Contribution of cytochrome P450 4A isoforms to renal functional response to inhibition of nitric oxide production in the rat.

Many studies have clearly indicated that products of the ω/ω-1 hydroxylase pathway of cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism are synthesized in the kidney and exert profound effects therein (McGiff & Quilley, 1999). The major ω-hydroxylation product of AA in tubular and vascular structures of the renal cortex and outer medulla of the rat is 20-hydroxyeicosatetraenoic acid (20-HETE) (Omata et al. 1992a 1992b; Imig et al. 1996), an important regulator of renal vascular tone, tubular reabsorption and the control of arterial pressure (see McGiff & Quilley, 1999). ω-Hydroxylation of fatty acids, including AA has been characterized and shown to be catalysed by enzymes of the CYP4A family. In the rat, four isoforms have been identified: CYP4A1, −4A2, −4A3 and −4A8, and mRNA for all four have been identified in the kidney (Kimura et al. 1989a 1989b; Stromstedt et al. 1990). These isoforms, although sharing 66–98 % homology and a common unique catalytic activity, i.e. hydroxylation at the ω-carbon, are localized to different renal structures. For example, CYP4A1, −4A3, and −4A8 are highly expressed in proximal tubules (Stromstedt et al. 1990; Hardwick, 1991; Omata et al. 1992a 1992b) and the renal microvasculature (Wang et al. 1999). On the other hand, CYP4A2, the constitutively expressed isoform especially in male rats (Kimura et al. 1989a 1989b; Sundseth & Waxman, 1993), is preferentially expressed in the outer medulla and thick ascending limb of the loop of Henle and is believed to be the major isoform in the kidney (Kimura et al. 1989a 1989b; Sundseth & Waxman, 1993). The differential expression of the AA ω-hydroxylating enzymes along the nephron may therefore contribute to the selective effects of 20-HETE on tubular function. The regulation of CYP4A is the subject of active interest and hypolipidaemic agents have been shown to selectively increase the expression of CYP4A1 and −4A3 isoforms (Hardwick, 1991). A potential exists also for a differential regulation of renal CYP4A by nitric oxide (NO) but this has not been actively explored. It has been demonstrated that NO inhibits CYP enzymes including the 4A family (Oyekan et al. 1999) by forming stable iron-nitrosyl complexes at the catalytic haeme binding site in this enzyme (Minamiyama et al. 1997; Mehl et al. 1999). This inhibition is corroborated by the observations that NO donors inhibit the synthesis of 20-HETE by renal microsomes (Alonso-Galicia et al. 1997; Oyekan et al. 1999) and that inhibition of NO production increased CYP4A expression and renal efflux of 20-HETE in the perfused rat kidney (Oyekan et al. 1999) and in the isolated proximal tubule of the normal rat (Escalante et al. 2002) and in the renal microvessels of the pregnant rat (Wang et al. 2002). In addition, incubation of recombinant CYP4A protein with NO donors revealed a differential formation of iron-nitrosyl complexes between different CYP4A isoforms (Wang et al. 2002). Since the capacity for 20-HETE production and therefore its renal effect are determined by the expression of specific CYP4A isoforms, for which expression differs between vascular and tubular sites in the kidney, we therefore hypothesize that the renal effect of NO inhibition (to increase CYP4A expression) will depend on the extent of NO regulation of specific CYP4A isoform(s). The availability of antisensense technology in the form of molecular probes has facilitated a definition of the functional role of each of the isoforms of the CYP4A family, permitting recognition of their separate and overlapping spheres of activity and, therefore, of the physiological significance of each isoform. Antisense technology has been used in other studies to demonstrate the roles of CYP4A1 versus -4A2 isoforms in the regulation of blood pressure in normotensive and spontaneously hypertensive rats (Wang et al. 1999, 2001). In the present study, we evaluated changes in renal haemodynamics and excretory function in rats that were treated with antisense oligonucleotides directed against CYP4A1, -A2 and -A3.