Upregulation of the angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas receptor axis in the heart and the kidney of growth hormone receptor knock-out mice

Abstract

Objective: Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR-/-) mouse.

Design: RAS components were evaluated in the heart and the kidney of GHR-/- and control mice by immunohistochemistry and Western blotting (n=12 for both groups).

Results: The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR-/- mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR-/- mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues.

Conclusions: The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR-/- mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.

Figure 1

Representative images showing the immunohistochemical staining of angiotensin II in the heart (A) and the kidney (B) from GHR−/− (n=12) and normal mice (n=12). Images are shown at x400 magnification; black arrows indicate specific staining in cardiomyocytes (A) as well as in glomerular and tubular sections in the kidney (B). Lower panels show cardiac (A) and renal (B) sections incubated with either PBS (control of non-specific staining) or with anti-Ang II antibody (Ab) previously blocked by preincubation with Ang II (control of antibody specificity).

Figure 2

Representative images showing the immunohistochemical staining of angiotensin-(1-7) in the heart (A) and the kidney (B) from GHR−/− (n=12) and normal mice (n=12). Images are shown at x400 magnification; black arrows indicate specific staining in cardiomyocytes (A) as well as in glomerular and tubular sections (B). Lower panels show cardiac (A) and renal (B) sections incubated with either PBS (control of non-specific staining) or with anti-Ang-(1-7) antibody (Ab) previously blocked by preincubation with Ang-(1-7) (control of antibody specificity).

Figure 3

Representative images showing the immunohistochemical staining of AT1 receptor in the heart (A) and the kidney (B) from GHR−/− and normal mice. Images are shown at x400 magnification; black arrows indicate positive staining in cardiomyocytes (A) and in tubular epithelial cells (B). Tissue sections were incubated with PBS instead of primary antibody as a control of non-specific staining. Results were confirmed by submitting tissue homogenates to western blot analysis. Representative images and bar charts showing the quantification of AT1 receptor in the heart (C) and the kidney (D) are shown for each group. Data are shown as mean ± S.D. * P < 0.05 vs. normal group (n=12); Mann Whitney test. The calculated molecular weight for the specific bands corresponding to the AT1 receptor and β-tubulin was approximately 44 and 50 kDa respectively in both tissues. IB: immunoblotting.

Figure 4

Representative images showing the immunohistochemical staining of AT2 receptor in heart (A) and kidney (B) from GHR−/− and normal mice. Images are shown at x400 magnification; black arrows indicate positive staining in cardiomyocytes (A) and in tubular epithelial cells (B). Tissue sections were incubated with PBS instead of primary antibody as a control of non-specific staining. Results were confirmed by submitting tissue homogenates to western blot analysis. Representative images and bar charts showing the quantification of AT2 receptor in heart (C) and kidney (D) are shown for each group. Data are shown as mean ± S.D. * P < 0.05 vs. normal group (n=12); Mann Whitney test. The calculated molecular weight for the specific band corresponding to the AT2 receptor was approximately 50 kDa in both tissues. IB: immunoblotting.

Figure 5

Representative images showing the immunohistochemical staining of Mas receptor in heart (A) and kidney (B) from GHR−/− and normal mice. Images are shown at x400 magnification; black arrows indicate positive staining in cardiomyocytes (A) as well as in both glomerular and tubular sections in the kidney (B). Tissue sections were incubated with PBS instead of primary antibody as a control of non-specific staining. Results were confirmed by submitting tissue homogenates to western blot analysis. Representative images and bar charts showing the quantification of Mas receptor in heart (C) and kidney (D) are shown for each group. Data are shown as mean ± S.D. * P<0.05 vs. normal group (n=12); Mann Whitney test. The calculated molecular weight for the specific band corresponding to the Mas receptor was approximately 45 kDa in both tissues. IB: immunoblotting.

Figure 6

Representative images showing the immunohistochemical staining of angiotensin-converting enzyme (ACE) in heart (A) and kidney (B) from GHR−/− and normal mice. Images are shown at x400 magnification; black arrows indicate positive staining in cardiomyocytes (A) and in both glomerular and tubular sections of the kidney (B). Tissue sections were incubated with PBS instead of primary antibody as a control of non-specific staining. Results were confirmed by submitting tissue homogenates to western blot analysis. Representative images and bar charts showing the quantification of ACE in heart (C) and kidney (D) are shown for each group. Data are shown as mean ± S.D. The calculated molecular weight for ACE was approximately 188 kDa in both tissues. IB: immunoblotting.

Figure 7

Representative images showing the immunohistochemical staining of angiotensin-converting enzyme type 2 (ACE2) in heart (A) and kidney (B) from GHR−/− and normal mice. Images are shown at x400 magnification; black arrows indicate positive staining in cardiomyocytes (A) and in tubular epithelial cells (B). Tissue sections were incubated with PBS instead of primary antibody as a control of non-specific staining. Results were confirmed by submitting tissue homogenates to western blot analysis. Representative images and bar charts showing the quantification of ACE2 in heart (C) and kidney (D) are shown for each group. Data are shown as mean ± S.D. * P < 0.05 vs. normal group (n=12); Mann Whitney test. The calculated molecular weight for ACE2 was approximately 89 kDa in both tissues. IB: immunoblotting.

Figure 8

Representative images and bar charts showing the quantification of angiotensinogen (AGT) in heart (A) and kidney (B) are shown for each group. Data are shown as mean ± S.D. (n=12); Mann Whitney test. The calculated molecular weight for the specific band corresponding to AGT was approximately 50 kDa in the heart and 52 kDa in the kidney. IB: immunoblotting.

Figure 9

Representative images and bar charts showing the quantification of endothelial nitric oxide synthase (eNOS) in heart (A) and kidney (B) are shown for each group. Data are shown as mean ± S.D. * P < 0.05 vs. normal group (n=12); Mann Whitney test. The calculated molecular weight for the specific band corresponding to eNOS was approximately 138 kDa in both tissues. IB: immunoblotting.