The Role of Hepatocyte Nuclear Factor 4a in Renal Proximal Tubule Development A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Molecular and Developmental Biology Graduate Program University of Cincinnati College of Medicine 2020 by Sierra S. Marable B.S. Valdosta State University, 2012 Committee Chair: Joo-Seop Park, PhD Elif Erkan, MD S. Steven Potter, PhD Katherine Yutzey, PhD Aaron Zorn, PhD i Abstract Nephron segmentation is a poorly understood process that forms four distinct regions of the nephron: the renal corpuscle, the proximal tubule, the loop of Henle, and the distal tubule. Each segment has a specialized function necessary for proper renal filtration. The proximal tubule is the main site of active reabsorption in the nephron, responsible for approximately 65% of total reabsorption. Proximal tubule dysfunction has been implicated in many nephrology disorders, such as renotubular acidosis and Fanconi renotubular syndrome (FRTS), which can lead to chronic kidney disease in adulthood. In order to provide better treatments, it is necessary to understand the molecular mechanisms underlying proximal tubule development. The goal of this dissertation is to determine the molecular mechanisms regulating proximal tubule development. Hepatocyte nuclear factor 4 alpha (Hnf4a) is a transcription factor that is only expressed in the proximal tubules of the kidney. A heterozygous mutation in the HNF4A gene has been identified in patients with FRTS. FRTS is defined as generalized proximal tubular dysfunction characterized by polyuria, polydipsia, glucosuria, proteinuria, and phosphaturia. This suggests that Hnf4a is a key regulator of proximal tubule development and function. However, the role of Hnf4a in proximal tubule development is unknown and there was no mouse model for kidney-specific Hnf4a deletion. Therefore, to investigate the role of Hnf4a in the kidney, we generated two mouse models of nephron-specific Hnf4a deletion. The first mouse model studied featured mosaic deletion of Hnf4a in Six2-expressing nephron progenitors. In this model, Hnf4a mutant mice showed a paucity of proximal tubules in the developing kidney. This paucity led to mutant mice developing FRTS-like symptoms. Hnf4a mutant cells were ii unable to mature into LTL-high proximal tubules cells, indicating that Hnf4a is required for proximal tubule maturation. In the second mouse model, we investigated the results of complete deletion of Hnf4a in the Osr2-expressing proximal portion of the nephron. In this model, there was complete loss of LTL-high mature proximal tubules causing early postnatal lethality in the mutant mice. These mutant mice showed arrested proximal tubule development with an increase in Cdh6+ progenitors. Transcriptomic and genomic analyses identified transporters genes and metabolism genes are the primary targets of Hnf4a in the kidney. Overall, these results showed that Cdh6+ cells in the developing kidney are proximal tubule progenitors and that Hnf4a is required for the transition of the Cdh6+ progenitors to LTL-high mature proximal tubules. Hnf4a regulates this differentiation via expression of transporter and fatty acid metabolism genes. iii iv Acknowledgements I would first like to thank my advisor, Joo-Seop Park PhD, for guiding me over the course of my graduate education. Your open-door policy and willingness to listen and discuss have been essential to my progress. Thank you to the members of the Park lab, Eunah Chung PhD and Patrick Deacon, who helped me design and troubleshoot experiments. Thanks to the members of the Reddy Lab, Pramod Reddy MD, Elizabeth Mann PhD, and Melissa Mogle, for their invaluable insights. I also am very grateful for the assistance of my thesis committee, for their constructive advice and their roles in my development as a scientist. I must also acknowledge previous mentors who have helped me along my journey to graduate school, especially Mark Blackmore PhD, Thomas Manning PhD, and Colin Bishop PhD. Many thanks to my fellow MDB students, especially Talia, Kelsey, Alex, Marshall, Joe, Sandra, and Valeria, who have been with me on this graduate journey from the beginning. I also thank the MDB program for the education I have received as graduate student. Last and most important, I must thank my family and friends for their unwavering love and support throughout the years. I come from a large extended family and it is wonderful to come from such a supportive group of people. To my best friends Jeana and Lacey, thank you for your support over the many years of our friendship. Thank you to my grandmothers for always spoiling me. And finally, thank you to my mother, Ginny, for your unconditional love and for everything you taught me. Though you are gone, your love and wisdom live on within me. v Table of Contents Abstract ........................................................................................................................................................... ii Acknowledgements .......................................................................................................................................... v List of Tables and Figures .................................................................................................................................. 3 CHAPTER 1: INTRODUCTION ............................................................................................................................. 4 Kidney Anatomy and Function................................................................................................................................... 4 Kidney Development .................................................................................................................................................. 4 Nephrogenesis ........................................................................................................................................................... 6 The Proximal Tubule .................................................................................................................................................. 7 Hepatocyte Nuclear Factor 4 Alpha (Hnf4a) ........................................................................................................... 11 Rationale ................................................................................................................................................................. 12 Scope of Dissertation ............................................................................................................................................... 13 CHAPTER 2: Hnf4a deletion in the mouse kidney phenocopies Fanconi renotubular syndrome ......................... 18 Abstract ................................................................................................................................................................... 19 Introduction ............................................................................................................................................................. 20 Results ..................................................................................................................................................................... 21 Discussion ................................................................................................................................................................ 29 Methods................................................................................................................................................................... 32 Acknowledgments ................................................................................................................................................... 37 Figures ..................................................................................................................................................................... 38 CHAPTER 3: Hnf4a-mediated regulation of proximal tubule progenitors in the mouse kidney ........... 47 Abstract ................................................................................................................................................................... 48 Significance .............................................................................................................................................................. 49 Introduction ............................................................................................................................................................. 50 Methods................................................................................................................................................................... 52 Results ..................................................................................................................................................................... 55 Discussion ................................................................................................................................................................ 62 Author contributions ............................................................................................................................................... 66 Acknowledgments ................................................................................................................................................... 66 Figures ....................................................................................................................................................................