal outer

medulla, without significantly affecting fluid balances in the body. Future experiments should

also examine the timing of administration of dDAVP, and also assess the efficacy of dDAVP in

other clinical settings such as surgery requiring cardiopulmonary bypass. In addition, direct

measurements should be made to assess the effect of dDAVP on renal medullary perfusion and

O2 delivery. Possible techniques that may examine whether dDAVP improves renal medullary

perfusion include BOLD imaging and measuring urinary PO2. The BOLD imaging can measure

the changes in blood flow and therefore can be used to assess the effect of dDAVP on the renal

medullary blood flow. The level of PO2 in the urine reflects the PO2 in the renal medullary

interstitium, since diffusion of O2 between renal medullary interstitial compartment and the

medullary CD allows equilibrium of PO2. Hence, measuring PO2 in the urine will be useful in

examining the effect of dDAVP in improving O2 delivery to the renal medulla.

The activity of plasma vasopressinase increased with exposure to hypoxia. It is currently

of interest to determine the site of increased vasopressinase activity. The possible candidate for

releasing plasma vasopressinase is the liver, since dysfunction of the liver is repeatedly

associated with release of vasopressin-degrading aminopeptides. However, the liver may not be a

good candidate for being an O2 detector because of its portal blood supply with relatively low

143

PO2 under normal conditions, unless it can create a signal to dilate hepatic arteries when hepatic

sinusoidal PO2 is dangerously low. Hence, another potential candidate for the source for

vasopressinase during hypoxia is the kidney, where vasopressinase is present at high levels in the

renal medullary tubular cells, regulating the local level of vasopressin. Therefore, the kidney may

be a potential source of vasopressinase that degrades vasopressin, leading to a large water

diuresis that was observed in rats exposed to hypoxia. Future studies should examine the

significant increase in the activity of plasma vasopressinase in nephrectomized rats, to exclude

the kidney as the possible source of hypoxia-induced release of vasopressinase.

This study has demonstrated that the optimum dose of furosemide that may reduce the

function of mTAL in the renal outer medulla is 0.8 mg/kg in rats, and this dose of furosemide is

also the dose in which the rates of excretion of electrolytes begin to increase significantly. It is

not yet certain whether low dose of furosemide can significantly reduce renal medullary O2

consumption. Therefore, it is of current interest to detect any changes in hypoxia markers such as

HIF-1α in the renal medulla following a low-dose (0.8 mg/kg) furosemide administration in rats.

Future experiments may also assess the efficacy of this dose of furosemide in combination with

dDAVP in preventing the renal medullary hypoxic injury in rats exposed to hypoxia, to examine

whether the combination of dDAVP (increasing O2 supply) and furosemide (decreasing O2

consumption) will be a superior therapy in preventing hypoxic injury in this model.

Future experiments should also aim at determining the dose of furosemide that may

significantly decrease the function of mTAL in the renal outer medulla in humans. The doses

used in this study may not be directly applicable to humans, since humans may require lower

doses of furosemide for the same diuretic effect seen in rats with 0.8 mg/kg of furosemide. This

study demonstrated that urine osmolality closely reflects the renal medullary osmolality, which

144

represents the active reabsorption of mTAL. Therefore future studies should determine the dose

of furosemide that significantly reduces the urine osmolality in humans, rather than the dose that

significantly increases the urine flow rate.

145

CHAPTER 6

CITATIONS

146

Reference List

1. Andersson LG, Ekroth R, Bratteby LE, Hallhagen S and Wesslen O. Acute renal

failure after coronary surgery--a study of incidence and risk factors in 2009 consecutive

patients. Thorac Cardiovasc Surg 41: 237-241, 1993.

2. Aravena C, Salas I, Tagle R, Jara A, Miranda R, McNab P, Rodriguez JA, Valdes G

and Valdivieso A. [Hypokalemia, hypovolemia and electrocardiographic changes due to

furosemide abuse. Report of one case]. Rev Med Chil 135: 1456-1462, 2007.

3. Aravindan N, Aravindan S, Riedel BJ, Weng HR and Shaw AD. Furosemide prevents

apoptosis and associated gene expression in a rat model of surgical ischemic acute renal

failure. Ren Fail 29: 399-407, 2007.

4. Aravindan N and Shaw A. Effect of furosemide infusion on renal hemodynamics and

angiogenesis gene expression in acute renal ischemia/reperfusion. Ren Fail 28: 25-35,

2006.

5. Arjamaa O and Nikinmaa M. Natriuretic peptides in hormonal regulation of hypoxia

responses. Am J Physiol Regul Integr Comp Physiol 296: R257-R264, 2009.

6. Armstead WM. Opioids and nitric oxide contribute to hypoxia-induced pial arterial

vasodilation in newborn pigs. Am J Physiol 268: H226-H232, 1995.

147

7. Assadi F. Hypomagnesemia: an evidence-based approach to clinical cases. Iran J Kidney

Dis 4: 13-19, 2010.

8. Azevedo JL, Carey JO, Pories WJ, Morris PG and Dohm GL. Hypoxia stimulates

glucose transport in insulin-resistant human skeletal muscle. Diabetes 44: 695-698, 1995.

9. Bachmann S, Le HM and Eckardt KU. Co-localization of erythropoietin mRNA and

ecto-5'-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates

that fibroblasts produce erythropoietin. J Histochem Cytochem 41: 335-341, 1993.

10. Baertschi AJ, Adams JM and Sullivan MP. Acute hypoxemia stimulates atrial

natriuretic factor secretion in vivo. Am J Physiol 255: H295-H300, 1988.

