Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Genetic Dissection of Hypertension Related Renal Disease Using the Dahl Salt-Sensitive Rat Submitted by: Michael R. Garrett In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Joseph Shapiro, M.D. Academic Advisory Committee: David Allison, M.D., Ph.D. Richard J. Roman, Ph.D. En-Bing Lin, Ph.D. Deepak Malhotra, M.D., Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: September 7, 2006 Genetic Dissection of Hypertension-Related Renal Disease Using the Dahl Salt-Sensitive Rat Michael R. Garrett University of Toledo 2006 ii DEDICATION I dedicate this thesis to my wife, Jean and my two sons, Noah and Parker. This work is not solely a personal achievement but a testament of their love, support, encouragement, and understanding. Thank you. iii ACKNOWLEDGEMENTS I wish to thank the many people who have helped me not only learn about research, but also more importantly what it means to be a scientist. In particular, I wish to acknowledge John P. Rapp, D.V.M., Ph.D. for his guidance, support and encouragement. I would like to thank my major advisor, Joseph Shapiro, M.D., for giving me the opportunity to work under his supervision and my other graduate committee members, David Allison, M.D., Ph.D., Deepak Malhotra, M.D., Ph.D., Richard Roman, Ph.D., and En-Bing Lin, Ph.D. for their time and support. I appreciate the guidance and encouragement provided by Sonia Najjar, Ph.D., Director of the Molecular Basis of Disease (MBD) program. I especially wish to thank Shane Yerga-Woolwine for his contributions to my work. His involvement in taking blood pressure measurements and genotyping is sincerely appreciated. I thank Tracy Radecki for her help in phenotyping congenic strains and Kris Farms for her help in maintaining the rat colony. I thank Howard Dene, Ph.D. for his involvement with blood pressure measurements and statistical calculations. The expertise of William Gunning, Ph.D. in light and electron microscopy is appreciated. Finally, I wish to thank Bina Joe, Ph.D. and Yasser Saad, Ph.D. for their helpful discussions and interest in my work. iv TABLE OF CONTENTS Dedication………………………………………………………………………………ii Acknowledgments……………………………………………………………………...iii Table of Contents………………………………………………………………………iv Introduction……………………………………………………………………………..1 Literature………………………………………………………………………………..4 Manuscript 1: Time-Course Genetic Analysis of Albuminuria in Dahl Salt-Sensitive Rats on Low Salt Diet………………………………………………….56 Manuscript 2: Genetic Linkage of Urinary Albumin Excretion in Dahl Salt-Sensitive Rats: Influence of Dietary Salt and Confirmation using Congenic Strains……………………………………………………….96 Manuscript 3 Dissection of a Genetic Locus Influencing Renal Function in the Rat and its Concordance with Kidney Disease Loci on Human Chromosome 1q21…………………………………………………….132 Summary………………………………………………………………………………193 Bibliography…………………………………………………………………………..198 Abstract………………………………………………………………………………..234 1 INTRODUCTION Chronic kidney disease (CKD) is an important healthcare problem with increasing incidence and prevalence worldwide (United States Renal Data System 2005). Chronic kidney disease is characterized by a gradual decline in kidney function and can culminate in end stage renal disease (ESRD) requiring expensive treatments of dialysis and renal transplantation (USRDS 2005). Most CKD cases are not associated with primary renal disease, but with systemic conditions like diabetes and hypertension. In fact, diabetes and hypertension account for about two-thirds of patients that progress to ESRD. Additionally, age, gender, race/ethnicity, and socioeconomic factors play a role in the onset and progression of the disease (Agodoa et al. 2005; Norris and Agodoa 2005). Analysis involving familial aggregation studies, comparison of incidence between different racial and ethnic populations, and linkage analysis have provided strong evidence that CKD is, in part, genetically determined (Bowden 2003). Genetic analysis of congenital and familial forms of kidney disease has led to the identification of genes required for proper functioning of the glomerular filtration barrier (Chow et al. 2005). While these studies have provided insight into mechanisms of proteinuria and glomerulosclerosis, they have not helped to explain common causes of kidney disease. A number of genetic analyses have been performed to identify genes involved in diabetes and hypertension related ESRD (Bowden et al. 2004; DeWan et al. 2001; Freedman et al. 2003, 2004). Hundreds of these studies have utilized the candidate gene approach, while only a handful have utilized the more systemic whole genome scan approach to identify genes involved