Altered endothelial mitochondrial Opa1-related fusion in mouse accelerates age-associated vascular and kidney damage

Abstract

Cardiovascular diseases are the major cause of death worldwide, and their frequency increases with age in association with kidney damage. As a reduction in fusion protein optic atrophy type 1 (Opa1) level in endothelial cells (ECs) decreases the vascular response to flow and increases oxidative stress in perfused kidneys, we hypothesized that reduced Opa1 expression contributes to vascular aging. We used male and female mice with ECs specific Opa1 knock-out (EC-Opa1), and littermate wild-type (EC-WT) mice aged 6 (young) and 20 months (old). Mesenteric resistance arteries (MRA) and kidneys were collected for vascular reactivity and western-blot analysis. In old EC-Opa1 mice, blood urea was greater than in EC-WT mice, and MRA showed reduced endothelium-dependent relaxation. In kidneys, the mitochondria fission protein fission-1 (Fis-1) and the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1α) were increased in old EC-Opa1 mice. The level of caveolin-1 expression was greater in old EC-Opa1 mice. Moreover, in kidneys from EC-Opa1 old mice, NADPH-oxidase subunit gp91 expression was greater than in age-matched EC-WT mice. Thus, reduced mitochondrial fusion in mouse ECs altered mesenteric vascular reactivity and increased markers of oxidative stress in aging kidneys. Thus, Opa1 might protect the vascular tree in target organs such as the kidney.

Keywords: aging; arteries; endothelial cell; kidney; mitochondrial fusion.

FIGURE 1

Blood urea nitrogen measurement. Blood urea nitrogen was measured in young and old EC‐Opa1 and EC‐WT mice. Data are expressed as means ± SD (n = 9 EC‐WT mice, 9 young EC‐Opa1 young mice, 20 old EC‐WT mice and 18 EC‐Opa1 old mice). Two‐way ANOVA and Bonferroni's multiple comparisons test.

FIGURE 2

Phenylephrine‐mediated contraction. Phenylephrine (1 nmol/L to 30 μmol/L)‐mediated cumulative concentration‐response curve was determined in mesenteric arteries isolated from young (a) and old (b) EC‐Opa1 and EC‐WT mice. Data are expressed as mean ± SD (n = 12 young EC‐Opa1 mice, 13 young EC‐WT mice, 19 old EC‐Opa1 mice and 21 old EC‐WT mice). Two‐way ANOVA for repeated measurements and Bonferroni's multiple comparisons test.

FIGURE 3

Acetylcholine‐mediated endothelium‐dependent relaxation. Acetylcholine (ACh, 1 nmol/L to 30 μmol/L)‐mediated endothelium‐dependent relaxation was determined in mesenteric arteries isolated from young (a) and old (b) EC‐Opa1 and EC‐WT mice. Data are expressed as means ± SD (n = 12 young EC‐Opa1 mice, 13 young EC‐WT mice, 19 old EC‐Opa1 mice and 21 old EC‐WT mice). Two‐way ANOVA for repeated measurements and Bonferroni's multiple comparisons test.

FIGURE 4

Sodium nitroprusside‐mediated endothelium‐independent relaxation. Sodium nitroprusside (SNP, 1 nmol/L to 30 μmol/L)‐mediated endothelium‐dependent relaxation was determined in mesenteric arteries isolated from young (a) and old (b) EC‐Opa1 and EC‐WT mice. Data are expressed as means ± SD (n = 12 young EC‐Opa1 mice, 13 young EC‐WT mice, 19 old EC‐Opa1 mice and 21 old EC‐WT mice). NS, Two‐way ANOVA for repeated measurements and Bonferroni's multiple comparisons test.

FIGURE 5

Protein expression level of fission‐1 protein (Fis1), proliferation‐activated receptor gamma co‐activator 1α (Pgc1α), mitofusin‐2 (Mfn2) and nuclear respiratory factor (Nrf‐1) in the kidney. Protein expression level of Fis1 (a), Mfn2 (b), Pgc1α (c) and Nrf‐1 (d) was determined in kidneys isolated from young and old EC‐WT and EC‐Opa1. Mean ± SD is shown (n = 21–27 mice per group). Young and old samples came from separate blots. Complete blot panels with marker proteins can be viewed in the Figure S8. Two‐way ANOVA and Bonferroni's multiple comparisons test.

FIGURE 6

Protein expression level of endothelial nitric oxide enzyme (eNos) and Caveoline‐1 (Cav‐1) in the kidney. Protein expression level of eNos (a) and Cav‐1 (b) was determined in kidneys isolated from young and old EC‐WT and EC‐Opa1 mice. Mean ± SD is shown (n = 21–27 mice per group). Young and old samples came from separate blots. Complete blot panels with marker proteins can be viewed in the Figure S8. Two‐way ANOVA and Bonferroni's multiple comparisons test.

FIGURE 7

Protein expression levels of Gp91^phox, p67^phox, p47^phox, 3‐nitrotyrosine, Mn superoxide dismutase (Sod) and Cu/ZnSod in the kidney. Protein expression levels of gp91^phox (a), p67^phox (b), p47^phox (c), 3‐nitrotyrosine (d), MnSod (e) and Cu/ZnSod (f) was determined in kidneys isolated from young and old EC‐WT and EC‐Opa1 mice. Mean ± SD is shown (n = 21–27 mice per group). Young and old samples came from separate blots. Complete blot panels with marker proteins can be viewed in the Figure S8. Two‐way ANOVA and Bonferroni's multiple comparisons test.