11. Bagshaw SM and Bellomo R. Early diagnosis of acute kidney injury. Curr Opin Crit

Care 13: 638-644, 2007.

12. Bagshaw SM and Gibney RT. Conventional markers of kidney function. Crit Care Med

36: S152-S158, 2008.

13. Bagshaw SM and Gibney RT. Conventional markers of kidney function. Crit Care Med

36: S152-S158, 2008.

148

14. Bagshaw SM, Gibney RT, McAlister FA and Bellomo R. The SPARK Study: a phase

II randomized blinded controlled trial of the effect of furosemide in critically ill patients

with early acute kidney injury. Trials 11: 50, 2010.

15. Bagshaw SM, Laupland KB, Doig CJ, Mortis G, Fick GH, Mucenski M, Godinez-

Luna T, Svenson LW and Rosenal T. Prognosis for long-term survival and renal

recovery in critically ill patients with severe acute renal failure: a population-based study.

Crit Care 9: R700-R709, 2005.

16. Bankir L. Antidiuretic action of vasopressin: quantitative aspects and interaction

between V1a and V2 receptor-mediated effects. Cardiovascular Research 51: 372-390,

2001.

17. Banks RO. Vasoconstrictor-induced changes in renal blood flow: role of prostaglandins

and histamine. Am J Physiol 254: F470-F476, 1988.

18. Barbey F, Bonny O, Rothuizen L, Gomez F and Burnier M. A pregnant woman with

de novo polyuria-polydipsia and elevated liver enzymes. Nephrol Dial Transplant 18:

2193-2196, 2003.

19. Barron WM, Cohen LH, Ulland LA, Lassiter WE, Fulghum EM, Emmanouel D,

Robertson G and Lindheimer MD. Transient vasopressin-resistant diabetes insipidus of

pregnancy. N Engl J Med 310: 442-444, 1984.

149

20. Beall CM, Strohl KP, Blangero J, Williams-Blangero S, Almasy LA, Decker MJ,

Worthman CM, Goldstein MC, Vargas E, Villena M, Soria R, Alarcon AM and

Gonzales C. Ventilation and hypoxic ventilatory response of Tibetan and Aymara high

altitude natives. Am J Phys Anthropol 104: 427-447, 1997.

21. Beltowski J. Hypoxia in the renal medulla: implications for hydrogen sulfide signaling. J

Pharmacol Exp Ther 334: 358-363, 2010.

22. Bennett M, Dent CL, Ma Q, Dastrala S, Grenier F, Workman R, Syed H, Ali S,

Barasch J and Devarajan P. Urine NGAL predicts severity of acute kidney injury after

cardiac surgery: a prospective study. Clin J Am Soc Nephrol 3: 665-673, 2008.

23. Berg JM, Tymoczko JL and Stryer L. Peptides Can Be Synthesized by Automated

Solid-Phase Methods. In: Biochemistry, New York: W H Freeman, 2002.

24. Bernhardt WM, Campean V, Kany S, Jurgensen JS, Weidemann A, Warnecke C,

Arend M, Klaus S, Gunzler V, Amann K, Willam C, Wiesener MS and Eckardt KU.

Preconditional activation of hypoxia-inducible factors ameliorates ischemic acute renal

failure. J Am Soc Nephrol 17: 1970-1978, 2006.

25. Bestle MH, Olsen NV, Poulsen TD, Roach R, Fogh-Andersen N and Bie P. Prolonged

hypobaric hypoxemia attenuates vasopressin secretion and renal response to

osmostimulation in men. J Appl Physiol 92: 1911-1922, 2002.

150

26. Bichet DG, Razi M, Lonergan M, Arthus MF, Papukna V, Kortas C and Barjon JN.

Hemodynamic and coagulation responses to 1-desamino[8-D-arginine] vasopressin in

patients with congenital nephrogenic diabetes insipidus. N Engl J Med 318: 881-887,

1988.

27. Bjornsson TD. Use of serum creatinine concentrations to determine renal function. Clin

Pharmacokinet 4: 200-222, 1979.

28. Blitzer ML, Lee SD and Creager MA. Endothelium-derived nitric oxide mediates

hypoxic vasodilation of resistance vessels in humans. Am J Physiol 271: H1182-H1185,

1996.

29. Boone M and Deen PM. Physiology and pathophysiology of the vasopressin-regulated

renal water reabsorption. Pflugers Arch 456: 1005-1024, 2008.

30. Bozic N and Vujcic Z. Detection and quantification of leucyl aminopeptidase after

native electrophoresis using leucine-p-nitroanilide. Electrophoresis 26: 2476-2480, 2005.

31. Brezis M, Agmon Y and Epstein FH. Determinants of intrarenal oxygenation. I. Effects

of diuretics. Am J Physiol 267: F1059-F1062, 1994.

32. Brezis M and Rosen S. Hypoxia of the renal medulla--its implications for disease. N

Engl J Med 332: 647-655, 1995.

151

33. Bruick RK and McKnight SL. A conserved family of prolyl-4-hydroxylases that

modify HIF. Science 294: 1337-1340, 2001.

34. Calesnick B, Christensen JA and Richter M. Absorption and excretion of furosemide-

S35 in human subjects. Proc Soc Exp Biol Med 123: 17-22, 1966.

35. Caretti A, Morel S, Milano G, Fantacci M, Bianciardi P, Ronchi R, Vassalli G, von

Segesser LK and Samaja M. Heart HIF-1alpha and MAP kinases during hypoxia: are

they associated in vivo? Exp Biol Med (Maywood ) 232: 887-894, 2007.