in kidney disease (Bowden 2003). These genome scans have identified 2 many genomic regions linked to ESRD, but none have culminated in gene identification. One reason is that the linkage analysis in humans suffer from several limitations that inherently make the process of gene identification difficult (Schork 1997). While some problems can be overcome with proper experimental design, an alternative approach is to utilize animal models of CKD (Jacob and Kwitek 2002). The rat provides a particularly fertile model to study disease and there are many well-defined inbred rat strains currently being used to study the genetics of CKD (Korstanje and DiPetrillo 2004). In particular, the Dahl salt-sensitive rat (S) was selectively bred as a model to study the genetics of salt-sensitive hypertension (Dahl et al. 1962; Rapp and Dene 1985). The S rat also has a unique early onset and marked propensity to develop proteinuria, glomerulosclerosis and progressive renal damage (Hampton et al. 1989; Sterzel et al. 1988). In contrast, the spontaneously hypertensive rat (SHR) is exactly opposite with regard to renal pathology. SHR have minimal proteinuria and a pronounced resistance to development of renal lesions in spite of their hypertension (Feld et al. 1977; Karlsen et al. 1997). In order to understand the genetic causes of ESRD, the focus of this work is to define the genetic components responsible for the development of the rapidly progressing renal pathology in the S rat. The assumption (as with all models of human disease) is that knowledge gained using an animal model will foster understanding and treatment of human disease. The aim of manuscript 1 was to conduct a genetic analysis of renal and cardiovascular traits using the S and SHR. Linkage analysis identified quantitative trait loci (QTL) on multiple chromosomes (1, 2, 6, 8, 9, 10, 11, 13, and 19) for urinary protein 3 excretion (UPE) and/or urinary albumin excretion (UAE) with variable time-course patterns. Manuscript 2 sought to perform a second linkage analysis to determine if QTL for UPE and/or UAE would be influenced by salt-loading either by altering the time-course pattern of known QTL or by identifying QTL that were not detected on low-salt. A second aim was to perform congenic strain analysis for several UPE and/or UAE QTL to confirm the linkage analysis (on rat chromosomes 2 6, 9, 11, and 13) and to demonstrate the magnitude of the effect of each QTL once isolated on the S background. The objective of manuscript 3 was to characterize the chromosome 2 congenic strain [S.SHR(2)] by conducting a time-course analysis and establishing onset and progression of renal disease in comparison to both parental strains. A comprehensive approach was employed that examined several renal parameters, histology, electron microscopy, gene expression analysis, and gene pathway analysis to characterize the strain. A second aim was to employ recombinant progeny testing (RPT) to reduce the QTL to a small genomic region to aid in gene identification. 4 LITERATURE Physiology of Kidney Disease Overview of Normal Kidney Physiology The kidney is an important organ involved in eliminating waste products produced by metabolism, such as urea (from protein), uric acid (from nucleic acid), and creatinine (from muscle creatine). Just as important, the kidney plays a role in regulating water and electrolyte balance, acid-base balance (pH), and the secretion of hormones that participate in the regulation of systemic and renal hemodynamics, red blood cell production, and mineral metabolism (Berne 2004). The kidney's ability to perform many of its functions depends on the three fundamental roles: (1) filtration, (2) reabsorption, and (3) secretion. Figure 1 shows a schematic drawing of the functional unit of the kidney, the nephron. Each nephron is composed of a renal corpuscle and a tubule extending from the renal corpuscle. The renal corpuscle contains a compact group of interconnected capillary loops, called the glomerulus and is surrounded by a hollow capsule known as the Bowman’s capsule. The glomerulus filters out large solutes from the blood (cells, proteins, and other large molecules), delivering water and small solutes (glucose, salt, amino acids, and urea) to the renal tubule and ultimately producing urine. The renal tubule consists of different 5 segments, the proximal tubule, the loop of Henle, and the distal tubule, each of which performs unique functions involved in reabsorption and secretion. Figure 1. Nephron Structure A simple diagram of a nephron illustrating its major structural components. The nephron is composed of a renal corpuscle, including the glomerulus and Bowman’s capsule and a