Over the past six years the NAOPH dependent cytochrome P-450 metabolism of arachidonic acid has assumed increasing importance. This Program Project Grant represents a unique collaboration designed to characterize the biochemistry of this pathway and its overall importance to the kidney. Six independent but complimentary projects are proposed: 1) Transport effects of arachidonate P-450 metabolites in defined nephron segments. There is now good reason to believe that P-450 metabolites have important functional effects on sodium transport in proximal tubule, organic ion secretion in proximal tubule, salt transport in the medullary thick ascending limb of Henle, and vasopressin modulated water flow in the cortical collecting tubule. Project 1 will pursue the sites and mechanisms of these direct tubular actions. 2) characterization of renal cytochrome P-450 arachidonate metabolism. This project will characterize the biochemistry of arachidonic acid oxidation in terms of enzymology, metabolite formation, structural characterization, regulation, and localization in the kidney. These studies are critical to the functional studies. 3) Role of cytochrome P-450 arachidonate metabolites in human platelets and their aggregation. Platelet aggregation and release of biologically active products plays an important role in renal disease. Based on the recent demonstration of potent effects of a P-450 metabolite on platelet aggregation, this project will identify P-450 oxygenation products in human platelets, determine their esterification in platelet lipids, examine their effects on agonist induced platelet aggregation, examine their release from activated platelet and determine the mechanism by which they inhibit platelet aggregation. 4) In vivo studies of P-450 metabolites. Studies will test their effects on renal function in vivo utilizing clearance and micropuncture studies in the rat. Preliminary data on profound modulation of P-450 metabolism in experimental models such as unilateral nephrectomy, diabetes, and pregnancy will be pursued. 5) Characterization of a model cell system to study cytochrome P-450 arachidonate metabolites to test their effects on signal transduction pathways, and to determine whether they serve as intracellular second messenger. 6) Characterization and synthesis of renal P-450 eicosnoids, development of strategies to synthesize the metabolites and inhibitors for biological evaluation. We feel that these projects represent an ideal integration of techniques and investigators to address the biological importance of renal cytochrome P-450 arachidonate metabolism.
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