36. Cheema-Dhadli S, Chong CK, Kim N, Kamel KS and Halperin ML. Importance of

Residual Water Permeability on the Excretion of Water during Water Diuresis in Rats.

Electrolyte Blood Press 8: 1-9, 2010.

37. Cheema-Dhadli S and Halperin ML. Influence of hypernatraemia and urea excretion

on the ability to excrete a maximally hypertonic urine in the rat. J Physiol 541: 929-936,

2002.

38. Chen J, Edwards A and Layton AT. A mathematical model of O2 transport in the rat

outer medulla. II. Impact of outer medullary architecture. Am J Physiol Renal Physiol

297: F537-F548, 2009.

152

39. Chen J, Layton AT and Edwards A. A mathematical model of O2 transport in the rat

outer medulla. I. Model formulation and baseline results. Am J Physiol Renal Physiol

297: F517-F536, 2009.

40. Chong ZZ, Kang JQ and Maiese K. Erythropoietin is a novel vascular protectant

through activation of Akt1 and mitochondrial modulation of cysteine proteases.

Circulation 106: 2973-2979, 2002.

41. Choong K and Kissoon N. Vasopressin in pediatric shock and cardiac arrest. Pediatr

Crit Care Med 9: 372-379, 2008.

42. Coca SG, Yalavarthy R, Concato J and Parikh CR. Biomarkers for the diagnosis and

risk stratification of acute kidney injury: a systematic review. Kidney Int 73: 1008-1016,

2008.

43. Cohen N, Almoznino-Sarafian D, Zaidenstein R, Alon I, Gorelik O, Shteinshnaider

M, Chachashvily S, Averbukh Z, Golik A, Chen-Levy Z and Modai D. Serum

magnesium aberrations in furosemide (frusemide) treated patients with congestive heart

failure: pathophysiological correlates and prognostic evaluation. Heart 89: 411-416,

2003.

44. Conger J, Robinette J, Villar A, Raij L and Shultz P. Increased nitric oxide synthase

activity despite lack of response to endothelium-dependent vasodilators in postischemic

acute renal failure in rats. J Clin Invest 96: 631-638, 1995.

153

45. Conlon PJ, Stafford-Smith M, White WD, Newman MF, King S, Winn MP and

Landolfo K. Acute renal failure following cardiac surgery. Nephrol Dial Transplant 14:

1158-1162, 1999.

46. Cowley AW, Jr. Control of the renal medullary circulation by vasopressin V1 and V2

receptors in the rat. Exp Physiol 85 Spec No: 223S-231S, 2000.

47. Cowley AW, Jr. Renal medullary oxidative stress, pressure-natriuresis, and

hypertension. Hypertension 52: 777-786, 2008.

48. Cowley AW, Jr., Mori T, Mattson D and Zou AP. Role of renal NO production in the

regulation of medullary blood flow. Am J Physiol Regul Integr Comp Physiol 284:

R1355-R1369, 2003.

49. Cowley AW, Jr., Skelton MM and Kurth TM. Effects of long-term vasopressin

receptor stimulation on medullary blood flow and arterial pressure. Am J Physiol 275:

R1420-R1424, 1998.

50. Cutler RE, Forrey AW, Christopher TG and Kimpel BM. Pharmacokinetics of

furosemide in normal subjects and functionally anephric patients. Clin Pharmacol Ther

15: 588-596, 1974.

154

51. Davison JM, Gilmore EA, Durr J, Robertson GL and Lindheimer MD. Altered

osmotic thresholds for vasopressin secretion and thirst in human pregnancy. Am J Physiol

246: F105-F109, 1984.

52. Davison JM and Lindheimer MD. Volume homeostasis and osmoregulation in human

pregnancy. Baillieres Clin Endocrinol Metab 3: 451-472, 1989.

53. Dery MA, Michaud MD and Richard DE. Hypoxia-inducible factor 1: regulation by

hypoxic and non-hypoxic activators. Int J Biochem Cell Biol 37: 535-540, 2005.

54. Devarajan P. Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol

17: 1503-1520, 2006.

55. Devarajan P. Emerging biomarkers of acute kidney injury. [Review] [44 refs].

Contributions to Nephrology 156:203-12, 2007.

56. Ebert EC. Hypoxic liver injury. Mayo Clin Proc 81: 1232-1236, 2006.

57. Eckardt KU, Bernhardt WM, Weidemann A, Warnecke C, Rosenberger C,

Wiesener MS and Willam C. Role of hypoxia in the pathogenesis of renal disease.

Kidney Int Suppl S46-S51, 2005.

58. Elin RJ. Magnesium: the fifth but forgotten electrolyte. Am J Clin Pathol 102: 616-622,

1994.

155

59. Engel A and Pagel H. Increased production of erythropoietin after application of

antidiuretic hormone. A consequence of renal vasoconstriction? Exp Clin Endocrinol

Diabetes 103: 303-307, 1995.

60. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR,

Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL,

Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ and

Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of

dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43-54, 2001.

61. Evans RG, Eppel GA, Anderson WP and Denton KM. Mechanisms underlying the

differential control of blood flow in the renal medulla and cortex. J Hypertens 22: 1439-

1451, 2004.

62. Fandrey J. Oxygen-dependent and tissue-specific regulation of erythropoietin gene

expression. Am J Physiol Regul Integr Comp Physiol 286: R977-R988, 2004.

63. Fenton RA and Knepper MA. Mouse models and the urinary concentrating mechanism

in the new millennium. Physiol Rev 87: 1083-1112, 2007.

