Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice - PubMed (original) (raw)

. 2007 Jul;117(7):1914-25.

doi: 10.1172/JCI28764.

Sabine Bardin, Sonia Verp, Véronique Baudrie, Annie Michaud, Sophie Conchon, Martine Muffat-Joly, Brigitte Escoubet, Evelyne Souil, Ghislaine Hamard, Kenneth E Bernstein, Jean Marie Gasc, Jean-Luc Elghozi, Pierre Corvol, Eric Clauser

Affiliations

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice

Sandrine Billet et al. J Clin Invest. 2007 Jul.

Abstract

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

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Figures

Figure 1

Figure 1. Targeted knockin of the Agtr1a gene by homologous recombination into ES cells and mouse.

(A) Schematic representation of the targeting vector and partial restriction map of the Agtr1a gene locus before and after homologous recombination. The top line shows the structure of AT1AWT locus containing exon 3 of the gene with coding sequence (black box) and the 5′ and 3′ untranslated sequences (gray boxes). Exon 3 is flanked by 3 kb of intron 2 (upstream) and 2 kb of noncoding sequence (downstream). The targeting construct containing AT1AMUT and a hygromycin cassette (hygro) was used to replace the WT BamHI (B) KpnI (K) fragment within AT1A locus (middle line). The lower line shows the targeted AT1A locus with the internal probe (probe 1) and 3′ external probe (probe 2) used for Southern blot analysis. (B) Southern blot analysis of targeted ES cell DNA using internal probe 1 with SpeI digestion (left) and using probe 2 with SacI digestion (right). C, control (targeting construct). –/–, WT cells; +/–, heterozygous recombinant cells. (C) Southern blot analysis of tail DNA samples from F2 offspring (generated by crossing heterozygous mice from the first generation [F1 mice]) digested with SacI and probed with the 3′ external probe (probe 2). –/–, WT mice; +/–, heterozygous transgenic mice; +/+, homozygous transgenic mice; S, SpeI; B, BamHI; G, BglII; X, XbaI; A, SacI; K, KpnI; N, NotI; E, EcoRI.

Figure 2

Figure 2. Quantitative RT-PCR of the Ang II receptors in target tissues.

RNA from liver, kidney, heart, adrenal gland, and aorta were retrotranscribed and amplified for AT1A (A), AT1B (B), and AT2 (C) mRNAs using the LightCycler technology in AT1AWT (white bars) and AT1AMUT (black bars) mice. Results are expressed in arbitrary units relative to the expression of cyclophilin gene in the same tissues. ND, not detectable.

Figure 3

Figure 3. Signaling properties of AT1A in tissues and primary hepatocyte cultures from AT1AWT and AT1AMUT mice.

(A) Basal phosphorylation of STAT1, STAT3, and ERK1/2 in the liver. Left: Representative Western blot for 3 AT1AWT and 3 AT1AMUT mice. Right: Histograms show mean ± SEM of the ratio of phosphorylated protein to total protein and expressed as a percentage of this ratio in AT1AWT corresponding tissue. White bars, AT1AWT; black bars, AT1AMUT. *P < 0.05 compared with AT1AWT. (B and C) Stimulation of ERK1/2 phosphorylation by Ang II (B) and Ang IV (C). Primary cultured hepatocytes from AT1AWT (black squares) and AT1AMUT (white triangles) mice were serum-starved for 3 hours and stimulated with Ang II (B) or Ang IV (C) for 5 minutes at 37°C. Left panels show representative immunoblots of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (ERK1/2). ERK1/2 phosphorylation was quantified by densitometry, normalized to the amount of total ERK1/2 in each lane and expressed as a percentage over basal phosphorylation of ERK1/2 as shown in the right panels. Results are mean ± SEM of 3 independent experiments.

Figure 4

Figure 4. Pressor response to Ang II of AT1AWT and AT1AMUT mice.

Effect of cumulative intravenous bolus doses of Ang II on BP in anesthetized mice. (A) Representative recordings, following Ang II injections (arrows), of systolic (red), mean (blue), and diastolic (green) BPs in AT1AWT and AT1AMUT mice. After each Ang II dose, BP stabilized at a significantly higher level in AT1AMUT mice than in control mice. (B) Ang II dose-response effect on systolic BP. The area under the curve (AUC; see Methods) was measured for each Ang II dose in AT1AWT (solid line) and AT1AMUT mice (dotted line). (C) Systolic BP response to increasing bolus doses of CGP42112A injected in anesthetized AT1AWT (white bars) and AT1AMUT (black bars) mice. *P < 0.05, **P < 0.01 compared with AT1AWT.

Figure 5

Figure 5. Systolic BP measured by the tail-cuff method.

(A) Systolic BP measured throughout the lives of AT1AWT mice (white bars) and AT1AMUT mice (black bars). **P < 0.01 compared with AT1AWT. (B) Systolic BP measurements after candesartan treatment. Animals were treated with candesartan in drinking water (10 mg/kg/d) for 25 days. Systolic BP was measured as above on the last 3 days of the treatment period. **P < 0.01 compared with untreated animals.

Figure 6

Figure 6. Renin-angiotensin-aldosterone system parameters.

Plasma renin (A), plasma immunoreactive angiotensin (B), and plasma aldosterone (E) concentrations were determined in AT1AWT mice (white bars) and AT1AMUT mice (black bars). (C) Representative in situ hybridization detection of renin expression in kidney sections from AT1AWT and AT1AMUT adult (C) and neonate (D) animals. The juxtaglomerular apparatus is indicated by arrows. Relative hyperaldosteronism was also evaluated by computing the aldosterone/renin ratio (F). *P < 0.05, ***P < 0.001 compared with AT1AWT.

Figure 7

Figure 7. Functional and morphological characterization of AT1AWT and AT1AMUT mouse kidney.

Kidney function was evaluated by measuring 24-hour urinary creatinine (A) and Na/K balance (B) for 3 days. All assays were performed in AT1AWT (white bars) and AT1AMUT (black bars) mice fed a normal-sodium diet. (C) Representative kidney histological sections from both mice genotypes stained with Sirius red. Scale bars: 10 μm. (D) Quantitative RT-PCR of collagen αI and PAI1 mRNA in AT1AWT (white bars) and AT1AMUT (black bars) kidneys. *P < 0.05 compared with AT1AWT.

Figure 8

Figure 8. Cardiovascular characterization of AT1AMUT and AT1AWT mice.

(A) Vascular morphology was assessed by morphometric analysis of the wall thickening ratio (S/L) in renal arteries in 5-month-old AT1AWT (white bar) and AT1AMUT (black bar) littermates. Three mice were studied in each group, and a mean of 20 arteries were measured in each mouse. *P < 0.05 compared with AT1AWT. (B) Cardiac hypertrophy was assessed by the ratio of heart weight to body weight (HW/BW) in 6- to 10-month-old AT1AWT (white triangles) and AT1AMUT (black circles) littermates. (C) Quantitative RT-PCR of ANF and BNP mRNA in AT1AWT (white bars) and AT1AMUT (black bars) hearts. (D) Myocardial sections from 5-month-old AT1AWT and AT1AMUT mice were stained with Sirius red. Scale bar: 10 μm. (E) Quantitative RT-PCR of collagen αI and PAI1 mRNA in AT1AWT (white bars) and AT1AMUT (black bars) hearts. **P < 0.01 compared with AT1AWT.

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