Increased blood pressure in mice lacking cytochrome P450 2J5

Abstract

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (-/-) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17beta-estradiol levels were reduced in female Cyp2j5 (-/-) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (-/-) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex-specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen-dependent mechanism.

Figure 1.

Development and initial characterization of Cyp2j5 (−/−) mice. A) Gene targeting strategy. The intron/exon organization of the Cyp2j5 gene and the locations of selected restriction sites, PCR primers and Southern probes are shown. In the targeting vector, exon 9 is replaced by the neomycin resistance gene. B) Genotyping by Southern blotting and PCR analysis of tail genomic DNAs. Wild-type (+/+), heterozygous (+/−), and homozygous null (−/−) genotypes are shown. C) Northern blot analysis showing absence of CYP2J5 transcripts in male and female Cyp2j5 (−/−) kidneys. D) RT-PCR analysis confirming absence of CYP2J5 mRNA in male and female Cyp2j5 (−/−) kidneys. E) Immunoblotting showing absence of CYP2J5 protein in male and female Cyp2j5 (−/−) kidneys. There were no differences in the expression of CYP4A, CYP2C, or CYP2F subfamily P450s, or in the expression of eNOS and sEH between the genotypes in either males or females.

Figure 2.

Cyp2j5 (−/−) female mice have increased blood pressure and left ventricular mass. A) Systolic blood pressure measured in conscious mice via tail cuff is significantly higher in Cyp2j5 (−/−) mice than in Cyp2j5 (+/+) littermate controls. When analyzed separately by sex, only female Cyp2j5 (−/−) mice have elevated blood pressure relative to their female Cyp2j5 (+/+) counterparts; blood pressure is similar in male Cyp2j5 (−/−) and male Cyp2j5 (+/+) mice. Values are means ± se; n = 26 Cyp2j5 (−/−) mice (12 females, 14 males); n = 28 Cyp2j5 (+/+) mice (18 females, 10 males). *P = 0.001 vs. all Cyp2j5 (+/+) mice; ^P = 0.0002 vs. female Cyp2j5 (+/+) mice. B) Systolic blood pressure measured by invasive methods is significantly higher in female Cyp2j5 (−/−) mice than in female Cyp2j5 (+/+) littermate controls. Values are means ± se; n = 7–8 mice/group. *P < 0.01 vs. Cyp2j5 (+/+) mice. C) Left ventricular mass measured by two-dimensional M-mode echocardiography is significantly higher in Cyp2j5 (−/−) female mice than in Cyp2j5 (+/+) female littermate controls. Values are means ± se; n = 6–7 mice/group. *P < 0.05 vs. female Cyp2j5 (+/+) mice.

Figure 3.

Cyp2j5 (−/−) female mice have increased proximal tubular transport rates. Proximal tubular transport rates from lumen to bath are significantly higher in Cyp2j5 (−/−) mice than in Cyp2j5 (+/+) littermate controls. When analyzed separately by sex, only female Cyp2j5 (−/−) mice have elevated tubular transport rates relative to their female Cyp2j5 (+/+) counterparts; transport rates are comparable in male Cyp2j5 (−/−) and male Cyp2j5 (+/+) mice. Values are means ± se; n = 14 Cyp2j5 (−/−) mice (7 females, 7 males); n = 14 Cyp2j5 (+/+) mice (7 females, 7 males). *P = 0.03 vs. all Cyp2j5 (+/+) mice; P̂ = 0.003 vs. female Cyp2j5 (+/+) mice.

Figure 4.

Cyp2j5 (−/−) female mice have increased afferent arteriolar responses to angiotensin II and endothelin I. Afferent arteriolar responses to increasing doses of angiotensin II (0.01–10 nM) and endothelin I (1–10 nM) in Cyp2j5 (−/−) mice (solid symbols) and Cyp2j5 (+/+) littermate controls (open symbols) are shown for females (A) and males (B). Results are reported as percentage of control diameter; values are means ± se, n = 8–11 mice/group. *P < 0.05 vs. vehicle, #P < 0.05 vs. Cyp2j5 (+/+) mice.

Figure 5.

Cyp2j5 (−/−) female mice have reduced circulating estrogen levels, and estrogen replacement normalizes their blood pressure and afferent arteriolar response. A) Plasma 17β-estradiol levels are significantly lower in female Cyp2j5 (−/−) mice than in female Cyp2j5 (+/+) littermates. There are no significant differences in 17β-estradiol levels in males and no significant differences in circulating testosterone levels in males or females of either genotype. Values are means ± se; n = 15–17 mice/group for 17β-estradiol; 8–12 mice/group for testosterone; *P = 0.008 vs. female Cyp2j5 (+/+) mice. B) Blood pressure was measured by tail cuff in intact female mice with (hatched bars) or without (dark gray bars) estrogen supplementation, and in ovariectomized mice with (open bar) or without (light gray bar) estrogen supplementation. Value are means ± se; n = 7–12 mice/group. *P < 0.05 vs. Cyp2j5 (+/+) mice of same treatment group; ^P < 0.05 vs. intact mice of same genotype. C) Effect of estrogen replacement on afferent arteriolar responsiveness to angiotensin II in female mice. Female Cyp2j5 (−/−) mice (squares) and Cyp2j5 (+/+) littermate controls (circles) were treated with placebo (open symbols) or 17β-estradiol containing pellets (solid symbols) and afferent arteriolar responsiveness to angiotensin II was determined. Values are means ± se; n = 7 mice/group; *P < 0.05 vs. vehicle; ^P < 0.05 vs. Cyp2j5 (+/+).

Figure 6.

Estrogen biosynthesis and metabolism is unchanged in female Cyp2j5 (−/−) mice. A) Ovarian expression of StAR, CYP11A1, CYP17A1, 3β-HSD1, CYP19A1, and 17β-HSD1 in Cyp2j5 (−/−) and Cyp2j5 (+/+) female mice. Values are means ± se; n = 8 mice/group. B) Metabolism of 17β-estradiol by liver and kidney microsomes form Cyp2j5 (−/−) and Cyp2j5 (+/+) female mice. Retention of authentic standards for 17β-estradiol and its hydroxylated derivatives is marked by arrows. Results are representative of 3 independent experiments with identical results.

Figure 7.

Arachidonic acid metabolism is unchanged in Cyp2j5 (−/−) mice. A) Renal microsomal AA epoxygenase, midchain hydroxylase and ω-hydroxylase activity is similar in Cyp2j5 (−/−) and Cyp2j5 (+/+) mice of either sex. Results are means ± se; n = 3–4 mice/group. B) Urinary levels of AA epoxygenase and hydroxylase metabolites are similar in Cyp2j5 (−/−) and Cyp2j5 (+/+) mice of either sex. Values are means ± se; n = 12 mice/group. C) Cyp2j5 (−/−) female mice have increased CYP2J9 mRNA levels compared to Cyp2j5 (+/+) littermate controls. Values are means ± se; n = 3 mice/group.