64. Fenton RA and Moeller HB. Recent discoveries in vasopressin-regulated aquaporin-2

trafficking. Prog Brain Res 170: 571-579, 2008.

156

65. Franchini KG and Cowley AW. Renal cortical and medullary blood flow responses

during water restriction: role of vasopressin. American Journal of Physiology -

Regulatory, Integrative and Comparative Physiology 270: R1257-R1264, 1996.

66. Franchini KG and Cowley AW, Jr. Sensitivity of the renal medullary circulation to

plasma vasopressin. Am J Physiol 271: R647-R653, 1996.

67. Franchini KG, Mattson DL and Cowley AW, Jr. Vasopressin modulation of medullary

blood flow and pressure-natriuresis-diuresis in the decerebrated rat. Am J Physiol 272:

R1472-R1479, 1997.

68. Fried W. The liver as a source of extrarenal erythropoietin production. Blood 40: 671-

677, 1972.

69. Gess B, Schricker K, Pfeifer M and Kurtz A. Acute hypoxia upregulates NOS gene

expression in rats. Am J Physiol 273: R905-R910, 1997.

70. Ghezzi P and Brines M. Erythropoietin as an antiapoptotic, tissue-protective cytokine.

Cell Death Differ 11 Suppl 1: S37-S44, 2004.

71. Gordge MP, Williams DJ, Huggett NJ, Payne NN and Nelld GH. Loss of biological

activity of arginine vasopressin during its degradation by vasopressinase from pregnancy

serum. Clin Endocrinol (Oxf) 42: 51-58, 1995.

157

72. Gowrishankar M, Lenga I, Cheung RY, Cheema-Dhadli S and Halperin ML.

Minimum urine flow rate during water deprivation: importance of the permeability of

urea in the inner medulla. Kidney Int 53: 159-166, 1998.

73. Haddad GG and Lister G. Tissue oxygen deprivation : from molecular to integrative

function. New York: M. Dekker , 1996.

74. Halperin ML, Cheema-Dhadli S, Lin SH and Kamel KS. Properties permitting the

renal cortex to be the oxygen sensor for the release of erythropoietin: clinical

implications. Clin J Am Soc Nephrol 1: 1049-1053, 2006.

75. Halperin ML, Kamel KS and Oh MS. Mechanisms to concentrate the urine: an

opinion. Curr Opin Nephrol Hypertens 17: 416-422, 2008.

76. Halperin ML, Oh MS and Kamel KS. Integrating Effects of Aquaporins, Vasopressin,

Distal Delivery of Filtrate and Residual Water Permeability on the Magnitude of Water

Diuresis. Nephron Physiol 114: 11-17, 2010.

77. Han WK and Bonventre JV. Biologic markers for the early detection of acute kidney

injury. Curr Opin Crit Care 10: 476-482, 2004.

78. Han WK, Wagener G, Zhu Y, Wang S and Lee HT. Urinary biomarkers in the early

detection of acute kidney injury after cardiac surgery. Clin J Am Soc Nephrol 4: 873-882,

2009.

158

79. Han WK, Waikar SS, Johnson A, Betensky RA, Dent CL, Devarajan P and

Bonventre JV. Urinary biomarkers in the early diagnosis of acute kidney injury. Kidney

Int 73: 863-869, 2008.

80. Hebert SC. Extracellular calcium-sensing receptor: implications for calcium and

magnesium handling in the kidney. Kidney Int 50: 2129-2139, 1996.

81. Hildebrandt W, Ottenbacher A, Schuster M, Swenson ER and Bartsch P. Diuretic

effect of hypoxia, hypocapnia, and hyperpnea in humans: relation to hormones and O(2)

chemosensitivity. J Appl Physiol 88: 599-610, 2000.

82. Holmes CL, Landry DW and Granton JT. Science Review: Vasopressin and the

cardiovascular system part 2 - clinical physiology. Crit Care 8: 15-23, 2004.

83. Honig A. Peripheral arterial chemoreceptors and reflex control of sodium and water

homeostasis. Am J Physiol 257: R1282-R1302, 1989.

84. Hooper NM. Families of zinc metalloproteases. FEBS Letters 354: 1-6, 1994.

85. Ivan M, Haberberger T, Gervasi DC, Michelson KS, Gunzler V, Kondo K, Yang H,

Sorokina I, Conaway RC, Conaway JW and Kaelin WG, Jr. Biochemical purification

and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-

inducible factor. Proc Natl Acad Sci U S A 99: 13459-13464, 2002.

159

86. Jamison RJ. The renal concentrating mechanism. Kidney Int Suppl 21: S43-S50, 1987.

87. Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev

72: 449-489, 1992.

88. Jonsson V, Bock JE and Nielsen JB. Significance of plasma skimming and plasma

volume expansion. J Appl Physiol 72: 2047-2051, 1992.

89. Jordens I, Molle D, Xiong W, Keller SR and McGraw TE. Insulin-regulated

Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of

GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles. Mol Biol Cell 21:

2034-2044, 2010.

90. Kaufmann JE and Vischer UM. Cellular mechanisms of the hemostatic effects of

desmopressin (DDAVP). J Thromb Haemost 1: 682-689, 2003.

91. Kikkoji T, Kamiya A, Inui K and Hori R. Urinary excretion and diuretic action of

furosemide in rats: increased response to the urinary excretion rate of furosemide in rats

with acute renal failure. Pharm Res 5: 699-703, 1988.

