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…………………………..