Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice

Abstract

Renal cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP-derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild-type littermate controls, an attenuated afferent arteriole constrictor response to endothelin-1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild-type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N-nitro-l-arginine methyl ester and indomethacin. In a separate experiment, a high-salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high-salt-induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild-type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension-induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.

Figure 1.

Development and initial characterization of Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice. A) Diagram of the Tr constructs. B, C) Expression of human CYP2J2 and CYP2C8 mRNA by quantitative RT-PCR in primary aortic and renal endothelial cells (B; n=1 endothelial cell pool/genotype group) and aorta and kidney tissue homogenates (C; n=11–12 mice/group) is significantly higher in Tr mice compared to wild-type (WT) littermates. *P < 0.001 vs. WT. D) Representative immunoblot of microsomal fractions isolated from whole-kidney homogenates from Tie2-CYP2J2-Tr (lines 0–8), Tie2-CYP2C8-Tr (lines 0–7), and WT littermates demonstrate higher CYP2J2 and CYP2C8 protein expression in Tr mice compared to WT littermates. Recombinant CYP2J2 and CYP2C8 protein are included as positive controls. E, F) Primary renal endothelial cells were isolated from Tie2-CYP2J2-Tr (lines 0–8), Tie2-CYP2C8-Tr (lines 0–7), and WT littermates (n=3 isolations/group) and stimulated with A23187. Concentrations of 11,12- and 14,15-EET and DHET (stable EET metabolite), and the regioisomer sum total, are significantly higher in Tr mice in primary endothelial cell media (E) and plasma (F) compared to WT littermates (n=5–6/group). *P < 0.05 vs. WT.

Figure 2.

Endothelial CYP2J2 and CYP2C8 expression in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice. A, B) Endothelial CYP2J2 (A) and CYP2C8 (B) mRNA expression was evaluated in coronal sections through the central region of the kidney by in situ hybridization in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice, respectively, and wild-type (WT) littermate controls. Panels show representative bright-field autoradiograph images from emulsion coated slides using antisense and sense CYP2J2 and CYP2C8 probes. Black dots indicate hybridization (binding) of the probe to tissue RNA. Blue arrows indicate the presence of a vessel. Red arrows identify autoradiographic grains indicative of endothelial cell labeling. Scale bars = 20 μm. C, D) CYP2J2 (C) and CYP2C8 (D) immunostaining was completed using the anti-CYP2J2pep1 and anti-CYP2C8 antibodies, respectively. Panels show images from representative sections of aorta and renal cortex. Arrows indicate endothelial cell staining. No immunostaining was observed with normal rabbit serum in Tie2-CYP2J2-Tr, Tie2-CYP2C8-Tr, or WT mice (not shown).

Figure 3.

Afferent arteriolar responses to acetylcholine and endothelin-1 in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice. Afferent arteriolar responses to acetylcholine (A), endothelin-1 (B), and endothelin-1 in the presence of 14,15-EEZE (C, D) in Tie2-CYP2J2-Tr, Tie2-CYP2C8-Tr, and wild-type (WT) littermate control mice. Arteriole diameters at each dose are expressed as percentage of baseline (control). Number of mice in each group is indicated. *P < 0.05 vs. WT; **P < 0.05 for both Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr vs. WT.

Figure 4.

Blood pressure in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice under basal conditions and in the presence of nitric oxide synthase and cyclooxygenase inhibition. A) Daily mean arterial pressure (MAP) in Tie2-CYP2J2-Tr (n=21), Tie2-CYP2C8-Tr (n=19), and wild-type (WT) littermate control (n=29) mice was invasively measured by an indwelling catheter under basal conditions and during administration of L-NAME (1 mg/ml) and indomethacin (20 μg/ml) in the drinking water. B) Averaged MAP in each group under basal (ANOVA; P=0.061) and L-NAME/Indo-treated (ANOVA; P=0.036) conditions. *P < 0.05 vs. WT.

Figure 5.

Blood pressure and renal injury in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr mice in the presence of angiotensin/high-salt-induced hypertension. A) In the angiotensin/high-salt (ANG/HS) hypertension model, systolic blood pressure was measured noninvasively by tail-cuff plethysmography. Changes (ΔBP) from baseline at 4 wk in Tie2-CYP2J2-Tr (ANG/HS, n=6), Tie2-CYP2C8-Tr (ANG/HS, n=7), and wild-type (WT) littermate mice (ANG/HS, n=10) are provided. Control WT mice treated with in high-salt (HS) alone (n=11) are also included. B, C) On d 28, urinary protein excretion over 24 h (B) and histological glomerular injury scores (C) were measured in each group (n=5–7/group). *P < 0.05 vs. WT mice treated with ANG/HS. D) Representative images of renal histological sections in each group demonstrate the relative degree of glomerular injury.