92. Kobayashi H, Nomura S, Mitsui T, Ito T, Kuno N, Ohno Y, Kadomatsu K,

Muramatsu T, Nagasaka T and Mizutani S. Tissue Distribution of Placental Leucine

Aminopeptidase/Oxytocinase During Mouse Pregnancy. Journal of Histochemistry &

Cytochemistry 52: 113-121, 2004.

160

93. Koller EA, Lesniewska B, Buhrer A, Bub A and Kohl J. The effects of acute altitude

exposure in Swiss highlanders and lowlanders. Eur J Appl Physiol Occup Physiol 66:

146-154, 1993.

94. Kone BC and Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal

kidney. Am J Physiol 272: F561-F578, 1997.

95. Korkeila M, Ruokonen E and Takala J. Costs of care, long-term prognosis and quality

of life in patients requiring renal replacement therapy during intensive care. Intensive

Care Med 26: 1824-1831, 2000.

96. Koury MJ and Bondurant MC. Maintenance by erythropoietin of viability and

maturation of murine erythroid precursor cells. J Cell Physiol 137: 65-74, 1988.

97. Koury ST, Bondurant MC and Koury MJ. Localization of erythropoietin synthesizing

cells in murine kidneys by in situ hybridization. Blood 71: 524-527, 1988.

98. Koyner JL, Bennett MR, Worcester EM, Ma Q, Raman J, Jeevanandam V, Kasza

KE, O'Connor MF, Konczal DJ, Trevino S, Devarajan P and Murray PT. Urinary

cystatin C as an early biomarker of acute kidney injury following adult cardiothoracic

surgery. Kidney Int 74: 1059-1069, 2008.

99. Krantz SB. Erythropoietin. Blood 77: 419-434, 1991.

161

100. Kusakabe Y, Matsushita T, Honda S, Okada S and Murase K. Using BOLD imaging

to measure renal oxygenation dynamics in rats injected with diuretics. Magn Reson Med

Sci 9: 187-194, 2010.

101. Kuwahira I, Heisler N, Piiper J and Gonzalez NC. Effect of chronic hypoxia on

hemodynamics, organ blood flow and O2 supply in rats. Respir Physiol 92: 227-238,

1993.

102. Lacombe C, Da Silva JL, Bruneval P, Fournier JG, Wendling F, Casadevall N,

Camilleri JP, Bariety J, Varet B and Tambourin P. Peritubular cells are the site of

erythropoietin synthesis in the murine hypoxic kidney. J Clin Invest 81: 620-623, 1988.

103. LaManna JC, Chavez JC and Pichiule P. Structural and functional adaptation to

hypoxia in the rat brain. J Exp Biol 207: 3163-3169, 2004.

104. Lenfant C and Sullivan K. Adaptation to high altitude. N Engl J Med 284: 1298-1309,

1971.

105. Liangos O, Perianayagam MC, Vaidya VS, Han WK, Wald R, Tighiouart H,

MacKinnon RW, Li L, Balakrishnan VS, Pereira BJ, Bonventre JV and Jaber BL.

Urinary N-acetyl-beta-(D)-glucosaminidase activity and kidney injury molecule-1 level

are associated with adverse outcomes in acute renal failure. J Am Soc Nephrol 18: 904-

912, 2007.

162

106. Lindheimer MD. Polyuria and pregnancy: its cause, its danger. Obstet Gynecol 105:

1171-1172, 2005.

107. Macdougall IC. Optimizing the use of erythropoietic agents-- pharmacokinetic and

pharmacodynamic considerations. Nephrol Dial Transplant 17 Suppl 5: 66-70, 2002.

108. Mackenzie R, Maxwell N, Castle P, Brickley G and Watt P. Acute hypoxia and

exercise improve insulin sensitivity (S(I) (2*)) in individuals with type 2 diabetes.

Diabetes Metab Res Rev 27: 94-101, 2011.

109. Majid DS and Navar LG. Nitric oxide in the control of renal hemodynamics and

excretory function. Am J Hypertens 14: 74S-82S, 2001.

110. Manotham K, Tanaka T, Ohse T, Kojima I, Miyata T, Inagi R, Tanaka H, Sassa R,

Fujita T and Nangaku M. A biologic role of HIF-1 in the renal medulla. Kidney Int 67:

1428-1439, 2005.

111. Maril N, Margalit R, Rosen S, Heyman SN and Degani H. Detection of evolving acute

tubular necrosis with renal 23Na MRI: studies in rats. Kidney Int 69: 765-768, 2006.

112. Martin PY, Bianchi M, Roger F, Niksic L and Feraille E. Arginine vasopressin

modulates expression of neuronal NOS in rat renal medulla. Am J Physiol Renal Physiol

283: F559-F568, 2002.

163

113. Masuda S, Hattori A, Matsumoto H, Miyazawa S, Natori Y, Mizutani S and

Tsujimoto M. Involvement of the V2 receptor in vasopressin-stimulated translocation of

placental leucine aminopeptidase/oxytocinase in renal cells. Eur J Biochem 270: 1988-

1994, 2003.

114. Mattson DL. Importance of the renal medullary circulation in the control of sodium

excretion and blood pressure. Am J Physiol Regul Integr Comp Physiol 284: R13-R27,

2003.

115. Maxwell P. HIF-1: an oxygen response system with special relevance to the kidney. J

Am Soc Nephrol 14: 2712-2722, 2003.

116. Maxwell PH, Pugh CW and Ratcliffe PJ. Inducible operation of the erythropoietin

3[prime] enhancer in multiple cell lines: evidence for a widespread oxygen-sensing

mechanism. Proc Natl Acad Sci USA 90: 2423-2427, 1993.

