A Dissertation Entitled Na/K-ATPase Mediates Renal Sodium Handling By Yanling Yan Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences Dr. Joseph Shapiro, Committee Chair Dr. Jiang Liu, Committee Member Dr. Nader Abraham, Committee Member Dr. Deepak Malhotra, Committee Member Dr. Bina Joe, Committee Member Dr. Zijian Xie, Committee Member Dr. Patricia Komuniecki, Dean College of Graduate Studies The University of Toledo August 2012 Copyright 2012, Yanling Yan This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Na/K-ATPase Mediates Renal Sodium Handling by Yanling Yan Submitted to the Graduate Faculty as partial fulfillment of the requirement for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo August 2012 Hypertension, affecting over one billion people worldwide, is one of the leading risk factors for heart attack and stroke. Kidney cross-transplantation studies between hypertensive and normotensive people and strains of rats provide the most compelling evidence for the fundamental role of the kidney in the pathogenesis of hypertension. Recently, there is growing evidence supporting Arthur Guyton’s hypothesis that a common characteristic feature of hypertension is impaired renal sodium excretion. However, the exact molecular mechanism responsible for the impaired renal sodium excretion is not clearly defined. The overall aim of this dissertation is to improve our understanding of Na/K-ATPase and sodium proton exchanger 3 (NHE3) trafficking regulation and determine molecular mechanisms of renal proximal tubular sodium iii handling, which might contribute to the impaired sodium excretion associated with hypertension and results may help to develop effective therapy for hypertension. Renal proximal tubules (RPTs) responsible for 65-70% of filtered sodium and water reabsorption have profound effects on renal and body fluid balance associated with hypertension. Studies from our lab were the first to demonstrate that in renal proximal tubular cells, binding of cardiotonic steroids (CTS) such as ouabain to Na/K-ATPase stimulates Na/K-ATPase signaling cascade and induces the redistribution of basolateral Na/K-ATPase and apical NHE3, leading to a net increase in urinary sodium excretion. The first manuscript entitled “Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells” improves our understanding of Na/K-ATPase and NHE3 trafficking regulation. Three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 cells in which the pig α1 was replaced by ouabain- resistant rat α1) were employed to compare ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular 22Na+ transport. Data indicate that our previous observations that ouabain-induced redistribution of Na/K-ATPase α1 subunit and NHE3 is not species-specific. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. The second manuscript entitled “Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension” defines a iv novel mechanism of salt-sensitive hypertension. Studies from Sprague Dawley and Dahl salt-resistant and Dahl salt-sensitive rats demonstrate that impaired Na/K- ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt induced hypertension in the Dahl S rat. Given there is no difference in the Na/K-ATPase α1 gene (Atp1a1) coding and α1 sensitivity to ouabain between Dahl salt-resistant and Dahl salt-sensitive rats, other factors must be present to affect the activation of Na/K-ATPase/c-Src signaling. Reactive oxygen species (ROS) play a critical role in the pathogenesis of hypertension. Manuscript 3 entitled “Redox modulation of the Na/K-ATPase signaling and renal proximal tubular sodium handling” present that ouabain- stimulated Na/K-ATPase signaling and subsequent redistribution are redox- sensitive, and ouabain induces direct carbonylation of proline/threonine residues in the actuator (A) domain of the Na/K-ATPase α1 subunit. These data indicate that a proper redox modulation of renal proximal tubular Na/K-ATPase signaling is critical in renal sodium handling and might play an important role in counteracting volume expansion mediated high blood pressure. All in all, this dissertation not only improves our understanding of Na/K- ATPase signaling regulation, but also defines a novel mechanism involved in salt- sensitive hypertension, and also demonstrates ouabain-activated Na/K-ATPase signaling is redox sensitive, which might play a crucial role in the regulation of renal sodium handling and blood pressure. v Dedication I dedicate this dissertation to my lovely family, particularly