Renal cytochrome P450 omega-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride

Abstract

Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.

Figure 1

Representative reverse-phase HPLC chromatograms of [¹⁴C]AA metabolites formed by renal microsomes in untreated control rats (top) and rats treated with L-NAME or NaCl only (middle) or L-NAME and 2% NaCl (bottom).

Figure 2

Concentration-dependent inhibition by sodium nitroprusside of production of HETEs, DHTs, and EETs in rat renal microsomes. Microsomes (300 μg) were incubated with NADPH (1 mM) and indomethacin (10 μM) in the presence or absence of sodium nitroprusside (10 μM, 30 μM, and 100 μM) or 8BrcGMP (100 μM) for 30 minutes at 37°C. Metabolites were analyzed by HPLC. Control enzyme activity was 0.29 ± 0.04 nmol/mg protein per 30 minutes. Results are presented as mean ± SEM; n = 5 separate experiments (n = 3 for 8BrcGMP). *P < 0.05, **P < 0.01. NP, sodium nitroprusside.

Figure 3

Effect of L-NAME on conversion of [¹⁴C]AA to HETEs, DHTs, and EETs. Microsomes from control rats and rats treated with L-NAME and/or 2% NaCl were incubated with [¹⁴C]AA in the presence of NADPH and indomethacin. Values are mean ± SEM obtained from renal microsomes prepared from 5 rats. CYP enzyme activity in rats treated with L-NAME was 0.36 ± 0.06 nmol/mg protein per 30 minutes. *P < 0.05 vs. control.

Figure 4

(a) Representative Western blot showing the expression of CYP4A protein (51 kDa mol wt) in renal microsomes prepared from normal rats (control, lanes 2 and 3) or in those treated with L-NAME (lanes 4, 5, and 6). Samples of liver microsomes treated with clofibrate provided the positive control (lane 1). The immunoblotting procedure used anti-rat CYP4A primary antibody from sheep. (b) Density of the blot as analyzed by densitometric scanning. *P < 0.05, L-NAME (n = 4) vs. control (n = 4).

Figure 5

Concentration of 20-HETE in renal effluents collected after addition of phenylephrine (PE; 7.5 × 10^–7 M) alone (L-NA–) or after combined addition of phenylephrine (2–4 × 10^–7 M) and L-NA (5 × 10^–5 M) to Krebs buffer perfusing isolated kidneys of Wistar rats. Perfusates collected were subjected to HPLC fractionation and quantified by GC/MS analysis as described in Methods. Data are presented as mean ± SEM. *P < 0.05 vs. L-NA–. PP, perfusion pressure after addition of PE with (+) or without (–) L-NA added to the perfusate.