The renal metabolism of arachidonic acid (AA) produces a diverse array of biologically active lipid mediators which potently regulate renal hemodynamics and ion transport along the nephron. Cyclooxygenase mediated prostanoid formation has been extensively studied and an important physiologic role, established. The kidney also possess a robust cytochrome P450 (CYP450) system which rapidly metabolizes arachidonate to several bioactive lipids including the epoxyeicosatrienoic acids (EETs) and omega/omega-1 AA metabolites (the hydroxyeicosatetraenoic acids or HETEs). Accumulating evidence supports an intimal association between renal CYP450 activity and the regulation of systemic blood pressure. Inhibition of renal CYP450 activity induces hypertension and the CYP450 AA metabolite, 20-HETE inhibits CI-absorption in the thick ascending limb (TAL). 20-HETE is actively formed by only a few CYP450s, and the CYP4A family appear to be the relevant isoforms in the kidney.
In specific aim #1 we propose to characterize the role of specific CYP4A isoforms and their omega/omega-1 AA metabolites in regulating ion transport to murine microperfused thick ascending limbs (TAL). Studies in mice with targeted disruption of CYP4A10 and 4A14 should allow us to define roles for specific CYP450 isoforms in regulating TAL ion transport. The expression of CYP4As has recently been shown to be under the control of a fatty acid activated transcription factor, known as peroxisome proliferator activated receptor alpha (PPARalpha). We have recently mapped the intra-renal expression of three PPAR isoforms (PPARalpha, gamma, and delta) and find PPARalpha is highly expressed in proximal tubule and TAL, whereas PPARgamma is primarily expressed in the collecting duct. Preliminary data suggests activation and TAL, whereas PPARgamma enhances Na+ absorption in collecting duct.
In specific aim #2, we propose to characterize the effects of PPAR activation on epithelial Na+ transport in cultured cortical collecting ducts (CCDs) as well as in whole animal Na+ balance studies. Finally, the production of the EETs is mediated by distinct families of CYP450s including CYP2C and CYP2B isoforms Evidence suggests 5,6-EET and 14,15-EET potently regulate transport in the renal collecting duct.
In specific aim #3 we propose to further examine the role of CYP450 AA epoxygenases in regulating ion transport along the nephron. Roles for the prostaglandin EP1 receptor in mediating the effect of 5,6-EET in microperfused CCDs will be tested, as will effects of adenoviral mediated CYP450 expression on transport in cultured CCDs. It is our hope these studies will help define the role of CYP450-AA metabolism in regulating renal ion transport, its impact on Na+ excretion, and ultimately the control of systemic blood pressure.
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