ROLE OF P450 MONOOXYGENASES IN RENAL HEMODYNAMICS

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The long term research goal is the elucidation of mechanisms by which arachidonic acid metabolites influence renal blood flow and glomerular filtration. Renal blood flow and glomerular filtration are efficiently regulated by afferent and efferent arterioles which respond in concert to hormonal and paracrine influences. Renal blood flow regulation arises from disparate signaling mechanisms utilized by pre- and postglomerular arterioles. We have demonstrated that exogenous applications of arachidonic acid vasoconstricts the afferent arteriole and does not influence efferent artiolar tone. This afferent arteriolar vasoconstriction is mediated, in part, by metabolism of arachidonic acid to P450 metabolites. Additionally, activation of arachidonic acid metabolites modulate renal and vascular responses to hormonal and paracrine agonists. Our hypothesis is that P450 metabolites are importantly involved in the renal microvascular responses to paracrine and hormonal agonists. The pre-glomerular vascular vasodilates to the P450 metabolites 11, 12-EET and 14,15-EET and vasoconstricts to 20-HETE and 5,6-EET. The direct actions of P450 metabolites on the postglomerular arterioles are not known. Thus, P450 metabolites may influence afferent arteriolar responses to paracrine and hormonal agonists to a greater extent than efferent arteriolar responses. The above stated hypothesis will be investigated utilizing an in vitro perfused renal vascular bed preparations, freshly isolated and cultured renal vascular smooth muscle cells. Experiments will be performed utilizing the in vitro perfused juxtamedullary nephron preparation which provides direct accessibility to both the pre- and postglomerular arterioles. We will evaluate the postglomerular responses to P450 metabolites and determine the contribution of P450 metabolites to the afferent and efferent arteriolar responses evoked by vasoactive agonists. These studies will employ newly developed stable analogues of P450 metabolites, isoform specific P450 inhibitors, and mice with specific kidney P450 enzymes knocked out. Additionally, isolated perfused vascular bed and vascular smooth muscle cell experiments will determine the cellular-signaling mechanisms by which P450 metabolites vasodilate and vasoconstrict the renal microvasculature. The role of P450 metabolites as intracellular signaling messengers for vasoactive agonists will also be determined. We will evaluate the ability of P450 metabolites to regulate agonist-induced intracellular calcium responses in freshly isolated and cultured renal microvascular smooth muscle cells. Expression vectors of specific P450 enzymatic isoforms will be inserted into cultured renal microvascular smooth muscle cells and the effect of over expression of stereoselective EETs and HETEs on agonist- induced calcium responses in renal microvascular smooth muscle cells will be determined. These vertically integrated studies will provide an improve understanding of the mechanisms by which cytochrome P450 arachidonic acid metabolites influence renal blood flow and glomerular filtration.