Cellular Regulation of prostaglandin Synthesis

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Renal glomerular mesangial cells (GMC) are important participants in glomerular injury. GMC apoptosis is the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. Resistance of GMC to apoptosis may account for progression of injury to end stage disease since cell proliferation is not counteracted by extra cells deletion. The long term objectives of this application are to elucidate mechanisms of resistance of GMC to apoptosis. This proposal will examine the hypothesis that cyclooxygenase 2 (Cox-2), which generate prostaglandins from arachidonic acid, protects GMC from apoptosis via up-regulation of genes, that inhibit activation activity of nitric oxide synthases (NOS) and regulate efflux preliminary data indicating that 1) Cox-2 inhibits apoptosis of pro-apoptotic Bcl-2 family proteins, control of xenobiotics. The hypothesis is based on our in several types of cells including GMC; 2) Cox-2 up-regulates expression of Protein Inhibitor of NOS (PIN), and multidrug resistance P-glycoprotein (MDR1); 3) apoptotic agents increase cellular NO production. The Bim-dependent Bax activation will be measured by immunofluorescence. The activity of NOS and generation of NO will be evaluated by measurement of arginine to citrullin conversion and by quantification of NO oxidation products with Sievers analyzer in GMC induced to apoptosis in the presence and absence of Cox-2 expression. We will identify products of cyclooxygenase activity in isolated glomeruli and in GMC infected with recombinant adenovirus encoding Cox-2 by Mass Spectrometer coupled with Liquid Chromatography (LC/MS) in the presence and absence of selective Cox-2 inhibitors. To evaluate the role of cyclooxygenase activity in the regulation of gene expression and anti-apoptotic action of Cox-2 we will test the ability of Cox-2 products (identified by LC/MS) to mimic Cox-2 effects and will evaluate the anti-apoptotic properties of Cox-2 mutants which lack the cyclooxygenase activity, but retain peroxidase activity. We will also investigate the effects of blocking the Cox-2 activity on progression of proliferative glomerulonephritis, hypertension and diabetic nephropathy in in vivo experimental models. The significance of this study is that it will provide new insights into the response of GMC to injury and help to understand why elimination of proliferating GMC by apoptosis in the course of progressive renal injury is often inefficient.