Molecular cloning, enzymatic characterization, developmental expression, and cellular localization of a mouse cytochrome p450 highly expressed in Kidney

A cDNA encoding a new cytochrome P450 was isolated from a mouse liver libra ry. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501- amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s an d is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cyto chrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microso mal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected ce lls metabolize arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrien oic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 degrees C); thus CYP 2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 tran scripts are most abundant in mouse kidney and present at lower levels in li ver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific p eptide detects a protein with the same electrophoretic mobility sis recombi nant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistoc hemical staining of kidney sections with anti-human CYP2J2 IBG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collect ing ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridizat ion confirms abundant CYP2J5 mRNA within tubules of the renal cortex and ou ter medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachi donic acid by the mouse renal epoxygenase(s). Based on these data, we concl ude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to t he oxidation of endogenous renal arachidonic acid pools. In light of the we ll documented effects of epoxyeicosatrienoic acids in modulating renal tubu lar transport processes, we postulate that CYP2J5 products play important f unctional roles in the kidney.