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Role of Vav2 in Podocyte Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia

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Role of Vav2 in Podocyte Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2016 Role of Vav2 in Podocyte Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia Sabena Conley VCU Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Immunopathology Commons, Laboratory and Basic Science Research Commons, Molecular Biology Commons, Pharmacology Commons, and the Physiological Processes Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/4628 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Role of Vav2 in Podocyte Inflammasome Activation and Glomerular Injury during Hyperhomocysteinemia A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University By Sabena M. Conley Bachelor of Science in Biology Fayetteville State University, 2011 Master of Science in Biology North Carolina Agricultural and Technical State University, 2013 Director: Pin-Lan Li, MD, PhD Professor, Pharmacology and Toxicology Virginia Commonwealth University Richmond, Virginia December 2016 III TABLE OF CONTENTS Acknowledgements .................................................................................................................... VIII Table of Figures ............................................................................................................................. X List of Abbreviations ................................................................................................................... XII Abstract ...................................................................................................................................... XIV Chapter One .................................................................................................................................... 1 Introduction ..................................................................................................................................... 1 1.1 Hyperhomocysteinemia ..................................................................................................... 2 1.1.1 Homocysteine Metabolism ............................................................................................. 3 1.1.2 Models of Hyperhomocysteinemia in Animals .............................................................. 6 1.2 NAPDH Oxidase in the Kidney ..................................................................................... 6 1.2.1 Pathophysiological Role of NADPH Oxidase in the Kidney ......................................... 8 1.2.2 Role of Rac-1 GTPase in NADPH Oxidase Regulation ................................................ 9 1.3 NLRP3 Inflammasome in the Glomeruli ....................................................................... 9 1.3.1 NLRP3 inflammasome ................................................................................................. 10 1.3.2 NLRP3 inflammasome activators ................................................................................ 11 1.3.3 Mechanisms Mediating NLRP3 Inflammasome Activation ........................................ 13 IV 1.3.4 Effector Response of Inflammasome Activation ......................................................... 16 1.4 NLRP3 Inflammasomes in Glomerular Diseases ............................................................ 18 1.4.1 Chronic glomerulonephritis (GN) ................................................................................ 18 1.4.2 Hyperhomocysteinemic nephropathy ........................................................................... 19 1.4.3 Diabetic nephropathy ................................................................................................... 21 1.4.4 Glomerular injury and sclerosis in obesity ................................................................... 22 1.5 The NLRP3 Inflammasome as a Therapeutic Target in Chronic Glomerular Diseases.. 23 1.6 Aims of Study .................................................................................................................. 25 Chapter Two.................................................................................................................................. 28 General Methods ........................................................................................................................... 28 2.1 Hyperhomocysteinemic Mouse Model ........................................................................... 28 2.1.1 In vivo Treatments ........................................................................................................ 28 2.1.2 Ultrasound-microbubble Assisted Plasmid Transfection ............................................. 29 2.2 Culture of Murine Podocytes .......................................................................................... 29 2.2.1 In vitro Treatments ....................................................................................................... 30 2.2.2 RNA interference ......................................................................................................... 30 2.3 High Performance Liquid Chromatography (HPLC) Analysis of Plasma Hcys ............. 31 V 2.4 Confocal Microscopy and Immunofluorescence of Frozen Tissue and Cells ................. 31 2.5 Immunohistochemistry .................................................................................................... 32 2.6 Urinary Protein and Albumin Measurements .................................................................. 33 2.7 Glomerular Morphological Examinations ....................................................................... 33 2.8 Cellular Protein Homogenates ......................................................................................... 34 2.9 Western blot analysis ....................................................................................................... 34 2.10 RNA Isolation and Real Time RT-Polymerase Chain Reaction (RT-PCR) ................. 35 2.11 Caspase-1 Activity, IL-1β Production and Vascular Endothelial Growth Factor (VEGF) Measurements ............................................................................................................... 35 2.12 Rac-1 GTPase Activation Assay ................................................................................... 36 .- 2.13 Electron Spin Resonance (ESR) Spectrophotometry of O2 Production ....................... 36 2.14 Statistical Analysis ........................................................................................................ 36 Chapter Three................................................................................................................................ 38 3.1 Rationale and Hypothesis ................................................................................................ 38 3.2 Results ............................................................................................................................. 40 3.2.1 Confirmation of hHcys-induced Model ....................................................................... 40 3.2.2 In vivo inhibition of Vav2 prevented glomerular NLRP3 inflammasome formation and activation ...................................................................................................................... 42 VI 3.2.3 Glomerular protection by Vav2 inhibition ................................................................... 45 3.2.4 Analysis of Vav2 expression in mouse podocytes ....................................................... 48 3.2.5 Role of Vav2 in Hcys-induced NLRP3 inflammasome formation in podocytes ......... 51 3.2.6 Activation of the NLRP3 inflammasome in vitro ........................................................ 53 3.2.7 Inhibition of Vav2 improves podocytes damage .......................................................... 55 3.2.8 Effect of Rac-1 activation on Hcys-induced NOX activity .......................................... 57 3.3 Summary ......................................................................................................................... 59 Chapter Four ................................................................................................................................. 60 4.1 Rationale and Hypothesis ................................................................................................ 60 4.1 Results ............................................................................................................................. 62 4.2.1 Rac-1 inhibition by NSC23766 ameliorated Hcys-induced inflammasome activation and podocyte injury in vitro ................................................................................................ 62 4.2.2 Mouse Nlrp3 gene deletion and Vav2 inhibition prevented hHcys-induced NLRP3 inflammasome formation and activation in glomeruli ................................................. 68 4.2.3 Administration of NSC23766 protected glomerular podocytes from hHcys-induced dysfunction and injury .................................................................................................. 72 4.3 Summary ......................................................................................................................... 74 Chapter Five .................................................................................................................................
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