Renal Brush Border Membrane Glucose Transport: Regulatory Mechanisms and Adaptation to Diabetic Hyperglycaemia
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RENAL BRUSH BORDER MEMBRANE GLUCOSE TRANSPORT: REGULATORY MECHANISMS AND ADAPTATION TO DIABETIC HYPERGLYCAEMIA. A thesis submitted by Joanne Marks For the degree of Doctor of Philosophy In Physiology In the Faculty of Science University of London Department of Physiology University College London Royal Free Campus Rowland Hill Street 2004 London NW3 2PF ProQuest Number: 10015825 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10015825 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract There is now substantial evidence that implicates hyperglycaemia in the progression of diabetic nephropathy. Studies using mesangial cells have demonstrated that overexpression of the facilitative glucose transporter, GLUT1, is a key factor that predisposes this cell type to glucose-induced damage. Diabetes is also reported to evoke changes in proximal tubule function, yet the underlying mechanisms involved have not been studied in detail. The studies described in this thesis were designed to investigate the effect of streptozotocin-induced diabetes on proximal tubular glucose transport and to determine the cellular mechanisms involved in its regulation. Streptozotocin-induced diabetes was found to increase facilitative glucose transport across the proximal tubule brush border membrane (BBM), a response that could be abolished by normalisation of the blood glucose levels. Changes in transport rate correlated with expression of GLUT2 at the proximal tubule BBM. Experiments investigating the regulation of renal glucose transport demonstrated that sodium-dependent and facilitative glucose transport, are regulated by different intracellular signaling events. The regulation of sodium-dependent glucose transport was found to occur via cAMP-induced insertion of SGLT1 protein into the proximal tubule BBM. In contrast, a pathway involving both protein kinase 0 and intracellular calcium was demonstrated to regulate facilitative glucose transport. The data reported herein provided evidence that GLUT2 expression at the proximal tubule BBM provides a dominant low affinity/high capacity route for glucose reabsorption during hyperglycaemia, which may culminate in glucose-induced damage of this nephron segment. The adaptation of renal glucose transport to experimentally-induced diabetes and the mechanisms involved in the regulation of renal glucose transport display striking similarities to those reported for the small intestine. Acknowledgments The work reported in this thesis was carried out in the Department of Physiology at the Royal Free and University College School of Medicine, London, UK. I am grateful to the St. Peters Research Trust of the Middlesex Hospital and the Wellcome Trust for financial support, without which this work could not have been performed. First and foremost I would like to thank my supervisors; Dr Ted Debnam for his excellent supervision and motivation, and Professor Robert Unwin for his continued financial support and encouragement with the pursuit of new techniques. In addition, I would like to thank the members of the Department of Physiology for their help and encouragement, specifically. Miss Linda Churchill for her outstanding friendship at moments of crisis and advice on immunohistochemistry, and Mr Melvyn Adams for his technical assistance with the brush border membrane vesicle experiments. Last but by no means least my thanks also go to my partner, Mr Alan Walker, my parents and Miss Rachael Wheeler for their patience and invaluable support. Table of Contents Abstract 2 Acknowledgments 3 Table of Contents 4 List of Tables and Figures 9 Abbreviation list 13 Chapter 1. Background 17 1.1. Introduction 18 1.2. Mechanisms of epithelial glucose transport 18 1.3. Sodium Glucose Linked Transporters (SGLTs) 22 1.3.1. Gene and protein properties 22 1.3.2. Transport properties of SGLT proteins 24 1.3.3. Regulation of SGLT transporter proteins 26 1.3.4. Defects in SGLT proteins 27 1.4. Facilitative glucose transport 29 1.4.1. Gene and protein properties 29 1.4.2. Transport properties of GLUT proteins 31 1.4.3. Regulation of facilitative glucose transport 35 1.4.4. Defects in GLUT proteins 38 1.5. Diabetes 39 1.5.1. Clinical implications 39 1.5.2. Pathogenesis of diabetic nephropathy 42 1.5.2.1. Diabetic glomerulosclerosis and tubulointerstitial fibrosis 43 1.5.2.2. Hyperglycaemia-induced glomerular and tubular cell damage. 