117. Mayeur N, Minville V, Jaafar A, Allard J, Al ST, Guilbeau-Frugier C, Fourcade O,

Girolami JP, Schaak S and Tack I. Morphologic and functional renal impact of acute

kidney injury after prolonged hemorrhagic shock in mice. Crit Care Med 2011.

118. McLaren AT, Marsden PA, Mazer CD, Baker AJ, Stewart DJ, Tsui AK, Li X, Yucel

Y, Robb M, Boyd SR, Liu E, Yu J and Hare GM. Increased expression of HIF-1alpha,

nNOS, and VEGF in the cerebral cortex of anemic rats. Am J Physiol Regul Integr Comp

Physiol 292: R403-R414, 2007.

164

119. Mehta RL and Chertow GM. Acute renal failure definitions and classification: time for

change? J Am Soc Nephrol 14: 2178-2187, 2003.

120. Mehta RL, Pascual MT, Soroko S, Savage BR, Himmelfarb J, Ikizler TA, Paganini

EP and Chertow GM. Spectrum of acute renal failure in the intensive care unit: the

PICARD experience. Kidney Int 66: 1613-1621, 2004.

121. Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, Ruff SM, Zahedi K,

Shao M, Bean J, Mori K, Barasch J and Devarajan P. Neutrophil gelatinase-

associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery.

Lancet 365: 1231-1238, 2005.

122. Mount PF and Power DA. Nitric oxide in the kidney: functions and regulation of

synthesis. Acta Physiol (Oxf) 187: 433-446, 2006.

123. Naitoh M, Suzuki H, Murakami M, Matsumoto A, Ichihara A, Nakamoto H,

Yamamura Y and Saruta T. Arginine vasopressin produces renal vasodilation via V2

receptors in conscious dogs. Am J Physiol 265: R934-R942, 1993.

124. Nakagawa NK, Donato-Junior F, Kondo CS, King M, Auler-Junior JO, Saldiva PH

and Lorenzi-Filho G. Effects of acute hypovolaemia by furosemide on tracheal

transepithelial potential difference and mucus in dogs. Eur Respir J 24: 805-810, 2004.

165

125. Nakamura H, Itakuara A, Okamura M, Ito M, Iwase A, Nakanishi Y, Okada M,

Nagasaka T and Mizutani S. Oxytocin stimulates the translocation of oxytocinase of

human vascular endothelial cells via activation of oxytocin receptors. Endocrinology 141:

4481-4485, 2000.

126. Nakanishi K, Mattson DL, Gross V, Roman RJ and Cowley AW, Jr. Control of renal

medullary blood flow by vasopressin V1 and V2 receptors. Am J Physiol 269: R193-

R200, 1995.

127. Navar LG, Inscho EW, Majid SA, Imig JD, Harrison-Bernard LM and Mitchell

KD. Paracrine regulation of the renal microcirculation. Physiol Rev 76: 425-536, 1996.

128. Nissenson AR. Novel erythropoiesis stimulating protein for managing the anemia of

chronic kidney disease. Am J Kidney Dis 38: 1390-1397, 2001.

129. Norman JT, Orphanides C, Garcia P and Fine LG. Hypoxia-induced changes in

extracellular matrix metabolism in renal cells. Exp Nephrol 7: 463-469, 1999.

130. O'Connor PM and Cowley AW, Jr. Vasopressin-induced nitric oxide production in rat

inner medullary collecting duct is dependent on V2 receptor activation of the

phosphoinositide pathway. Am J Physiol Renal Physiol 293: F526-F532, 2007.

166

131. Ogilvie M, Yu X, Nicolas-Metral V, Pulido SM, Liu C, Ruegg UT and Noguchi CT.

Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts. J

Biol Chem 275: 39754-39761, 2000.

132. Olsen NV. Effect of hypoxaemia on water and sodium homeostatic hormones and renal

function. Acta Anaesthesiol Scand Suppl 107: 165-170, 1995.

133. Pallone TL, Robertson CR and Jamison RL. Renal medullary microcirculation.

Physiol Rev 70: 885-920, 1990.

134. Pallone TL, Silldorff EP and Turner MR. Intrarenal blood flow: microvascular

anatomy and the regulation of medullary perfusion. Clin Exp Pharmacol Physiol 25: 383-

392, 1998.

135. Pallone TL, Turner MR, Edwards A and Jamison RL. Countercurrent exchange in the

renal medulla. Am J Physiol Regul Integr Comp Physiol 284: R1153-R1175, 2003.

136. Papandreou I, Cairns RA, Fontana L, Lim AL and Denko NC. HIF-1 mediates

adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption.

Cell Metab 3: 187-197, 2006.

137. Parikh CR, Abraham E, Ancukiewicz M and Edelstein CL. Urine IL-18 is an early

diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit.

J Am Soc Nephrol 16: 3046-3052, 2005.

167

138. Parikh CR and Devarajan P. New biomarkers of acute kidney injury. Crit Care Med

36: S159-S165, 2008.

139. Parikh CR, Mishra J, Thiessen-Philbrook H, Dursun B, Ma Q, Kelly C, Dent C,

Devarajan P and Edelstein CL. Urinary IL-18 is an early predictive biomarker of acute

kidney injury after cardiac surgery. Kidney Int 70: 199-203, 2006.

140. Park F, Zou AP and Cowley AW, Jr. Arginine vasopressin-mediated stimulation of

nitric oxide within the rat renal medulla. Hypertension 32: 896-901, 1998.

