Diabetic nephropathy progressing to chronic renal failure develops in 30- 40% of individuals with Type I diabetes (5-10% of individuals with Type II diabetes). As such, diabetic nephropathy is one of the major, perhaps the major determinant of premature mortality in the Type I diabetic. In addition to the mortality, a devastating toll is exacted from the survivors in the form of accelerated visual loss, lower extremity amputation and painful neuropathy. The renal disease characteristic of diabetes mellitus is marked by glomerular hypertension and hypertrophy and mesangial cell proliferation. These phenomena are likely mediated by vasoactive peptides which activate cells in phospholipase C protein kinase C-dependent and independent manners. We have found that these peptides also stimulate tyrosine phosphorylation in mesangial cells. We propose that these peptides mediate the early steps of diabetic nephropathy. Since the molecular mechanisms by which vasoactive peptides such as endothelin, angiotensin and vasopressin act are unknown, we propose to study the molecular mechanisms of vasoactive peptide signaling; specifically the cascade of protein phosphorylation triggered by endothelin, which couples a heterotrimeric G-protein-linked receptor to Tyr and eventually Ser/Thr phosphorylation. Our hypothesis is that endothelin, acting through its receptors, initiates a protein phosphorylation cascade which couples the receptor to distal effector systems. This cascade begins with the activation of protein kinase C (PKC). PKC, or another Ser/Thr kinase, then phosphorylates and activates a Tyrosine phosphatase specific for inhibitory phosphotyrosine 527 of the product of the proto-oncogene c-scr. c-Src so activated, can, in turn, phosphorylate and activate the focal adhesion kinase (FAK). FAK, c-Src, PKC, or all three, then activate the product of the proto-oncogene c-ras. By an unknown mechanism, Ras then elicits activation of the proto oncogene c-raf-1. c-Raf-1 then activates MAP kinase-kinase, which activates MAP kinases. MAP kinases are coupled to the activation of a number of cellular effector systems including gene expression mediated by the AP-1 transcription factor. Activation by endothelin of, and interactions of individual signalling components (i.e. c-Src, and other Tyrosine kinases, mitogen-activated protein (MAP) kinases, Ras, Raf-1 will be studied using techniques of protein biochemistry and molecular biology in order to confirm their positions in the signalling cascade. Immunoprecipitated or purified components will be used at all times. We will focus our studies of vasoactive peptide signalling in the more homogeneous mesangial cell and NIH3T3 cell systems. These studies will greatly enhance our understanding of a signalling system which, in spite of the clinical toll exacted by renal hypertrophy, has not received as much attention as mitogen signaling. These studies will help in the design of specific, effective treatments for diabetic nephropathy, and for other pathologic conditions characterized by renal hypertrophy.
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