44 1.5.2.2.1. Role of reactive oxygen species (ROS) in the pathogenesis of diabetic nephropathy. 45 1.5.2.2.2. Involvement of the polyol pathway in the pathogenesis of diabetic nephropathy. 46 1.5.2.2.3. Involvement of PKC activation in diabetic nephropathy. 48 1.5.2.2.4. Formation of Advanced Glycation End Products (AGE’s) as a consequence of hyperglycaemia. 53 1.5.2.2.5. Pathway interactions 55 1.6. Effects of streptozotocin-induced diabetes on intestinal glucose transport 57 1.7. Effect of high glucose concentrations on mesangial cells 58 1.8 Effect of streptozotocin-induced diabetes on renal tubular glucose transport 59 1.9. Aims of thesis 60 Chapter 2. The effect of streptozotocin-induced diabetes on renal glucose transport. 61 2.1. Introduction 62 2.2. Materials and Methods 63 2.2.1. Induction of diabetes 63 2.2.2. Analysis of plasma glucose levels 63 2.2.3. Brush border membrane (BBM) vesicle preparation 64 2.2.3.1. Validation of BBM Vesicle purity 65 2.2.4. Glucose uptake studies 67 2.2.4.1. Validation of BBM vesicle transport capacity 67 2.2.5. Calculations 68 2.2.6. Statistics 69 2.3. Results 70 2.3.1. Animal parameters 70 2.3.2. BBM vesicle validation data 71 2.3.3. Streptozotocin-induced changes in renal BBM glucose transport. 74 2.3.4. Inhibition of GLUT-mediated glucose transport 76 2.4. Discussion 77 2.5. Conclusions 84 Chapter 3. The effects of streptozotocin-induced diabetes on renal facilitative glucose transporter expression and distribution. 85 3.1. Introduction 86 3.2. Materials and Methods 87 3.2.1. Induction of diabetes (as 2.2.1) 87 3.2.2. BBM vesicle preparation 87 3.2.3. Western blotting 87 3.2.4. Localisation of GLUT transporters using immunohistochemistry. 88 3.2.4.1. Tissue preparation for immunohistochemistry without in vivo fixation. 88 3.2.4.2. Preparation of periodate-lysine-paraformaldehyde (PLP) fixative 89 3.2.4.3. Surgical protocol for in vivo fixation 89 3.2.4.4. Immunohistochemistry 90 3.2.4.5. Immunofluorescence using cyanine-2 (Cy2) for confocal imaging. 90 3.2.5. Statistics 91 3.3. Results 92 3.3.1. Western blotting results 92 3.3.2. Immunohistochemistry 95 3.3.2.1. Immunohistochemistry performed on unfixed tissue 95 3.3.2.2. Immunohistochemistry performed on kidneys fixed in vivo with periodate-lysine-paraformaldehyde. 96 3.3.2.3. Confocal imaging of kidney fixed in vivo with PLP 97 3.3.2.4. Immunohistochemistry performed on kidneys perfused with 7 mM glucose prior to in vivo fixation with PLP 101 3.3.2.5. Identification of tubular structures using eosin and Vector Red. 103 3.4. Discussion 106 3.5. Conclusion 115 Chapter 4. Involvement of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways in the differential regulation of SGLT and GLUT-mediated glucose transport. 116 4.1. Introduction 117 4.2. Materials and Methods 119 4.2.1. Incubation of cortical suspension 119 4.2.2. BBM vesicle preparation (as 2.2.3.) and uptake studies (as 2.2.4.) 120 4.2.3. Western blotting (as 3.2.3.) 120 4.2.4. Statistics 120 4.3. Results 120 4.3.1. Viability of cortical preparation 120 4.3.2. BBM vesicle validation 120 4.3.3. Regulation of SGLT-mediated glucose transport by cAMP 121 4.3.4. Regulation of GLUT-mediated transport by the PKC signalling pathway 127 4.4. Discussion 130 4.5. Conclusion 134 Chapter 5. Detection of glucagon and glucagon-like peptide-1 receptor in the rat proximal tubule: Potential role for glucagon in the control of renal glucose transport during STZ-induced diabetes. 136 5.1. Introduction 137 5.1.1. Synthesis of glucagon and its structurally related peptides 137 5.1.2. Physiological function 139 5.1.3. Physiological functions in the kidney 141 5.1.4. The glucagon receptor 142 5.1.4.1 Sites of expression 142 5.1.4.2 Regulation 142 5.1.4.3 Signalling properties 143 5.1.4.4 Binding properties 144 5.1.5. GLP-1 and GLP-2 144 5.1.5.1. Sites of expression 144 5.1.5.2. Regulation 145 5.1.5.3. Signalling properties 146 5.1.5.4. Binding properties 146 5.1.6. Abnormalities in glucagon and glucagon-like-peptide during diabetes 147 5.2. Materials and Methods 149 5.2.1 Effect of glucagon on renal glucose transport 149 5.2.1.1. Incubation of cortical suspension 149 5.2.1.2. BBM vesicle preparation (as 2.2.3.) and uptake studies (as 2.2.4). 149 5.2.1.3. Western blotting (as 3.2.3.) 149 5.2.1.4. Statistics 150 5.2.2. Microdissection study 150 5.2.2.1. Induction of diabetes 150 5.2.2.2. Measurement of plasma cAMP and glucagon levels 150 5.2.2.3.