TXNIP AND THE GAPDH-SIAH1 SIGNALLING PATHWAY IN DIABETIC NEPHROPATHY by Hui Ze (Lexy) Zhong A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medicine, Graduate Department of Institute of Medical Sciences University of Toronto © Copyright by Hui Ze (Lexy) Zhong (2019) TXNIP AND THE GAPDH-SIAH1 SIGNALLING PATHWAY IN DIABETIC NEPHROPATHY Hui Ze (Lexy) Zhong Master of Science (2019) Graduate Department of Institute of Medical Sciences University of Toronto ABSTRACT Thioredoxin-interacting protein (TXNIP) is markedly upregulated by high glucose (HG) and contributes to Diabetic Nephropathy (DN) development partly by inhibiting the endogenous antioxidant thioredoxin. We postulate that this contributes to the nitrosylation and oxidation of GAPDH, which has been found in neuronal cells to promote GAPDH-Siah1 binding and nuclear translocation, leading to apoptosis. The goal of this study was to determine if TXNIP regulates GAPDH-Siah1 signalling and if DN can be prevented by blocking this pathway. In vitro results show that HG caused significant nuclear localization of both GAPDH and Siah1 and upregulation of apoptotic markers in cultured wildtype mesangial cells (MCs), but not TXNIP-/- (KO) MCs. In vivo, deprenyl, an inhibitor of GAPDH-Siah1 binding, protected diabetic mice from developing various structural and functional markers of DN. These data suggest that the GAPDH-Siah1 pathway has a pathogenic role in DN and is downstream of TXNIP signalling. ii ACKNOWLEDGEMENTS My successes during my graduate studies would not be possible without the help and support of my supervisor, colleagues, friends, and family. I am forever indebted to my supervisor, Dr I. George Fantus, who helped open my eyes to the holistic nature of the medical sciences and pushed me along this journey of self-discovery to appreciate both the small picture and big picture at large—from interactions at the molecular level to implications in other fields. Thank you for your patience, your support, and your guidance. You have inspired me to become a better scientist and an overall better thinker. Furthermore, I would also like to thank my program advisory committee members, Dr James Dennis and Dr Adria Giacca, for their continual support and expertise. The insightful feedback they provided has helped me better focus my project and keep me on track. I would also like to thank the previous and current members of the Fantus lab who have helped me throughout my journey and made this experience enjoyable. I would like to thank Dr Anu Shah for helping me get started on my MSc project and for performing some of the preliminary experiments that laid the foundations for this project. I am also deeply grateful for Dr Ling Xia for her technical support, guidance, and company in the lab. In addition to the Fantus Lab, I would also like to acknowledge the other labs at the University Health Network (Toronto, ON), Mount Sinai Hospital (Toronto, ON), and McGill University Health Centre (Montreal, QC), for their technical support and experimental protocols. Last but not least, I would like to thank my family and friends for their support and encouragement throughout my years of study. This accomplishment would not have been possible without them. Thank you all. iii STATEMENT OF CONTRIBUTIONS The experiments in this thesis were conducted by Hui Ze (Lexy) Zhong with the help of colleagues. Dr Ling Xia assisted with mouse colony maintenance, animal harvests, and some immunohistochemistry staining and analyses. Dr I George Fantus helped with the design of studies and interpretation of results. iv TABLE OF CONTENTS CHAPTER 1: INTRODUCTION 1.1. Normal Kidney Anatomy and Physiology .............................................................................................. 2 1.1.1. General Structure and Function ................................................................................................. 2 1.1.2. Glomerular Filtration Barrier ...................................................................................................... 3 1.1.3. Mesangial Cells ........................................................................................................................... 7 1.2. Diabetic Nephropathy ........................................................................................................................... 8 1.2.3. Histopathological Presentation in Diabetic Nephropathy .......................................................12 1.3. Thioredoxin-interacting Protein in Diabetes Mellitus ......................................................................... 15 1.3.1. Background ..............................................................................................................................15 1.3.2. TXNIP is elevated in diabetes ...................................................................................................16 1.3.3. TXNIP is implicated in Diabetic Nephropathy ..........................................................................17 1.4. Thioredoxin-interacting Protein in Diabetic Nephropathy ................................................................. 18 1.4.1. Oxidative and Nitrosative Stress ..............................................................................................18 1.4.2. Fibrosis .....................................................................................................................................22 1.4.3. Inflammation ............................................................................................................................23 1.4.4. Endoplasmic Reticulum Stress .................................................................................................24 1.4.5. Apoptosis ..................................................................................................................................25 1.5. TXNIP and the GAPDH/Siah1 Pathway ................................................................................................ 27 1.5.1. GAPDH Background ..................................................................................................................27 1.5.2. GAPDH/SIAH1 Pathway ............................................................................................................29 1.5.3. Regulation by the Thioredoxin and Glutathione Systems ........................................................33 1.5.4. Experimental Inhibition of the GAPDH/Siah1 Pathway ...........................................................36 1.5.5. Experimental techniques for the study of TXNIP function .......................................................40 1.6. Project rationale, hypothesis, and specific aims ................................................................................. 42 1.6.1. Rationale ..................................................................................................................................42 1.6.2. Hypothesis ................................................................................................................................42 1.6.3. Specific Aims.............................................................................................................................42 CHAPTER 2: METHODS 2.1. Glomeruli Isolation and Culturing of Primary Mesangial Cells ........................................................... 45 2.2. Cell Culture .......................................................................................................................................... 46 2.3. Nuclear/Cytoplasmic Fractionation and Extraction ............................................................................ 47 v 2.4. Mice and Metabolic Studies ................................................................................................................ 47 2.5. Blood Profiling and Urinalysis ............................................................................................................. 49 2.6. Electron Microscopy............................................................................................................................ 49 2.7. Tissue Histology and Immunohistochemistry ..................................................................................... 50 2.8. Western Blotting ................................................................................................................................. 51 2.9. Statistical Analyses .............................................................................................................................. 51 CHAPTER 3: RESULTS 3.1. TXNIP and the GAPDH/Siah1 Pathway ................................................................................................ 54 3.1.1. TXNIP, GAPDH, and Siah1 protein levels in total cell lysates ...................................................54 3.1.2. GAPDH and Siah1 nuclear translocation ..................................................................................55 3.1.3. Caspase-3 cleavage ..................................................................................................................57 3.2. Effects of deprenyl on nephropathy in STZ-induced diabetic mice .................................................... 57 3.2.1. Metabolic profiles of the DBA/2J mice ....................................................................................57 3.2.2. Histological Analyses ................................................................................................................62