141. Pirker R and Smith R. Darbepoetin alfa: potential role in managing anemia in cancer

patients. Expert Rev Anticancer Ther 2: 377-384, 2002.

142. Pohl U. Endothelial cells as part of a vascular oxygen-sensing system: hypoxia-induced

release of autacoids. Experientia 46: 1175-1179, 1990.

143. Prandota J. Extrarenal effects of furosemide on distribution of salt and water: an indirect

mechanism of the drug's action? Int Urol Nephrol 9: 347-352, 1977.

144. Rasmussen TE, Pedraza-DÃ-az S, Hardré R, Laustsen PG, CarrÃ-on AG and

Kristensen T. Structure of the human oxytocinase/insulin-regulated aminopeptidase gene

and localization to chromosome 5q21. Eur J Biochem 267: 2297-2306, 2000.

168

145. Richardson DW, Kontos HA, Raper AJ and Patterson JL, Jr. Modification by beta-

adrenergic blockade of the circulatory respones to acute hypoxia in man. J Clin Invest 46:

77-85, 1967.

146. Rogi T, Tsujimoto M, Nakazato H, Mizutani S and Tomoda Y. Human placental

leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc

metallopeptidase family. J Biol Chem 271: 56-61, 1996.

147. Romero JC, Lahera V, Salom MG and Biondi ML. Role of the endothelium-

dependent relaxing factor nitric oxide on renal function. J Am Soc Nephrol 2: 1371-1387,

1992.

148. Rosenberger C, Griethe W, Gruber G, Wiesener M, Frei U, Bachmann S and

Eckardt KU. Cellular responses to hypoxia after renal segmental infarction. Kidney Int

64: 874-886, 2003.

149. Rosenberger C, Heyman SN, Rosen S, Shina A, Goldfarb M, Griethe W, Frei U,

Reinke P, Bachmann S and Eckardt KU. Up-regulation of HIF in experimental acute

renal failure: evidence for a protective transcriptional response to hypoxia. Kidney Int 67:

531-542, 2005.

150. Rosenberger C, Rosen S and Heyman SN. Renal parenchymal oxygenation and

hypoxia adaptation in acute kidney injury. Clin Exp Pharmacol Physiol 33: 980-988,

2006.

169

151. Rosner MH and Okusa MD. Acute kidney injury associated with cardiac surgery. Clin

J Am Soc Nephrol 1: 19-32, 2006.

152. Schmidt HH, Pollock JS, Nakane M, Forstermann U and Murad F.

Ca2+/calmodulin-regulated nitric oxide synthases. Cell Calcium 13: 427-434, 1992.

153. Schrier RW, Hoggard JG, Hunt JM and Durr JA. Diabetes Insipidus in Pregnancy

Associated with Abnormally High Circulating Vasopressinase Activity. The New

England journal of medicine 316: 1070-1074, 1987.

154. Scott GR and Milsom WK. Control of breathing and adaptation to high altitude in the

bar-headed goose. Am J Physiol Regul Integr Comp Physiol 293: R379-R391, 2007.

155. Sharples EJ, Patel N, Brown P, Stewart K, Mota-Philipe H, Sheaff M, Kieswich J,

Allen D, Harwood S, Raftery M, Thiemermann C and Yaqoob MM. Erythropoietin

protects the kidney against the injury and dysfunction caused by ischemia-reperfusion. J

Am Soc Nephrol 15: 2115-2124, 2004.

156. Shimizu K, Nakao A, Nonaka T and Oka H. Identification of vasopressin and

determination of its corticomedullary levels in rat kidney tissue. Kidney Int 26: 785-790,

1984.

157. Sinagowitz E, Baker R, Strauss J and Kessler M. Renal tissue oxygenation during

hypoxic hypoxia. Adv Exp Med Biol 75: 441-447, 1976.

170

158. Small CW and Watkins WB. S-benzyl-L-cysteine-p-nitroanilide. A new substrate for

determination of oxytocinase with improved specificity. Biochem Med 9: 103-112, 1974.

159. Smith RE, Jr. and Tchekmedyian S. Practitioners' practical model for managing

cancer-related anemia. Oncology (Williston Park) 16: 55-63, 2002.

160. Suzuki Y, Shibata K, Kikkawa F, Kajiyama H, Ino K, Nomura S, Tsujimoto M and

Mizutani S. Possible Role of Placental Leucine Aminopeptidase in the Antiproliferative

Effect of Oxytocin in Human Endometrial Adenocarcinoma. Clinical Cancer Research 9:

1528-1534, 2003.

161. Sytkowski AJ. Erythropoietin: Blood, Brain and Beyond. Weinheim: Wiley-VCH,

2005.

162. Takamata A, Nose H, Kinoshita T, Hirose M, Itoh T and Morimoto T. Effect of acute

hypoxia on vasopressin release and intravascular fluid during dynamic exercise in

humans. Am J Physiol Regul Integr Comp Physiol 279: R161-R168, 2000.

163. Tanaka T and Nangaku M. The role of hypoxia, increased oxygen consumption, and

hypoxia-inducible factor-1 alpha in progression of chronic kidney disease. Curr Opin

Nephrol Hypertens 19: 43-50, 2010.

171

164. Terada Y, Tomita K, Nonoguchi H and Marumo F. Polymerase chain reaction

localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger

RNAs in microdissected rat nephron segments. J Clin Invest 90: 659-665, 1992.

165. Thadhani R, Pascual M and Bonventre JV. Acute renal failure. N Engl J Med 334:

1448-1460, 1996.

