Cytochrome P450 arachidonic acid omega-hydroxylation in the proximal tubule of the rat kidney.

Abstract

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a major cytochrome P450-dependent arachidonate metabolite in the rat kidney. In the present study we characterized the formation of 20-HETE in the proximal tubule, the nephron segment with the highest concentration of cytochrome P450 activities, including P450 arachidonic acid metabolism. Freshly isolated tubules showed a basal formation of 20-HETE, implying that it is an endogenous constituent of the proximal tubule. Conversion of exogenous arachidonic acid to 20-HETE in proximal tubule homogenates was enzymatic and NADPH-dependent (i.e., 0 and 65.5 +/- 1.1 pmol/mg/min in the absence and presence of NADPH, respectively). That its formation was not affected by indomethacin but inhibited following preincubation with 17-ODYA (17-octadecynoic acid) and 7-ER (7-ethoxyresorufin) suggested that a P450 monooxygenase activity was involved in its synthesis. This was further strengthened by the demonstration that antibody raised against the rat cytochrome P450 4A1, a major fatty acid omega-hydroxylase isozyme, inhibited 20-HETE formation, suggesting the involvement of a P450 4A1 or P450 4A1-like activity in this reaction. Pretreatment of rats with clofibrate and dexamethasone, inducers of the P450 4A gene family, yielded a twofold increase in the proximal tubular synthesis of 20-HETE as well as an increase in P450 4A1 mRNA. These results, together with previous demonstrations that 20-HETE vasoconstricts isolated blood vessels, namely, renal microvessels, and affects tubular ion transport, suggest a role for 20-HETE in the regulation of renal vascular tone and transport functions and further stress the importance of understanding the regulation of 20-HETE synthesis in the kidney.