CYP2C11 played a significant role in down-regulating rat blood pressure under the challenge of a high-salt diet

Abstract

Background: Arachidonic acid (AA) is oxidized by cytochrome P450s (CYPs) to form epoxyeicosatrienoic acids (EETs), compounds that modulate ion transport, gene expression, and vasorelaxation. Both CYP2Cs and CYP2Js are involved in kidney EET epoxidation.

Methods: In this study, we used a CYP2C11-null rat model to explore the in vivo effects of CYP2C11 on vasorelaxation. For 2 months, CYP2C11-null and wild-type (WT) Sprague-Dawley rats were either fed normal lab (0.3% (w/w) sodium chloride) or high-salt (8% (w/w) sodium chloride) diets. Subsequently, an invasive method was used to determine blood pressure. Next, western blots, quantitative PCR, and immunohistochemistry were used to determine renal expression of CYPs involved in AA metabolism.

Results: Among CYP2C11-null rats, a high-salt diet (females: 156.79 ± 15.89 mm Hg, males: 130.25 ± 16.76 mm Hg, n = 10) resulted in significantly higher blood pressure than a normal diet (females: 118.05 ± 8.43 mm Hg, P < 0.01; males: 115.15 ± 11.45 mm Hg, P < 0.05, n = 10). Compared with WT rats under the high-salt diet, western blots showed that CYP2C11-null rats had higher renal expression of CYP2J2 and CYP4A. This was consistent with the results of immunohistochemistry and the qPCR, respectively. The two rat strains did not differ in the renal expression of CYP2C23 or CYP2C24.

Conclusion: Our findings suggested that CYP2C11 plays an important role in lowering blood pressure under the challenge of a high-salt diet.

Keywords: Blood pressure; CYP2C11-null rats; CYP2J2; Epoxyeicosatrienoic acids; High-salt diet.

Figure 1. High-salt diets elevated blood pressure in CYP2C11-null rats.

A high-salt diet significantly increased blood pressure among CYP2C11-null rats compared with a normal diet (female, P < 0.01, male, P < 0.05; n = 10). The increase in CYP2C11-null females was significantly higher than in CYP2C11-null males and WT females (P < 0.01, n = 10). All data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01.

Figure 2. Western blots of CYP2J2 and CYP4A in rat kidney microsomes.

Representative western blots showing 57-kDa CYP2J2 and 50-kDa CYP4A protein bands in CYP2C11-null rats and WT rats in the normal (A and B) and high-salt (C and D) diet. Proteins were loaded in duplicate adjacent lanes. Under the normal diet, the expression of CYP2J2 (E) in CYP2C11-null male rats was significantly higher than that in WT male rats. In addition, the expression of CYP4A (G) in CYP2C11-null female rats was significantly lower than that in WT female rats (P < 0.01). The high-salt diet significantly increased renal CYP2J2 (F) and CYP4A (H) expression in both male and female CYP2C11-null rats compared with WT ones. Integrated density of the WT rats was regarded as 100%. All data were expressed as means ± standard deviation. **P < 0.01.

Figure 3. Quantitative PCR of renal mRNA expression.

After knocking out the CYP2C11 gene, renal CYP2C23 (A and B), and CYP2C24 (C and D) mRNA did not differ between CYP2C11-null and WT rats of either sex (n = 9). The results showed that the high-salt diet treatment had no effects on renal CYP2C23 or CYP2C24 mRNA expression. Under the normal diet, the mRNA expression of CYP2J2 (E) in CYP2C11-null male rats was significantly higher than that in WT male rats (P < 0.01, n = 9), and the mRNA expression of CYP4A1 (G) in CYP2C11-null female rats was significantly lower than that in WT female rats (P < 0.01, n = 9). Under the high-salt diet, renal expression of CYP2J2 (F) and CYP4A1 (H) mRNA in CYP2C11-null male rats was significantly higher than that in WT male rats (P < 0.01, n = 9). All data were expressed as means ± standard deviation. **P < 0.01.

Figure 4. Immunohistochemistry (cytoplasmic, positive) of CYP2J2 and CYP4A (magnification, ×200).

(A) Positive staining for CYP2J2 (A–H) was observed in the renal cortex and outer medulla of all groups. Under the normal diet, the positive reaction of D group was enhanced compared to B group. Under the high-salt diet, the positive reaction and staining intensity of G and H groups were increased compared to E and F groups. In tissue sections with arrow marks, the arrow a indicated the cortical staining area and the arrow b indicated the outer medulla stained area. Renal sections of all groups were observed to be positively stained for CYP4A (I–P) in interlobar, arcuate, and interlobular arteries. However, no positive enhancement was observed in the sections. (B) Under the normal diet, renal CYP2J2 expression (Q) was significantly elevated in CYP2C11-null females compared with WT females (P < 0.01, n = 5). We observed the same between-strain difference under the high-salt diet, except in both sexes (P < 0.01, n = 5). No significant difference was found for CYP4A expression (R). Density (mean) = integrated optical density (IOD)/Area. All data were expressed as means ± standard deviation. **P < 0.01.