Disruption of the Ang II type 1 receptor promotes longevity in mice

Abstract

The renin-angiotensin system plays a role in the etiology of hypertension and the pathophysiology of cardiac and renal diseases in humans. Ang II is the central product of this system and is involved in regulating immune responses, inflammation, cell growth, and proliferation by acting through Ang II type 1 receptors (AT1 and AT2). Here, we show that targeted disruption of the Agtr1a gene that encodes AT1A results in marked prolongation of life span in mice. Agtr1a-/- mice developed less cardiac and vascular injury, and multiple organs from these mice displayed less oxidative damage than wild-type mice. The longevity phenotype was associated with an increased number of mitochondria and upregulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney. In cultured tubular epithelial cells, Ang II downregulated Sirt3 mRNA, and this effect was inhibited by an AT1 antagonist. These results demonstrate that disruption of AT1 promotes longevity in mice, possibly through the attenuation of oxidative stress and overexpression of prosurvival genes, and suggests that the Ang II/AT1 pathway may be targeted to influence life span in mammals.

Figure 1. Knocking-out AT_1A prolongs life span in the mouse.

A Kaplan-Meier analysis of survival in male Agtr1a^–/– animals and wild-type littermates is shown (log-rank test, 20.32; P < 0.0001 versus wild-type mice). The average life span of Agtr1a^–/– animals and wild-type littermates was 31.20 ± 2.31 and 24.81 ± 3.10 months, respectively.

Figure 2. Body weight and blood glucose levels in Agtr1a^–/– and wild-type mice.

(A) Body weight in Agtr1a^–/– animals and wild-type littermates. No significant difference in weight gain was observed. (B) Fasting blood glucose levels were comparable in Agtr1a^–/– animals (n = 9) and wild-type littermates (n = 6) at month 24. Data are mean ± SD.

Figure 3. AT_1A deficiency protects heart from hypertrophy and fibrosis.

Paraffin-embedded hearts (Agtr1a^–/– mice, n = 7, 26 to 30 months old; wild-type mice, n = 4, 26 to 29 months old) were sectioned at 5 μm for hematoxylin-eosin for morphometric studies and at 10 μm for collagen content stained with picrosirius solution. (A) Neither the ratio of heart weight/body weight, (B) nor the estimated number of left ventricular cardiomyocytes were effected by the absence of AT_1A. (C) The cross-sectional area of cardiomyocytes, an index of hypertrophy, and (D) interstitial collagen, a marker of cardiac fibrosis, were significantly lower in Agtr1a^–/– mice. Data are mean ± SD. *P < 0.01 versus wild-type mice by Student’s t test for impaired data.

Figure 4. Lack of AT_1A protects mice from aorta damage.

(A and B) Representative images of aorta sections from wild-type mice (26 to 29 months old), showing fragmentation of lamina elastica along all the perimeter of the vessel wall (A) and intraparietal accumulation of foamy cells destroying layers of elastic tunica media (B). (C) Aorta section from Agtr1a^–/– mice (26 to 30 months old) showing regular elastic lamina. Original magnification, ×20.

Figure 5. Absence of AT_1A limits oxidative damage.

(A) Staining scores of nitrotyrosine, a marker of peroxynitrite, in heart, aorta, and kidney were increased in aged wild-type mice (26 to 29 months old) with respect to young wild-type mice (2 months old). Deficiency of AT_1A resulted in less oxidative stress. ^†P < 0.05 versus wild-type 2-month-old mice; *P < 0.05 versus wild-type 26- to 29-month-old mice. Data are mean ± SD. (B) Representative images of kidney sections from wild-type 2-month-old, wild-type 26- to 29-month-old, and Agtr1a^–/– 26- to 30-month-old animals. Peroxynitrite was localized with a specific anti-nitrotyrosine antibody in the tubulointerstitial area. Peroxynitrite staining was increased in wild-type 26- to 29-month-old animals with respect to wild-type 2-month-old animals. Very weak staining was found in Agtr1a^–/– mice, which was comparable to wild-type 2-month-old animals. Original magnification, ×400.

Figure 6. AT_1A deficiency prevents loss of mitochondria.

(A) Representative transmission electron micrographs of the ultrastructure of mouse proximal tubular cells obtained from resin-embedded kidney sections from young wild-type mice (2 months old), aged wild-type mice (26 to 29 months old), and Agtr1a^–/– animals (26 to 30 months old). Scale bar: 1 μm. (B) The number of mitochondria per volume in proximal tubular cells in aged wild-type animals was decreased with respect to wild-type 2-month-old and Agtr1a^–/– mice. Agtr1a^–/– animals showed the same numerical density as wild-type 2-month-old animals. *P < 0.01 versus wild-type 2-month-old and Agtr1a^–/– animals by ANOVA corrected with Bonferroni coefficient. Mean mitochondria volume evaluated on the same transmission electron micrographs through morphometrical analysis did not differ among groups. Data are mean ± SD.

Figure 7. Lack of AT_1A is associated with increased levels of Nampt and Sirt3 in the kidney.

By real-time PCR, whole kidney expression of both Nampt and Sirt3 was increased in Agtr1a^–/– (26 to 30 months old) with respect to wild-type (26 to 29 months old) mice. Renal expression of Sirt1 mRNA was comparable in the 2 strains. Data are mean ± SD of 4 animals. *P < 0.05 versus wild-type by ANOVA corrected with Bonferroni coefficient.

Figure 8. Effect of Ang II on prosurvival genes in mouse cultured PTECs.

(A) Ang II downregulates Nampt and Sirt3 mRNA via AT₁. After 24-hour incubation with serum-free DMEM, PTECs were incubated with candesartan (0.1 μM) or medium for 1 hour and then exposed to Ang II (1 μM) or medium (control) for 3 hours. The black column represents unstimulated cells maintained in medium (control) for 4 hours. Nampt and Sirt3 mRNA levels were evaluated by real-time PCR. Data are mean ± SD of 3 experiments. *P < 0.01 versus control, ^†P < 0.01 versus Ang II by ANOVA corrected with Bonferroni coefficient. (B) Nampt modulates Sirt3 gene transcription in response to Ang II. After 6 hour transfection with Nampt siRNA, cells were maintained in medium for 45 hours and then incubated 3 hours with Ang II. Data are mean ± SD of 3 experiments. *P < 0.01 versus control, ^‡P < 0.01 versus siRNA control plus Ang II by Student’s t test for unpaired data (left panel) or ANOVA corrected with Bonferroni coefficient (right panel).