A major manifestation of diabetic nephropathy is increased expression of transforming growth factor-beta (TGFBeta), concomitant with glomerular cell including mesangial cell hypertrophy and increased synthesis of matrix proteins such as fibronectin. The mechanism by which hyperglycemia and/or TGFB result in hypertrophy and increased expression of fibronectin is poorly understood. Our preliminary data provide the first evidence that in mesangial cells, TGFBeta activates phosphatidylinositol 3-kinase (PI 3-K) and Akt kinase in a tyrosine phosphorylation-dependent manner. Furthermore, we demonstrate that Akt kinase regulates mesangial cell hypertrophy and fibronectin expression. Moreover, high concentration of glucose or TGFBeta reduces expression of the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten), which inhibits PI 3-K signaling. To explore the role of this signaling pathway in diabetic nephropathy, we studied rats with streptozotocin-induced diabetes. We demonstrate that diabetes is associated with reduced expression of PTEN in renal cortex including glomeruli. In this proposal, using mesangial cells in culture and renal tissues from rats with streptozotocin-induced diabetes, we will test the hypothesis that PTEN as well as concerted actions of downstream targets of Akt kinase regulate mesangial hypertrophy and fibronectin expression. In the first specific aim, we plan to investigate the Src tyrosine kinase as a candidate signaling molecule that regulates PI 3-K and Akt activity as well as mesangial cell fibronectin expression. The role of NFKappaB as a target of Akt kinase, induced by TGFB or high glucose in regulating fibronectin expression will be studied. In the second specific aim, we will examine the role of a recently identified downstream target of Akt, a tumor suppressor protein, tuberin, along with mTOR (mammalian target of rapamycin) and ribosomal $6 kinase (S6K) in TGFBeta- and hyperglycemia-induced hypertrophy. In the specific aim 3, we will study the role of PTEN in regulating mesangial cell hypertrophy and fibronectin expression in mesangial cells and in a rat model of streptozotocin-induced diabetes. To address these specific aims, techniques including immunoprecipitation, immunoblotting, reporter: ransfection assays, adenovirus-mediated gene transfer of mutant kinases and conditional expression of proteins will be used.
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