A major manifestation of mesangioproliferative glomerulonephritis in human and experimental model is increased expression of platelet-derived growth factor receptor-2 (PDGFR) in mesangial cells with concomitant proliferation and migration. The mechanism by which increased PDGFR results in the activation of mesangial cells is poorly understood. Our results provide first evidence that in mesangial cells PDGFR-activated phosphatidylinositol (PI) 3 kinase/Akt kinase regulates proliferation and migration. Furthermore, we demonstrate an increase in PDGFR tyrosine kinase activity, resulting in PI 3 kinase and Akt kinase activities in anti-Thy-1-induced mesangioproliferative glomerulonephritis in the rat. Moreover, the non-receptor tyrosine kinase c-Src regulates Ras-GTPase and associates with PI 3 kinase, which regulates Akt kinase activity. In this proposal, using mesangial cells in culture and glomeruli from rats with anti-Thy-1-induced glomerulonephritis, we will test the hypothesis that signal relay from c-Src to PI 3 kinase/Akt through Ras regulates the state of mesangial cell activation. In the preliminary data, we show that PDGF-stimulated Akt kinase activates I:B kinases 1 and 2, resulting in NF:B activation. Importantly, we provide the first evidence that PDGF increases expression of Akt kinase effector Rheb (Ras homolog enriched in brain). We hypothesize that Akt-dependent NF:B-mediated expression of Rheb regulates mesangial cell activation. In the first specific aim, we plan to investigate the molecular mechanisms of action of c-Src and Ras on PDGFR-stimulated PI 3 kinase/Akt signaling in mesangial cells and in glomeruli of rats with mesangioproliferative glomerulonephritis. Also a cross-talk between c-Src and Ras will be studied in mesangial cell activation. In the second specific aim, the roles of I:B kinases 1 and 2 and NF:B in mesangial cell proliferation and migration will be studied. Phosphorylation of I:B kinase isotypes and phosphorylation of their substrates in anti-Thy-1-induced glomerulonephritis will be investigated. In the specific aim 3, role of PI 3 kinase/Akt signaling and its upstream regulators Ras and c-Src in expression of Rheb will be examined. Contribution of NF:B to the Rheb expression will be investigated. To address these specific aims, techniques including immunohistochemistry/immunofluorescence, immunoprecipitation, immunoblotting, reporter transfection assays, electrophoretic mobility shift assays, chromatin immunoprecipitation asays, adenovirus-mediated gene transfer of mutant enzymes, si/shRNA expression and conditional expression of proteins will be used.
Glomerulonephritis is the third most common case of end stage renal disease in the population at large and in veterans. Many forms of proliferative glomerulonephritis are associated with mesangial cell proliferation. Platelet-derived growth factor-mediated signal transduction pathways contribute to the pathology of the disease. The mechanism of the signaling pathways will be studied using mesangial cells in culture and in a rat model of mesangioproliferative glomerulonephritis. The results obtained from the experiments described in the proposal will help designing therapeutic modalities targeting many common forms of proliferative glomerulonephritis.