Growth Hormone (GH) and the Glomerular Podocyte A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Alison L. Brittain May 2019 © 2019 Alison L. Brittain. All Rights Reserved. 2 This dissertation titled Growth Hormone (GH) and the Glomerular Podocyte by ALISON L. BRITTAIN has been approved for the Department of Biological Sciences and the College of Arts and Sciences by John J. Kopchick Goll-Ohio Professor of Molecular Biology Joseph Shields Interim Dean, College of Arts and Sciences 3 ABSTRACT BRITTAIN, ALISON L., Ph.D., May 2019, Biological Sciences Growth Hormone (GH) and the Glomerular Podocyte Director of Dissertation: John J. Kopchick Growth Hormone (GH) is a peptide hormone secreted from the anterior pituitary that has numerous effects on the kidney. In mice, excess GH is associated with the development of glomerular disease. The glomerulus is the most proximal part of the nephron and contains several different cell types, including the podocyte, which forms part of the filtration barrier between the blood and urine. Previous literature suggests that in vitro, GH may damage the podocyte when administered at high levels. Whether these damaging effects exist in vivo has yet to be determined and is part of the focus of this dissertation. Discussed within this work is a thorough review of the role of GH in the renal system and podocyte physiology. These chapters are followed by a study examining the effects of differing GH concentrations on mouse and human podocytes in vitro, which highlight an ability for GH to induce a moderate amount of epithelial-to-mesenchymal transition (EMT) at 50 ng/mL while reducing apoptosis and cellular viability. The next chapter of this dissertation details the characterization of a novel mouse model: the podocyte-specific GHR gene “knockdown” mouse (PodGHR-/-). The PodGHR-/- mouse was developed and studied under three conditions: the normal aging C57BL6/J phenotype, transgenic bovine (b) GH overexpression, and streptozotocin (STZ)-induced diabetes. Studies of the PodGHR-/- mouse over the course of normal aging showed detrimental changes to the kidney in male mice in the form of increased glomerular 4 filtration rate (GFR), glomerular volume and albumin to creatinine ratio (ACR). When crossed with the bovine GH (bGH) overproducing mouse, PodGHR-/- double transgenic mice (bPodGHR-/-) exhibited decreased gene expression markers of renal damage along with decreased fluid retention, suggestive of some renoprotection. However, female bPodGHR-/- mice exhibited none of these changes but showed increased kidney hydroxyproline content at young and old age which was not associated with other markers of renal decline. When induced to diabetes with STZ, male PodGHR-/- mice did not exhibit overt changes in renal physiology, but also did not exhibit the decreases in podocyte foot process number or increases in foot process width that were seen in wild- type (WT) diabetic mice. The final study of this dissertation examined datasets to determine GH receptor (GHR) gene expression levels in chronic kidney diseases. This study concluded that GHR and insulin-like growth factor 1 (IGF1) gene expression levels are generally decreased in human and mouse models of chronic kidney disease, while IGFBP6 gene expression levels are generally increased. Cumulatively, the studies detailed in this dissertation suggest GH action through the podocyte plays a nuanced role in normal renal physiology and in the development of renal disease, being beneficial to the podocyte in some cases and detrimental in others. 5 PREFACE Many of the chapters of this dissertation are manuscripts in preparation for submission, including chapters 4, 5 and 6. Each of these chapters has a corresponding section in the appendix with supplemental materials, appendices A-C. Chapters 1, 2, 3 and 7 represent stand-alone bodies of work. Chapter 1 and 2 provide a thorough background into growth hormone, its impacts on general renal function and on specific podocyte function. 6 ACKNOWLEDGMENTS To my mentor and director of our laboratory, John Kopchick. I am grateful for your encouragement, kindness and endless support in achieving my goals. To Darlene Berryman, thanks for your friendship and your example as a leader at OUHCOM. To Nick Okada, who I appreciate for his broad knowledge of medical endocrinology and our chats about the difficulties of learning and teaching medicine. To Ed List, whose insight into both scientific and non-scientific predicaments is invaluable. To my fellow graduate students in the Kopchick Laboratory: Silvana Duran-Ortiz, Jonathan Young, Elizabeth Jensen, Kevin Funk, Colin Kruse, Prateek Kulkarni, Mat Buchman, Katie Troike. You made getting a Ph.D. fun… I can’t imagine a better group of people to work with. To post-doctoral researchers Reetobrata Basu and Yanrong Qian, I am grateful for not only your superior scientific insight and technical laboratory skills, but your kindness and friendship over the last four years. I would also like to thank the staff at the Edison Biotechnology Institute. To Lorianne Abdella, Rachel Beha, and the army of students at the front desk at EBI, thank you for everything you do day in and day out to keep our laboratory running. Thanks also goes to the staff of Laboratory Animal Resources, Ed, Eric, Nate, Darla and Tammy. I appreciate your efforts in keeping our mice happy and healthy. Another group of individuals essential to completion of this dissertation were the staff and faculty at OU and OU-HCOM. Thanks go to Julie Buckley at the Histopathology CORE, who assisted in preparing many of my samples for histology, and to Michelle Pate for her assistance with FACS sorting. Thanks also to Ramiro Malgor 7 and Mark Berryman for their histology expertise and guidance. Special thanks also to Karen Coschigano for her feedback and support on this project, and Jeff Thuma for his microscopy expertise. Thank you is also due to Ram Menon of University of Michigan, who supported this project with his clinical insight and expertise in podocyte biology. I would also like to extend a huge thanks to Ryan Woodyard, who worked with me to collect a large portion of the data presented in this project. This project would not have been completed without his help. To my mom, for her support and encouragement during all times, good and bad. To my dad, for encouraging my love of science from a young age. Also, to my sister Melissa and my brother Chris for their lighthearted support. And finally, to my wife Laura, whose intelligence improves my work and whose love improves my life. 8 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Preface................................................................................................................................. 5 Acknowledgments............................................................................................................... 6 List of Tables .................................................................................................................... 10 List of Figures ................................................................................................................... 11 Chapter 1: Growth Hormone (GH) and Renal Function ................................................... 15 Physiology of GH ......................................................................................................... 15 GH and General Renal Function ................................................................................... 21 The Glomerulus ............................................................................................................ 25 GH and Glomerular Function ....................................................................................... 30 GH and Diabetic Nephropathy ..................................................................................... 32 Concluding Remarks ..................................................................................................... 37 Chapter 2: GH and the Podocyte ...................................................................................... 39 The Podocyte ................................................................................................................ 39 Known Mechanisms of GH-Induced Podocyte Damage .............................................. 45 Potential Mechanisms of GH-Induced Podocyte Damage ........................................... 48 Concluding Remarks ..................................................................................................... 57 Chapter 3: Specific Aims .................................................................................................. 59 Chapter 4: The Effect of Varying GH Concentration on Podocyte Health In Vitro ......... 61 Abstract ......................................................................................................................... 61 Introduction ................................................................................................................... 62 Methods ........................................................................................................................ 64 Results ........................................................................................................................... 70 Discussion ....................................................................................................................