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

Abstract

20-Hydroxyeicosatetraenoic acid (20-HETE), a major renal eicosanoid, regulates renal function and contributes to renal responses following withdrawal of nitric oxide (NO). However, the role of 20-HETE-synthesizing isoforms in renal function resulting from NO inhibition is unknown. The present study evaluated the role of cytochrome (CYP)4A1, -4A2 and -4A3 isoforms on renal function in the presence and absence of NO. Antisense oligonucleotides (ASODN) to CYP4A1, -4A2 and -4A3 reduced 20-HETE synthesis and downregulated the expression of CYP4A isoforms in renal microsomes. Nomega-L-nitromethyl arginine ester (L-NAME, 25 mg kg(-1)), an inhibitor of NO production, increased mean arterial blood pressure (MABP, Delta = +18 to 26 mmHg), reduced renal blood flow (RBF, Delta = -1.8 to 2.9 ml min(-1)), increased renal vascular resistance (RVR, Delta = +47 to 54 mmHg ml(-1) min(-1)), reduced glomerular filtration rate (GFR), but increased sodium excretion (UNaV). ASODN to CYP4A1 and -4A2 but not -4A3 reduced basal MABP and RVR and increased basal GFR, while ASODN to CYP4A2 significantly reduced basal UNaV suggesting a differential role for CYP4A isoforms in the regulation of renal function. ASODN to CYP4A2 but not -4A1 or -4A3 blunted the increase in MABP by L-NAME (38 +/- 9 %, P < 0.05). ASODN to CYP4A1, -4A2 and -4A3 attenuated the reduction in RBF and the consequent increase in RVR by L-NAME with a potency order of CYP4A2 = CYP4A1 > CYP4A3. ASODN to CYP4A1 and -4A2 but not -4A3 attenuated L-NAME-induced reduction in GFR, but ASODN to all three CYP4A isoforms blunted the L-NAME-induced increase in UNaV (CYP4A3 > CYP4A1 >> CYP4A2). We conclude from these data that CYP4A isoforms contribute to different extents to basal renal function. Moreover, CYP4A2 contributes greatest to haemodynamic responses while CYP4A3 contributes greatest to tubular responses following NO inhibition. We therefore propose that NO differentially regulates the function of CYP4A1, -4A2, and -4A3 isoforms in the renal vasculature and the nephron.

Figure 1. ω-Hydroxylase activity and CYP4A expression in rats treated with sense (S) or antisense (AS) oligonucleotides to CYP4A isoforms

A,ω-hydroxylase activity in whole renal microsomes from rats treated with scrambled (sense) or antisense (antisense) oligonucleotides to CYP4A1, −4A2 and −4A3. Activity was measured as conversion of [1-¹⁴C]arachidonic acid (AA) to 20-HETE. B, (upper panel) a typical immunoblot of microsomal samples from rats with scrambled (sense) or antisense (antisense) to CYP4A1 (4A1), −4A2 (4A2) and −4A3 (4A3). The lower panel illustrates a densitometric analysis of the blots obtained from samples from rats treated with antisense oligonucleotides to CYP4A1 (CYP4A1-AS), −4A2 (CYP4A2-AS) and −4A3 (CYP4A3-AS). †P < 0.05 versus sense.

Figure 4. L-NAME-induced increase in renal vascular resistance in rats treated with scrambled (S) or antisense (AS) oligonucleotides (ODNs)

Responses in male rats to treatment with ODNs to CYP4A1 (CYP4A1-S and CYP4A1-AS) (A) and CYP4A2 (CYP4A2-S and CYP4A2-AS) (B) and with scrambled or antisense oligonucleotides to CYP4A2 (CYP4A3-S, CYP4A3-AS) in female rats (C). *P < 0.05 versus scrambled ODN-treated rats.

Figure 3. Reductions in renal blood flow evoked by acute administration of L-NAME (25 mg kg⁻¹) in rats treated with scrambled (S) or antisense (AS) oligonucleotides (ODNs)

Responses in male rats to treatment with ODNs to CYP4A1 (CYP4A1-S and CYP4A1-AS)(A) and CYP4A2 (CYP4A2-S and CYP4A2-AS) (B) and to scrambled or antisense oligonucleotides to CYP4A2 (CYP4A3-S and CYP4A3-AS) in female rats (C). *P < 0.05 versus scrambled ODN-treated rats.

Figure 2. Changes in mean arterial blood pressure in response to L-NAME (25 mg kg⁻¹) in rats treated with scrambled (S) or antisense (AS) oligonucleotides (ODNs)

Responses in male rats to treatment with ODNs to CYP4A1 (CYP4A1-S and CYP4A1-AS) (A) and CYP4A2 (CYP4A2-S and CYP4A2-AS) (B) and in female rats to treatment with ODNs to CYP4A2 (CYP4A3-S and CYP4A3-AS) (C). The arrows show the point of injection of L-NAME. *P < 0.05 versus scrambled ODN-treated rats.

Figure 5. Reduction in GFR and natriuresis in rats treated with sense (S) or antisense (AS) oligonucleotides to CYP4A isoforms

Glomerular filtration rate (GFR) (A) and sodium excretion (U_NaV) (B) in rats treated with scrambled ODNs to CYP4A isoforms (sense) before (basal) or after acute administration of L-NAME (25 mg kg⁻¹) in rats treated with scrambled (S) or antisense (AS) oligonucleotides (ODNs) to CYP4A1 (CYP4A1) and CYP4A2 (CYP4A2) in male rats or of scrambled or antisense oligonucleotides to CYP4A2 in female rats (CYP4A3). †P < 0.05 versus sense *P < 0.05 versus basal.