Pathogenesis of Diabetic Nephropathy

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DESCRIPTION (provided by applicant): Diabetic nephropathy is one of the major complications of diabetes, where cellular dysfunctions induced by hyperglycemia have certain degree of similarities in different compartments of the kidney, i.e., glomerular vs tubulo-interstitial or vascular. The cells affected may be derived from epithelial or mesenchymal progenitors; and at times hyperglycemia may induce a phenotypic change with epithelial-mesenchymal transformation. In target cells the high glucose activates various intracellular pathways that are similar except for minor variations. These pathways have been mainly studied in various cell types of the glomerulus, while the information available in the literature for the tubular compartment is limited. Intriguingly, our suppression subtraction analyses of kidneys of "neonatal" diabetic mice indicate that the majority of the differentially up- regulated genes are expressed in the renal tubules, e.g., GTP binding protein Rap1b, guanine nucleotide exchange factor Epac1 and ubiquitin fusion protein UbA52 and etc. The Rap1b was found to exert downstream effects leading to up-regulation of extracellular matrix (ECM) proteins. Interestingly, the co- expressed Epac1 happens to positively regulate the Rap1b activity. A recently described negative regulator of Rap1b is Cbl, a proto-oncogene. All the three molecules are modulated by high glucose ambience. Conceivably, both Cbl and Epac1 exert their effects on Rap1b to strike a balance in its activity to dampen the complications of diabetes related to ECM biology. To attest to this contention various experiments are proposed under the following specific aims: I. Modulation of Rap1b, Cbl and Epac1 expression in animal models of diabetes will be investigated and correlated with the disease activity. Attempts will be made to gauge the activity of Rap1b on the expression of tubulo-interstitial ECM proteins. II. Effect of high glucose ambience on their expression will be investigated in cell culture systems. Efforts will be devoted to tease out the modulation of Rap1b by Cbl/Epac1 under high glucose by employing various agonists, inhibitors, dominant constructs & shRNA/siRNA oligos. III. Mechanism(s) by which high glucose activate, directly or indirectly, Cbl and Epac1, will be investigated by carrying out promoter analysis, gel shift and chromatin immunoprecipitation assays. Role of glycative, oxidant/antioxidant and osmotic stresses in their activation will be investigated. IV. Other molecules that could activate Rap1b will be identified by using co-immuno- precipitation procedures and yeast-two-hybrid systems. V. Since Epac1 is up-regulated in the "neonatal" diabetic mice and exhibits differential expression during development, its modulation of nephrogenesis under high glucose ambience will be investigated. It is hoped that these studies would enhance our understanding of the "Renal Tubulo-interstitial ECM Pathobiology" relevant to diabetic nephropathy.