20-tIETE, an -hydroxylated metabolite of arachidonic acid (AA), is a potent vasoconstrictor and Na+/K+-ATPase inhibitor in the kidney. Renal 20-HETE formation is catalyzed by P450 enzymes of the CYP4A gene subfamily in animals, but it is not known whether the same P450s produce this eicosanoid in humans. Thus, we examined AA -hydroxylation in microsomes from human kidney cortex, and characterized the P450s involved. RenM microsomes oxidized AA to a single major metabolite, namely 20-HETE, at rates of 0.42 0.2 nmol/min/mg protein (n = 9). Western blotting revealed abundant expression in renal microsomes of two CYP4A gene family enzymes, CYP4All and while other AA-oxidizing P450s, including CYP2C8, CYP2C9, and CYP2EI, were not detected. Incubation of kidney microsomes with anti-CYP4All (IgG:microsomal protein ratio = 0.4 mg/mg) inhibited 20-HETE formation by 31.4) whereas incubation with anti-CYP4F2 at the same IgG:protein ratio inhibited this reaction by 56.5% (n = 2). Immunocytochemical analysis of normal kidney sections showed specific expression of CYP4All and CYP4F2 in 2 and 3 segments of proximal tubule epithelia in each of the 6 subjects examined. Our results demonstrate that CYP4All as well as CYP4F2 mediate AA whydroxylation to the vasoactive and natriuretic eicosanoid 20-HETE in human kidney. Considering their proximal tubular localization, these CYP4 enzymes may partake in pivotal renal functions, including the regulation of salt and wter balance.
|State||Published - 1998|