Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II

Abstract

The classically recognized functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b -/-) and both AT1A and AT1B receptors (Agtr1a -/-Agtr1b -/-). Agtr1b -/- mice are healthy, without an abnormal phenotype. In contrast, Agtr1a -/-Agtr1b -/- mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt-/-) and angiotensin-converting enzyme-deficient (Ace -/-) mice that are unable to synthesize angiotensin II. Agtr1a -/-Agtr1b -/- mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a -/- Agtr1b -/- mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin-angiotensin system.

Figure 1

Targeted disruption of the Agtr1b gene. (A) The endogenous Agtr1b gene locus. The intronless coding region is depicted by the black box. (B) Targeting construct. neo, neomycin resistance cassette; TK, thymidine kinase. (C) Structure of the disrupted gene. The PCR primers (indicated by arrowheads and numbers), the lengths of diagnostic restriction fragments, and the probe used for Southern analysis are shown. H, HindIII; M, Msc1; E, EagI; S, Spe1; P, PstI; C, ClaI B, BamHI. Enzyme sites in brackets are destroyed during ligation. (D) Southern blot of offspring of an Agtr1b +/− × Agtr1b +/− cross. A 10.2-kb PstI/HindIII fragment indicates the wild-type allele, and a 2.7-kb fragment identifies the targeted locus.

Figure 2

Assessment of AT_1B mRNA expression by RT-PCR. Total RNA was isolated from adrenal glands of two wild-type and two Agtr1b −/− mice and each specimen was subjected to RT-PCR to amplify AT_1B receptor mRNA. In tissues from the wild-type mice (lanes 1 and 2), a PCR product of the expected size (700 bp) was detected on an ethidium bromide stained gel (A) and was confirmed to be AT_1B receptor mRNA by hybridization (B). This band was not detected in RNA from the Agtr1b −/− mice (lanes 3 and 4).

Figure 3

Kidney histomorphology of wild-type and Agtr1a −/−Agtr1b −/− mice. (A and B): Axial sections from wild-type (A) and Agtr1a−/−Agtr1b −/− mice (B). (×5.) There is marked atrophy of the papilla in the AT₁-deficient kidney compared with the control. (C and D): Cross sections of representative small arteries in kidneys from wild-type (C) and Agtr1a −/−Agtr1b −/− (D) mice. The media of the vessels from the mutant animals was markedly thickened and many of the vessels were surrounded by inflammatory cells as shown. (×150.) (E and F): Sections of renal cortex from wild-type (E) and Agtr1a−/−Agtr1b −/− (F) mice. In the AT₁-deficient mice there were focal areas with tubular dropout and inflammatory cell infiltration. (×100.)

Figure 4

Urine osmolality following 12 hours of water deprivation. The experimental groups are represented as follows: open bar is Agtr1a−/−Agtr1b −/− group, gray bar is theAgtr1b⁻ −/− group, hatched bar is the Agtr1a⁻ −/−group, and the black bar is the wild-type group. (∗P < 0.0003 vs. wild-type or Agtr1b −/−; ^†P < 0.0001 vs. wild-type or Agtr1b −/−, P = 0.004 vs. Agtr1a −/−.

Figure 5

Hemodynamic effects of angiotensin II in enalapril-pretreated, anesthetized wild-type, Agtr1b −/−, and Agtr1a −/− Agtr1b −/− mice. The changes in MAP at 120 seconds after injection of vehicle, 0.1, 1.0, and 10 μg/kg of angiotensin II, or 10 μg/kg epinephrine are shown. The experimental groups are represented as follows: open bars are the Agtr1a −/−Agtr1b −/− group, gray bars are the Agtr1b −/− group, and black bars are the wild-type group (∗P < 0.0003 vs. wild-type or Agtr1b−/− groups).

Figure 6

The effect of chronic ACE inhibition on systolic blood pressure in conscious mice. Systolic blood pressures were measured daily for 10 days (Baseline) followed by an additional 10-day period while mice were treated with enalapril 30 mg/kg per day in drinking water (Enalapril). Data are mean ± SEM. The black squares represent the wild-type group and the open circles represent the Agtr1a −/−Agtr1b −/− group. (∗P < 0.0001 vs. wild-type at baseline; ^†P < 0.002 vs. baseline for wild-type; ^‡P < 0.03 vs. baseline for Agtr1a −/−Agtr1b −/−).