The podocyte synthesizes a major portion of the glomerular basement membrane (GBM) to which it anchors via the alphabeta31 integrin, and it maintains the integrity of the filtration barrier that restricts the permeability of proteins. Podocyte dysfunction contributes greatly to the pathogenesis of diabetic glomerulopathy, as reflected by altered GBM composition, decreased podocyte number, and albuminuria. The mechanisms of these changes are incompletely understood, but up-regulation of Vascular Endothelial Growth Factor (VEGF) production by the podocyte may be implicated. The target cell of podocyte-derived VEGF is believed to be the glomerular endothelium, but exciting recent data showing that podocytes possess functional VEGF receptor(s) strongly suggest that VEGF can act on the podocyte itself in an autocrine loop. In fact, over activity of this VEGF loop can exert untoward effects on the podocyte by stimulating production of alpha3 (IV) collagen and decreasing expression of the alpha3 integrin subunit. These cell-matrix interactions may contribute to GBM thickening, podocyte detachment/loss, and altered macromolecular permeability. Our hypothesis is that high levels of intrarenal Angiotensin II and Transforming Growth Factor-( TGF-beta), two of the principal culprits in diabetic kidney disease, cause podocyte overproduction of VEGF that in turn acts in an autocrine loop to promote GBM thickening, cell detachment and albuminuria.
Aim 1 will examine the mechanisms whereby diabetic mediators, notably Angiotensin II, stimulate VEGF expression in differentiated mouse podocytes and assess the intermediary role of the up-regulated TGF-beta/Smad pathway in accentuating VEGF expression.
Aim 2 will delve into the mechanisms of how the up-regulated EGF autocrine loop causes collagen IV stimulation and a3(1 integrin suppression in podocytes; the VEGF receptor(s) involved and the downstream signal transduction pathways will be elucidated.
Aim 3 will explore with in vivo experiments the extent to which individual cytokine systems participate in the development of albuminuria and other lesions of diabetic glomerulopathy; kidney structure and function will be examined in diabetic vs. non-diabetic transgenic mice produced by Cre/LoxP recombination to yield podocyte-specific knockouts of candidate signaling receptors that engage Angiotensin II, TGF-beta, and VEGF (i.e., AT1A, TGF-beta type II receptor, and VEGFR-l/Flt-1, respectively). Selectively ablating the ability of glomerular podocytes to respond to each of these relevant stimuli will allow us to dissect the relative contribution of these systems to podocyte dysfunction in diabetes.
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