Pathologic manifestations of diabetic nephropathy (DN) include glomerular and tubular hypertrophy and matrix protein fibronectin expression. These changes occur concomitant with increased expression of TGFb (transforming growth factor-b) that contributes to the pathogenesis of human and experimental DN. We interrogate the molecular signaling events by which high glucose and TGFb drive the pathologies and provide new potential therapeutic strategies for complications of DN. We have reported that Akt kinase/mTORC1 (mechanistic target of rapamycin complex 1) axis contributes to renal hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial (PTE) cells, and in kidneys of type 1 and type 2 diabetic mice. Recently, a novel longer translational variant of PTEN (phosphatase and tensin homolog deleted in chromosome 10), PTEN-Long, a negative regulator of Akt kinase, has been identified as a secretory and membrane permeable protein. Our preliminary data show markedly reduced levels of PTEN-Long along with increased Akt kinase activity in the kidney cortex of type 1 and type 2 diabetic mice and in high glucose- or TGFb-treated mesangial and PTE cells. Moreover, we show that high glucose and TGFb activate PDGFRb (platelet-derived growth factor-b). We find PDGFRb as a substrate for PTEN-Long; thus downregulation of PTEN-Long results in PDGFRb activation by high glucose and TGFb. Furthermore, in the kidney and in mesangial and PTE cells, we identify Akt-2 as the predominant isotype of Akt kinase that acts downstream of PTEN-Long/PDGFRb. Also, Akt-2 is activated in kidneys of type 1 and type 2 diabetic mice kidneys. We plan to exploit the intrinsic protective function of PTEN-long during the progression of DN. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that hyperglycemia/TGFb-induced inappropriate downregulation of PTEN-Long results in PDGFRb/Akt-2 activation that contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the novel negative regulatory function of PTEN-Long, we will use it as a therapeutic agent for DN. In the first specific aim, we will determine the role of PTEN-Long in hypertrophy and, fibronectin and PAI-1 (plasminogen-activator inhibitor-1) expression. In the second aim, how PTEN-Long forces activation of PDGFRb to regulate mesangial and PTE cell hypertrophy, and matrix protein expression will be investigated. In specific aim 3, we will study the contribution of Akt-2 isotype to hypertrophy and fibronectin/PAI-1 expression in response to high glucose and TGFb in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, morphometry, immunohistochemistry, transfection of expression vectors and siRNAs, administration of recombinant therapeutic protein and small molecule compound will be employed. Our study will estab...