DESCRIPTION (provided by applicant): Vascular endothelial growth factor (VEGF)-dependent albuminuria has been demonstrated in animal models of both type 1 and type 2 diabetes (de Vriese, JASN 2001 and Flyvbjerg, Diabetes 2002). Instead of targeting the VEGF ligand with a neutralizing antibody as in the cited studies above, we decided to target the VEGF signaling receptors with an inhibitor, SU5416, to see if this would replicate the benefits of anti-VEGF antibody therapy. Our Specific Aim 1 was to test if inhibition of VEGF receptor signaling ameliorates albuminuria and restores nephrin expression in diabetic db/db mice. When administered at 2 mg/kg of body weight twice-a-week for eight weeks, SU5416 almost completely prevented albuminuria, reversed glomerular basement membrane thickening, and partly restored nephrin expression toward the non-diabetic baseline. However, because SU5416 blocks all three VEGF receptor kinases and was given systemically, we could not pinpoint which receptor signaling in which renal cell type might have mediated the VEGF-dependent diabetic albuminuria. We hypothesize that the VEGFR-1 receptor in the podocyte was predominantly responsible for the manifestations of diabetic nephropathy that were improved by SU5416 treatment. Thus, Specific Aim 2 tests if deletion of VEGFR-1 specifically in the podocyte will ameliorate albuminuria, GBM thickening, and nephrin protein reduction in the streptozotocin (STZ)-induced diabetic mouse. Targeted deletion of podocyte VEGFR-1 will be accomplished with Cre/loxP technology and the tetracycline-regulatable gene expression system to create a conditional knockout mouse that will be induced with diabetes; this approach should avoid the developmental problems that a conventional allelic knockout might cause. Finally, we will examine the most novel finding from the db/db-SU5416 experiment in that diabetes- induced changes in nephrin expression can be partly corrected by a specific inhibitor of VEGF signaling. Specific Aim 3 will elucidate the mechanisms underlying the VEGF-modulated expression of nephrin by 1) defining the role of VEGF receptors in regulating nephrin expression in cultured podocytes, 2) identifying specific tyrosine residues on the VEGF receptor(s) that are activated by VEGF and the diabetic environment, 3) characterizing the downstream pathways that potentially mediate VEGF-stimulated changes in nephrin expression, including those involving the phosphatidylinositol 3-kinase and protein kinase C pathways, and 4) determining whether VEGF decreases nephrin translation efficiency or increases its protein degradation, as a possible mechanism for the reduced nephrin protein in the face of increased nephrin mRNA (Prelim. Data).