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

Chemicals and reagents

Normal (0.3% (w/w) sodium chloride) and high-salt (8% (w/w) sodium chloride) rat grains were purchased from Xietong Organism (Nanjing, China). PageRuler^™ Prestained Protein Ladder and RevertAid First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Immobilon^™ Western Chemiluminescent horseradish peroxidase (HRP) Substrate (ECL) was purchased from the EMD-Millipore (Billerica, MA, USA). CYP2J2 Rabbit Polyclonal antibody (Catalog number: 13562-1-AP) was purchased from Proteintech Group (Rosemont, IL, USA). The anti-CYP4A rabbit polyclonal antibody (catalog number: ab3573) was purchased from Abcam (Cambridge, UK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, enhanced bicinchoninic acid protein assay kit, and HRP-labelled goat anti-rabbit IgG (H+L) (catalog number: A0208) were purchased from Beyotime (Jiangsu, China). The HRP-polymer anti-mouse/rabbit antibody (catalog number: KIT-5030) and 3,3′-diaminobenzidine substrate were purchased from Maixin Biotech (Fuzhou, China). The UltraSYBR Mixture was purchased from ComWin Biotech (Beijing, China). DNase/RNase-free water was purchased from Solarbio Science & Technology (Beijing, China). Sodium phenobarbital (>98.5% purity) and all other reagents were purchased from Sinopharm Chemical Reagent (Shanghai, China).

Animals

The CYP2C11-null (knockout) rat model was generated using the CRISPR/Cas9 method to insert a two bp GT fragment into exon 6 of CYP2C11 (Wei et al., 2018). Rats were cultured at the Laboratory Animal Center of Jiangsu University (SPF grade). WT S-D rats (male and female, mean body weight 220 ± 10 g) were purchased from the Laboratory Animal Center of Jiangsu University (permit number SKXK (Su) 2013-0011). The breeding room environment was 22–25 °C with a relative humidity of 65% and a 12-h light/dark cycle. All subjects were allowed ad libitum access to water. Animal reproduction and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Jiangsu University.

Animal experiments for measuring blood pressure

For the control (normal diet) condition, 2-month-old rats weighing 220 ± 10 g were divided into four groups, each containing 10 rats. Both sexes and strains (WT and CYP2C11-null) were represented equally. The same grouping method was used for the high-salt condition (Matrougui et al., 1998). Feeding duration was 2 months.

Blood pressure was measured using a classic invasive method (Schenk, Hebden & McNeill, 1992) with modifications. First, 150 mg/kg sodium phenobarbital was administered through an intraperitoneal injection. After disinfection, a small incision was made in the neck to reach the carotid artery. The artery’s upper part (near the brain) was tightened with a surgical line before inserting a thin tube with a three-way valve filled with heparin sodium solution (25 U/mL). The other end of the three-way valve was connected to a bio-signal collector. Blood pressure signals were collected using Chart version 5.0.1 (ADInstruments, NSW, Australia).

Western blots with rat kidney microsomes

Rats were euthanized with carbon dioxide after measuring their blood pressure. Renal microsomes were obtained with differential centrifugation (Ding & Coon, 1990). In brief, 0.5 g of kidney tissue were obtained and rinsed with ice-cold 0.9% sodium chloride solution. The tissue was then homogenized with six mL of KCl-sucrose buffer for 3 × 15 s and centrifuged at 9,000×g for 20 min at 4 °C. The resultant supernatant was then centrifuged at 100,000×g for 1 h at 4 °C. The resulting microsomal pellet was ground to a particle-free state after adding one mL of glycerol-phosphate buffer to a glass homogenizer and stored at −80 °C for use. The microsomal protein concentration was determined using a bicinchoninic acid kit (Beyotime, Jiangsu, China). Proteins (five µg) (Gu et al., 2003) were then subjected to 10% SDS-PAGE with duplicate adjacent lanes, and transferred to an Immobilon-P Transfer Membrane (EMD-Millipore, Burlington, MA, USA). Duplicate SDS-PAGE was performed and Ponceau S was used to ensure that sample proteins were loaded equally (Wang et al., 2017). The common reference proteins, β-actin and GAPDH, were not used because they are absent from renal microsomes. Membranes were blocked in 5% skim milk (Murraygoulburn Cooperative, Brunswick, VIC, Australia), probed with primary antibodies (1:1,000), and then incubated with species-specific HRP-conjugated secondary antibodies (Beyotime, 1:1,000). Signals were detected using an ECL system (Clinx Science Instruments, Shanghai, China). The western blotting analysis was repeated three times and images were analyzed with Image J software (version 1.46, National Institutes of Health, Bethesda, MD, USA).

