End stage renal disease producing proteinuria and glomerular sclerosis can be initiated by both immune, e.g., inflammation, and non-immune, e.g., hypertension, injuries. The early events in these injuries are associated with changes in the individual cells of the glomeruli. Whether there is a common mechanism through which these injuries lead to progressive nephron destruction is the basis of this project. We hypothesize that both types of injuries affect glomerular synthesis of arachidonic acid metabolites by the cyclooxygenase pathway and these metabolites contribute to progression. We propose to examine in vitro the effects of immune and non-immune perturbations on the cyclooxygenase pathway in glomerular endothelial and epithelial cells. Specifically, we want (1) to characterize and quantitate the cyclooxygenase products of these cells as a result of conditions which mimic immune (thrombin, IL-1, PDGF and complement activation) and non- immune (stretching/relaxation) injuries; (2) to determine the effects of these conditions on the synthesis and activation of the cyclooxygenase enzyme; (3) to investigate the parameters of enzyme recovery in both cells after irreversible inhibition with aspirin; (4) to evaluate the effect f arachidonate metabolites produced by one cell on another cell; (5) to determine if the glomerular cells communicate with one another through these cyclooxygenase products and if this could be a form of self regulation of cyclooxygenase activation; (6) to examine the signal transduction mechanisms involved with activation of cyclooxygenase and those activated by cyclooxygenase products; and (7) to determine if another endothelial cell located outside the glomerulus, i.e., the endothelial cell of the afferent arteriole displays heterogeneity with respect to cyclooxygenase metabolites. These experiments will require the use of several different experimental methods, including cell culture techniques, biochemical analyses of the cyclooxygenase enzyme and its products, analyses of extracellular matrix components, growth studies, morphological analyses, RNA analyses, permeability studies, fluorescent techniques, and biochemical analyses of intracellular events. This project will rely upon the expertise of the three Cores in the Center Grant: B - the analytical core (quantitative and structural analysis of cyclooxygenase metabolites); C - the morphology and imaging core; and D - the extracellular matrix analysis core. These experiments are all in vitro and are designed to complement the in vivo studies in Projects 1 and 2. It is hoped that the results of these experiments will help in devising interventions to reduce the impact of these injuries and prevent progressive nephron destruction.
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