Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

Abstract

Background and purpose: ApolipoproteinE-deficient [apoE (-/-)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (-/-) mice.

Experimental approach: ApoE (-/-) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg(-1) ·h(-1) ) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg(-1) ·h(-1) ) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys.

Key results: Ang-(1-7)-treated apoE (-/-) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (-/-) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (-/-) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased.

Conclusion and implications: Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.

Figure 1

(A) The carbachol-induced endothelial-dependent vasorelaxation was impaired in isolated kidneys of apoE (−/−) mice (n = 7), compared with that in kidneys from apoE (+/+) mice (n = 6). Chronic Ang-(1-7) treatment (82 mg·kg⁻¹·h⁻¹) improved endothelium-dependent relaxation in kidneys of apoE (−/−) mice (n = 7). Combining Ang-(1-7) with D-Ala-Ang-(1-7) (125 mg·kg⁻¹·h⁻¹), a specific Mas receptor antagonist, attenuated the beneficial effects of Ang-(1-7) (n = 6). (B) Smooth muscle cell-dependent renal vasorelaxation, tested with the NO donor GSNO, did not differ between apoE (+/+) (n = 6), apoE (−/−) (n = 9) and Ang-(1-7)-treated apoE (−/−) (n = 9) mice. (C) D-Ala-Ang-(1-7) treatment alone showed no effects on carbachol-induced vasorelaxation in apoE (+/+) or apoE (−/−) mice. Data represent means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus apoE (−/−). #P < 0.05, ##P < 0.01 versus apoE (−/−) [Ang-(1-7)]. ‡P < 0.05, ‡‡P < 0.01, ‡‡‡P < 0.001 versus apoE (−/−) [Ang-(1-7) + D-Ala-Ang-(1-7)]. One-way anova for repeated measurements followed by Student's t-test. Ang, angiotensin; apoE, apolipoproteinE; GSNO, S-nitrosoglutathione; NO, nitric oxide.

Figure 2

(A) cGMP production was measured, in the presence of IBMX, in cortical renal slices from apoE (−/−) mice, with or without Ang-(1-7) treatment. Basal and carbachol (CCh)-induced cGMP production was significantly increased in Ang-(1-7)-treated apoE (−/−) (n = 4) mice, compared with untreated apoE (−/−) mice (n = 4). Exogenous NO (IBMX + NO) was provided by DEA-NO (100 µM). Data represent means ± SEM; *P < 0.05, **P < 0.01 versus apoE (−/−), Student's t-test. (B) Urinary excretion (over 24 h) of nitric/nitrate (NO_x) in apoE (+/+) (n = 14), Ang-(1-7)-treated apoE (+/+) (n = 8), apoE (−/−) (n = 7) and Ang-(1-7)-treated apoE (−/−) (n = 7) mice. Ang-(1-7) treatment significantly increased NO_x excretion in apoE (−/−) mice. *P < 0.05 versus apoE (+/+), #P < 0.01, versus apoE (−/−). ‡P < 0.05 versus apoE (+/+) [Ang-(1-7)]. Kruskal–Wallis test followed by Mann–Whitney U-test. (C) Relative expression of eNOS mRNA, respectively, in relation to GAPDH mRNA in isolated preglomerular arteries of apoE (+/+) (n = 12), Ang-(1-7)-treated apoE (+/+) (n = 4), apoE (−/−) (n = 9) and Ang-(1-7)-treated apoE (−/−) (n = 9) measured by quantitative real-time PCR. *P < 0.05, **P < 0.01 versus apoE (+/+), #P < 0.05, versus apoE (−/−), ‡P < 0.01 versus apoE (+/+) [Ang-(1-7)]. Kruskal–Wallis test followed by Mann–Whitney U-test. (D) representative immunoblots of renal cortical eNOS and phosphorylated eNOS in samples from apo (−/−) and Ang-(1-7)-treated apoE (−/−) mice. Ang-(1-7) treatment significantly increased eNOS protein levels (n = 4), expressed as eNOS/β-actin levels, but did not change the ratio between phosphorylated eNOS and total eNOS levels in apoE (−/−) mice (n = 4). *P < 0.05 versus apoE (−/−). Kruskal–Wallis test followed by Mann–Whitney U-test. Ang, angiotensin; apoE, apolipoproteinE; IBMX, 3-isobutyl-1-methylxanthine; NO, nitric oxide.

Figure 3

(A) carbachol-induced endothelial-dependent vasorelaxation was impaired in kidneys of apoE (−/−) mice (n = 7). In the presence of tempol (1 mM), endothelial-dependent vasorelaxation improved in kidneys of apoE (−/−) mice (n = 5). No additional effect of tempol on carbachol-induced vasorelaxation was observed in kidneys of Ang-(1-7)-treated apoE (−/−) mice (n = 6). Data represent means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 versus apoE (−/−) + tempol [Ang-(1-7)]. #P < 0.05, ##P < 0.01 versus apoE (−/−) [Ang-(1-7)]. ‡P < 0.05, ‡‡P < 0.01 versus apoE (−/−) + tempol. One-way anova for repeated measurements followed by Student's t test. (B) H₂O₂ production in renal cortex of apoE (+/+) (n = 9), apoE (−/−) (n = 7) and Ang-(1-7)-treated apoE (−/−) (n = 9) was measured using the Amplex Red assay. H₂O₂ production was increased in renal cortex of apoE (−/−) compared with apoE (+/+) mice. Ang-(1-7) treatment significantly decreased H₂O₂ production in apoE (−/−) mice. The fluorescence values were normalized to protein content in the tissue probes. Urinary 8-isoprostane levels (over 24 h) were measured in apoE (+/+) (n = 16), apoE (−/−) (n = 10) and Ang-(1-7)-treated apoE (−/−) mice (n = 9). Urinary 8-isoprostane levels were increased in apoE (−/−) mice compared with apoE (+/+). Chronic Ang-(1-7) treatment reduced urinary 8-isoprostane levels significantly. Data represent means ± SEM; *P < 0.01 versus apoE (+/+), #P < 0.01 versus apoE (−/−). Kruskal–Wallis test followed by Mann–Whitney U-test. Ang, angiotensin; apoE, apolipoproteinE.

Figure 4

Relative expression of catalase (A), gp91phox (B), p47phox (B), Nox1 (C), Nox4 (C) and p22phox (C) mRNA, respectively, in relation to GAPDH mRNA in isolated preglomerular arteries of apoE (+/+) (n = 12), Ang-(1-7)-treated apoE (+/+) (n = 4), apoE (−/−) (n = 11) and Ang-(1-7)-treated apoE (−/−) (n = 11) measured by quantitative PCR. *P < 0.05, **P < 0.01 versus apoE (+/+). #P < 0.05 versus apoE (−/−), ‡P < 0.05, ‡P < 0.01 versus apoE (+/+) [Ang-(1-7)]. Kruskal–Wallis test followed by Mann–Whitney U-test. Ang, angiotensin; apoE, apolipoproteinE.