Quantitative polymerase chain reaction analysis

Because CYP2C23 and CYP2C24 antibodies were unavailable, renal mRNAs were analyzed to determine expression of these CYPs. Renal mRNA levels of CYP2J2 and CYP4A1 were also analyzed. Total RNA was isolated using a TRIzol reagent (ComWin Biotech, Beijing, China) following the manufacturer’s protocol. Complementary DNA was synthesized from five μg of RNA using a cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and eluted in a final volume of 20 μL. Quantitative polymerase chain reaction (qPCR) was performed using the LightCycler96 Real-time PCR system (Roche, Basel, Switzerland). Primers (Table 1) for CYP2C23, CYP2C24, and β-actin were synthesized by Sangon Biotech (Shanghai, China). The thermocycling profile was 95 °C for 600 s, followed by 46 cycles of 95 °C for 15 s, 61 °C for 20 s, and 72 °C for 30 s. The 2^−ΔΔCt method was used to calculate the relative changes in target gene expression from the qPCR results, using β-actin as the reference gene.

Immunohistochemistry

Fixed kidney sections were de-waxed and rehydrated following published methods (Becker et al., 2006). Endogenous peroxidase was inactivated through 0.3% hydrogen peroxide treatment for 10 min. Treatment with phosphate-buffered saline for 5 min blocked non-specific binding. Sections were then incubated with primary antibodies (CYP2J2, 1:100; CYP4A, 1:300) overnight at 4 °C, followed by the secondary antibody (Maixin, 1:20) for 20 min. Sections were stained for 2 min with 3,3′-diaminobenzidine substrate, then rinsed with distilled water before being dehydrated and covered in a neutral resin/xylene mixture. Section images were obtained under 200× magnification in five randomly selected microscopic fields and analyzed in Image-Pro Plus version 6.0 (Media Cybernetics, Rockville, MD, USA). The integral optical density (IOD) and total area of the positive reaction region in each field of vision were automatically generated by the software. Their ratio was the mean density (IOD/area).

Statistical analysis

All data are expressed as means ± standard deviation. Student’s t-test was used to determine differences in means. Analyses were performed in Prism 6.0 (GraphPad, La Jolla, CA, USA), and P < 0.05 was considered statistically significant.

High-salt diets elevated blood pressure in CYP2C11-null rats

Under a normal lab diet, CYP2C11-null and WT rats did not differ in blood pressure (n = 10). Among CYP2C11-null rats, male and female average blood pressure was 115.2 ± 11.5 mm Hg and 118.1 ± 8.4 mm Hg, respectively. Among WT, male and female blood pressure was 120.4 ± 11.4 mm Hg and 120.0 ± 16.5 mm Hg. However, a high-salt diet significantly increased blood pressure (male: 130.3 ± 16.8 mm Hg, female: 156.8 ± 15.9 mm Hg; n = 10) among CYP2C11-null rats compared with a normal diet (male: 115.2 ± 11.5 mm Hg, P < 0.05, female: 118.1 ± 8.4 mm Hg, P < 0.01; n = 10). The increase in CYP2C11-null females was significantly higher than in CYP2C11-null males and WT females (128.5 ± 21.8 mm Hg, n = 10, P < 0.01) (Fig. 1).

Western blots of CYP2J2 and CYP4A in rat kidney microsomes

Under the normal diet, the expression of CYP2J2 in CYP2C11-null male rats was significantly higher than that in WT male rats. In addition, the expression of CYP4A 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 and CYP4A expression in both male and female CYP2C11-null rats compared with WT rats (P < 0.01) (Fig. 2).

Renal mRNA expression

After knocking out the CYP2C11 gene, renal CYP2C23, and CYP2C24 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. The mRNA levels of these genes in the two rat strains under the normal diet were consistent with the results of western blotting. Under the high-salt diet, renal expression of CYP2J2 and CYP4A1 mRNA in CYP2C11-null male rats was significantly higher than that in WT male rats (P < 0.01, n = 9) (Fig. 3).

Immunohistochemistry

Positive staining for CYP2J2 was observed in the renal cortex and outer medulla of all groups. The positive reaction of CYP2C11-null female rats treated with the normal diet was enhanced compared to WT female rats. The positive reaction and staining intensity of the CYP2C11-null rats treated with a high-salt diet were increased compared to WT rats. Renal sections of all groups were observed to be positively stained for CYP4A in interlobar, arcuate, and interlobular arteries. However, no positive enhancement was observed in the sections (Fig. 4A). Under a normal diet, renal CYP2J2 expression 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 (Fig. 4B).