to my understanding and patient husband, Dengyue, who has put up with my so many years of research overseas and has supported my every step of the way, and to our precious daughter Shuyan, who is the joy of our lives, and to my loving parents who have never failed to give me moral support and encouragement. vi Acknowledgments I wish to extend my sincere appreciation to all the persons who made my dissertation possible. I would never have been able to finish my dissertation without the guidance of my committee members, help from friends and support from my family. I would like to express my sincere gratitude to Dr. Joseph Shapiro, my major advisor for giving me a chance to join his excellent lab and providing me a fascinating research topic. I cannot thank him enough for his patience, his solicitude, his kindness, his wisdom and support. His inspiring words and excellent guidance will sway my life deeply and make me achieve what I have dreamt of. I am very grateful to Dr. Jiang Liu, for his thoughtfulness and generosity with time and energy, for his sharing meticulous research and insights with me that support and expand my thesis work, for risking his health to help me with those critical data. I am also indebted to my committee members, Dr. Nader Abraham, Dr. Bina Joe, Dr. Deepak Malhotra, and Dr. Zijian Xie, for serving on my dissertation committee, for their expert suggestions, contributions and good-natured support throughout the production of my research and dissertation. vii I would also like to thank my fellow lab members, past and present, for their assistance and support over the years: Dr. Steven Haller, Dr. David J. Kennedy, Dr. Jiang Tian, Dr. Christopher Drummond, Sandeep Vetteth, Shalini Gupta, Mohammad Taleb, Vinai Katragadda, Anna Shapiro, Chiamaka Mbaso, Georgy Budnyy, Imad Hariri, Sayed Moustafa Helmy, Eric Kim. I really enjoy being with all of you in the lab. Special thanks go to Dr. Sankaridrug Periyasamy, our previous lab manager, and his wife for their support and assistance when I first came to the lab. Many thanks must also go to Ms. Carol Woods, and Ms. Jenelle Thurn for their research assistance; to Dr. Robert Blumenthal for his timely help with my graduate admission process, to Dr. Randall Ruch for his support and assistance as an assistant dean for Admissions, Biomedical Graduate Program, to Dr. Andrew Beavis for his course and research help as a director of CVMD track; to Dr. Lijun Liu, Dr. Jean H. Overmeryer and Dr. John David Dignam for their kind, unselfish help to make my experiments go well, to Dr. Nancy Collins, my mentor in Graduate Student Professional Mentoring Program, for her valuable advice in many different ways, to Madge Levinson, my English tutor, for her encouragement and guidance with my English language study. I would like to gratefully acknowledge the support, encouragement and friendship of my good friends, classmates and co-workers over the years: Lauren Stanoszek, Erin Crawford, Xiaolu Zhang, Jiyoun Yeo, Jieying Wang, Shuai Dong, Ran Lu, Shuo Geng, Marjie Gable, Jian Wu, Yan Bai, Daxiang Li, Yiliang Chen, Zhichuan Li, Qiying Ye, Zhen Jiang, Fangfang Lai, Katye Smedlund, Jean-Yves Tano, Joshua Waldman, Qiang Mei, Jun Lu, Jie Zhou. viii Additionally, I wish to thank the members of Dr. Nader Abraham, Dr. Bina Joe, Dr. Lijun Liu and Dr. Zijian Xie’s lab for sharing their valuable thoughts with me, helping me get through difficult experiment. I would like to thank my parents, my family and all my friends for their steadfast support and encouragement throughout the years. Lastly, but most importantly, I would like to thank my loving husband, Dengyue Sun, and daughter, Shuyan Sun for never failing to lift my spirits. Your love and support have been with me through all the good times and bad, and helping to keep me sane. ix Contents Abstract iii Dedication vi Acknowledgments vii Contents x List of Abbreviations xiii Chapter 1 – Literature Review 1 Chapter 2 – “Ouabain-Stimulated Trafficking Regulation of the Na/K-ATPase and NHE3 in Renal Proximal Tubule Cells” (Manuscript 1accepted for publication) 55 2.1 Abstract 56 2.2 Introduction 57 2.3 Experimential Methods 58 2.4 Results 64 2.5 Discussion 68 2.6 Manuscript References 73 2.7 Figure Legends 79 x 2.8 Tables and Figures 82 Chapter 3– “Impairment of Na/K-ATPase Signaling in Renal Proximal Tubule Contributes to Dahl Salt-Sensitive Hypertension” (Manuscript 2) 90 3.1 Abstract 91 3.2 Introduction 92 3.3 Materials and Methods 92 3.4 Results 97 3.5 Discussion