166. Treschan TA and Peters J. The vasopressin system: physiology and clinical strategies.

Anesthesiology 105: 599-612, 2006.

167. Tumkur SM, Vu AT, Li LP, Pierchala L and Prasad PV. Evaluation of intra-renal

oxygenation during water diuresis: a time-resolved study using BOLD MRI. Kidney Int

70: 139-143, 2006.

168. Urena P. [Treatment of anemia in chronic renal failure by a long-activing activator of

erythropoiesis]. Presse Med 31: 505-514, 2002.

169. Vallet B, Curtis SE, Winn MJ, King CE, Chapler CK and Cain SM. Hypoxic

vasodilation does not require nitric oxide (EDRF/NO) synthesis. J Appl Physiol 76: 1256-

1261, 1994.

170. van der Voort PH, Boerma EC, Koopmans M, Zandberg M, de RJ, Gerritsen RT,

Egbers PH, Kingma WP and Kuiper MA. Furosemide does not improve renal recovery

172

after hemofiltration for acute renal failure in critically ill patients: a double blind

randomized controlled trial. Crit Care Med 37: 533-538, 2009.

171. van BF, Sola A, Roman C and Rudolph AM. Role of nitric oxide in the regulation of

the cerebral circulation in the lamb fetus during normoxemia and hypoxemia. Biol

Neonate 68: 200-210, 1995.

172. Veeraveedu PT, Watanabe K, Ma M, Palaniyandi SS, Yamaguchi K, Kodama M

and Aizawa Y. Effects of V2-receptor antagonist tolvaptan and the loop diuretic

furosemide in rats with heart failure. Biochem Pharmacol 75: 1322-1330, 2008.

173. Vincent JL and Su F. Physiology and pathophysiology of the vasopressinergic system.

Best Pract Res Clin Anaesthesiol 22: 243-252, 2008.

174. Vogel JA and Harris CW. Cardiopulmonary responses of resting man during early

exposure to high altitude. J Appl Physiol 22: 1124-1128, 1967.

175. Wagener G, Jan M, Kim M, Mori K, Barasch JM, Sladen RN and Lee HT.

Association between increases in urinary neutrophil gelatinase-associated lipocalin and

acute renal dysfunction after adult cardiac surgery. Anesthesiology 105: 485-491, 2006.

176. Walser M. Creatinine excretion as a measure of protein nutrition in adults of varying

age. JPEN J Parenter Enteral Nutr 11: 73S-78S, 1987.

173

177. Wang X, Thomas SR and Wexler AS. Outer medullary anatomy and the urine

concentrating mechanism. Am J Physiol 274: F413-F424, 1998.

178. Ward ME, Toporsian M, Scott JA, Teoh H, Govindaraju V, Quan A, Wener AD,

Wang G, Bevan SC, Newton DC and Marsden PA. Hypoxia induces a functionally

significant and translationally efficient neuronal NO synthase mRNA variant. J Clin

Invest 115: 3128-3139, 2005.

179. Warnecke C, Griethe W, Weidemann A, Jurgensen JS, Willam C, Bachmann S,

Ivashchenko Y, Wagner I, Frei U, Wiesener M and Eckardt KU. Activation of the

hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of

prolyl hydroxylase inhibitors. FASEB J 17: 1186-1188, 2003.

180. Watanabe S, Fukumoto S, Chang H, Takeuchi Y, Hasegawa Y, Okazaki R,

Chikatsu N and Fujita T. Association between activating mutations of calcium-sensing

receptor and Bartter's syndrome. Lancet 360: 692-694, 2002.

181. Waters SB, D'Auria M, Martin SS, Nguyen C, Kozma LM and Luskey KL. The

amino terminus of insulin-responsive aminopeptidase causes Glut4 translocation in 3T3-

L1 adipocytes. J Biol Chem 272: 23323-23327, 1997.

182. Whang R, Hampton EM and Whang DD. Magnesium homeostasis and clinical

disorders of magnesium deficiency. Ann Pharmacother 28: 220-226, 1994.

174

183. Wilcox CS. Role of macula densa NOS in tubuloglomerular feedback. Curr Opin

Nephrol Hypertens 7: 443-449, 1998.

184. Yim S, Choi SM, Choi Y, Lee N, Chung J and Park H. Insulin and hypoxia share

common target genes but not the hypoxia-inducible factor-1alpha. J Biol Chem 278:

38260-38268, 2003.

185. Zamboni WC and Stewart CE. An overview of the pharmacokinetic disposition of

darbepoetin alfa. Pharmacotherapy 22: 133S-140S, 2002.

186. Zappitelli M, Washburn KK, Arikan AA, Loftis L, Ma Q, Devarajan P, Parikh CR

and Goldstein SL. Urine neutrophil gelatinase-associated lipocalin is an early marker of

acute kidney injury in critically ill children: a prospective cohort study. Crit Care 11:

R84, 2007.

187. Zhai XY, Fenton RA, Andreasen A, Thomsen JS and Christensen EI. Aquaporin-1 is

not expressed in descending thin limbs of short-loop nephrons. J Am Soc Nephrol 18:

2937-2944, 2007.

188. Zhang W and Edwards A. Oxygen transport across vasa recta in the renal medulla. Am

J Physiol Heart Circ Physiol 283: H1042-H1055, 2002.

189. Zierath JR, Tsao TS, Stenbit AE, Ryder JW, Galuska D and Charron MJ.

Restoration of hypoxia-stimulated glucose uptake in GLUT4-deficient muscles by

175

176

muscle-specific GLUT4 transgenic complementation. J Biol Chem 273: 20910-20915,

1998.