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PACSIN2 and Septin 7 in Diabetic Kidney Disease: Roles in Intracellular Trafficking and Actin Cytoskeleton Organisation in Podocytes

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PACSIN2 and Septin 7 in Diabetic Kidney Disease: Roles in Intracellular Trafficking and Actin Cytoskeleton Organisation in Podocytes Research Program for Clinical and Molecular Metabolism and Department of Pathology, Faculty of Medicine, University of Helsinki, Helsinki, Finland. Doctoral School in Health Sciences Doctoral Programme in Biomedicine PACSIN2 and septin 7 in diabetic kidney disease: Roles in intracellular trafficking and actin cytoskeleton organisation in podocytes Vincent Dumont ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine of the University of Helsinki, for public examination in the PIII lecture hall in Porthania building, on Saturday the 18th of January 2020 at 12 noon. Helsinki 2020 Supervised by Professor Sanna Lehtonen Department of Pathology Research Program for Clinical and Molecular Metabolism Faculty of Medicine, University of Helsinki Helsinki, Finland Reviewed by Professor Jaakko Patrakka Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Center Department of Laboratory Medicine Karolinska Institutet at Karolinska University Hospital Huddinge Stockholm, Sweden and Professor Jari Ylänne Department of Biological and Environmental Sciences University of Jyväskylä Jyväskylä, Finland Official opponent Professor Nicole Endlich Greifswald Medical School University of Greifswald Greifswald, Germany ISBN 978-951-51-5720-1 (paperback) ISBN 978-951-51-5721-8 (PDF) https://ethesis.helsinki.fi/en Unigrafia Oy © Cover image by Omar Escalante CONTENTS LIST OF ORIGINAL PUBLICATIONS ........................................9 ABBREVIATIONS ................................................................... 10 ABSTRACT ............................................................................. 12 1. INTRODUCTION ................................................................. 14 2. REVIEW OF THE LITERATURE ......................................... 15 2.1. KIDNEY ................................................................................................................... 15 2.1.1. Structure and function of the kidney .............................................................. 15 2.1.2. Glomerulus and glomerular filtration barrier ............................................... 16 2.1.2.1. Structure and function of the glomerulus ................................................ 16 2.1.2.2. Glomerular endothelium .......................................................................... 17 2.1.2.3. Glomerular basement membrane ............................................................ 17 2.1.2.4. Glomerular mesangial cells ...................................................................... 17 2.1.2.5. Glomerular visceral epithelial cells or podocytes ................................... 18 2.1.2.5.1. The slit diaphragm .............................................................................. 19 2.1.2.5.2. Actin cytoskeleton in podocytes ........................................................ 20 2.1.2.5.3. Intracellular trafficking in podocytes ................................................ 21 2.1.3. Kidney tubules .................................................................................................. 23 2.2. DIABETES MELLITUS ............................................................................................. 23 2.2.1. Types of diabetes .............................................................................................. 23 2.2.2. Insulin: the key player in the development of diabetes ................................ 24 2.2.2.1. Insulin signalling ....................................................................................... 25 2.2.2.2. Glucose transporter (GLUT) 4 trafficking .............................................. 25 2.2.2.3. Insulin resistance ...................................................................................... 26 2.2.4. Complications of diabetes ............................................................................... 26 2.3. DIABETIC KIDNEY DISEASE .................................................................................. 27 2.3.1. Typical course of diabetic kidney disease and clinical features.................... 27 2.3.2. Risk factors for the progression of diabetic kidney disease ......................... 27 2.3.3. Pathology of diabetic kidney disease .............................................................. 28 2.3.3.1. Glomerular alterations .............................................................................. 28 2.3.3.2. Tubular changes ........................................................................................ 29 2.3.3.3. Vascular defects......................................................................................... 30 2.3.4. Mechanisms underlying the development of diabetic kidney disease ........ 30 2.3.4.1. Molecular pathways deregulated in diabetic kidney disease ................. 30 2.3.4.2. Inflammation ............................................................................................ 30 2.3.4.3. Hypoxia ...................................................................................................... 31 2.3.4.4. Alterations of the glomerular cell crosstalk ............................................ 31 2.3.4.5. Mechanisms driving podocyte injury in diabetic kidney disease .......... 31 2.3.4.5.1. Downregulation or mislocalisation of nephrin ................................. 32 2.3.4.5.2. Actin cytoskeleton deregulation ........................................................ 32 2.3.4.5.3. Defects in insulin signalling .............................................................. 32 2.3.6. Animal models of diabetes and diabetic kidney disease ............................... 33 2.3.6.1. Induction of diabetes by streptozotocin .................................................. 33 2.3.6.2. Naturally occurring diabetes in rodents .................................................. 34 2.3.6.3. Transgenic mouse models ........................................................................ 35 2.4. PACSIN2 ............................................................................................................... 35 2.4.1. Identification and tissue distribution of PACSINs ........................................ 35 2.4.2. Protein domains of PACSINs .......................................................................... 35 2.4.3. Functions of PACSINs ..................................................................................... 37 2.4.3.1. Role of PACSIN2 in endocytosis and intracellular trafficking ............... 37 2.4.3.2. Regulation of the cytoskeleton by PACSINs ........................................... 38 2.4.3.3. Regulation of receptor signalling by PACSIN2 ....................................... 38 2.4.4. PACSIN2 in the kidney .................................................................................... 39 2.4.5. PACSIN2 in diseases ........................................................................................ 39 2.5. SEPTIN 7 ................................................................................................................. 40 2.5.1. Septin family and structure of septin 7 ........................................................... 40 2.5.2. Function of septin 7 in the kidney .................................................................. 41 2.5.3. Other functions of septin 7 and its association with diseases ...................... 41 3. AIMS OF THE STUDY ......................................................... 43 4. MATERIALS AND METHODS .............................................44 4.1. RODENT MODELS (STUDIES I–IV) ...................................................................... 44 4.2. HUMAN MATERIAL (STUDIES II–IV) .................................................................. 44 4.3. METABOLIC MEASUREMENTS .............................................................................. 45 4.3.1. Blood glucose and FFA measurements (studies I–III) ................................. 45 4.3.2. Albumin excretion and estimation of the glomerular filtration rate (studies I and II) ....................................................................................................................... 45 4.4. CELL CULTURE ...................................................................................................... 45 4.4.1. Mouse podocytes (studies II and IV) .............................................................. 45 4.4.2. Human podocytes (study III) ......................................................................... 46 4.5. OVEREXPRESSION AND KNOCKDOWN STRATEGIES ........................................... 47 4.5.1. Transient overexpression (studies II–IV) ...................................................... 47 4.5.2. Silencing gene expression using siRNA (studies II and IV) ......................... 48 4.6. TREATING PODOCYTES WITH HIGH GLUCOSE, PALMITATE, PKC INHIBITOR OR INSULIN (STUDIES II–IV) ...................................................................................... 49 4.7. LYSIS OF CELLS AND TISSUES,WESTERN BLOTTING ......................................... 49 4.7.1. Isolation and lysis of glomeruli (studies II–IV) ............................................. 49 4.7.2. Lysis of cells (studies II–IV)............................................................................ 49 4.7.3. Western blotting (studies II–IV) .................................................................... 50 4.8. HISTOLOGY, IMMUNOHISTOCHEMISTRY AND IMMUNOFLUORESCENCE......... 50 4.8.1. Histological stainings (studies I, II and IV) ..................................................
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