VEGF-A is essential for angiogenesis but its non-vascular roles are not well understood. Excessive VEGF-A plays a pathogenic role in diabetic nephropathy and glomerular disorders, the leading causes of end-stage renal disease. Podocyte VEGF-A is required for the development and maintenance of the glomerular filtration barrier. However, the molecular mechanisms whereby the dysregulation of podocyte VEGF-A induces proteinuria are unknown. We determined that VEGF-A regulates podocyte phenotype, cell-cell interactions and glomerular basement membrane (GBM) homeostasis in vivo. Moderate podocyte VEGF164 overexpression in adult mice mimics diabetic nephropathy, whereas in newborn mice it causes a minimal change-like disease. Moreover, podocyte VEGF-A silencing in adult mice induces a dramatic phenotype involving all components of the glomerular filtration barrier. Our data also raise the possibility that VEGF-A acts directly in podocytes; these cells express VEGFR2 in vivo, and VEGF-A regulates slit-diaphragm protein expression in vitro. The objectives of this proposal are to elucidate the mechanisms of VEGF-A control of podocyte phenotype and cell-cell interactions, and to understand the molecular basis of glomerular filtration barrier homeostasis. Our findings led us to hypothesize that 1) moderate overexpression of podocyte VEGF164 induces podocyte effacement; 2) podocyte VEGF164 overexpression in adult mice is sufficient to induce a diabetic nephropathy phenotype; 3) VEGF-A plays a role in the regulation of the slit-diaphragm signaling complex and GBM composition; 4) VEGF-A exocytosis in podocytes is a regulated process. To test our hypotheses, we will examine phenotypes from transgenic mice that overexpress or silence podocyte VEGF-A in a tetracycline regulated manner, focusing on their reversibility, and age-dependent features. We identified VEGFR2 interacting partners in podocytes and will determine whether VEGF-A signaling alters their expression, protein-protein interactions and actin binding properties. We will define the kinetics of VEGF-A secretion by pulse-chase labeling, and examine the regulation of VEGF-A exocytic pathway by Ca+2 and nitric oxide. The proposed experiments should provide novel and important data on the pathogenesis of diabetic nephropathy, and on the signals that control podocyte phenotype and VEGF-A exocytosis. Understanding the molecular basis of renal diseases should enable us to design new strategies for treatment.
The molecular mechanisms of proteinuria in diabetic kidney disease, the leading cause of renal failure, are poorly understood. This proposal focuses on the role of an important angiogenic factor called vascular endothelial growth factor (VEGF-A), in the control of the structure and behavior of the kidney filters. The proposed experiments in transgenic mice should provide new information to improve our understanding of diabetic nephropathy in adults and kidney diseases in children, as well as enable us to design new strategies for treatment of diabetic nephropathy and glomerular diseases. ? ? ?
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