Increased Susceptibility of Mice Lacking Renin-b to Angiotensin II-Induced Organ Damage

Abstract

Several cardiac and renal diseases are attributed to a dysregulation of the renin-angiotensin system. Renin, the rate-limiting enzyme of the renin-angiotensin system, has 2 isoforms. The classical renin isoform (renin-a) encoding preprorenin is mainly confined to the juxtaglomerular cells and released into the circulation upon stimulation. Alternatively, renin-b is predicted to remain intracellular and is expressed in the brain, heart, and adrenal gland. In the brain, ablation of renin-b (Ren-b^Null mice) results in increased brain renin-angiotensin system activity. However, the consequences of renin-b ablation in tissues outside the brain remain unknown. Therefore, we hypothesized that renin-b protects from hypertensive cardiac and renal end-organ damage in mice. Ren-b^Null mice exhibited normal blood pressure at baseline. Thus, we induced hypertension by using a slow pressor dose of Ang II (angiotensin II). Ang II increased blood pressure in both wild type and Ren-b^Null to the same degree. Although the blood pressure between Ren-b^Null and wild-type mice was elevated equally, 4-week infusion of Ang II resulted in exacerbated cardiac remodeling in Ren-b^Null mice compared with wild type. Ren-b^Null mice also exhibited a modest increase in renal glomerular matrix deposition, elevated plasma aldosterone, and a modestly enhanced dipsogenic response to Ang II. Interestingly, ablation of renin-b strongly suppressed plasma renin, but renal cortical renin mRNA was preserved. Altogether, these data indicate that renin-b might play a protective role in the heart, and thus renin-b could be a potential target to treat hypertensive heart disease.

Keywords: aldosterone; angiotensin II; blood pressure; exons; heart; renin; renin-angiotensin system.

Figure 1:. Baseline Blood Pressure.

A) Systolic blood pressure (SBP), B) diastolic blood pressure (DBP), and C) heart rate was measured by radiotelemetry in Ren-b^Null and wildtype (WT) mice. D) Renal sympathetic nerve activity (RSNA) was measured in anesthetized animals and expressed as v*sec/min (left) or spikes/sec (right). Data are plotted as individual points, the bar height reflects the mean, and the error bars reflect SEM. The N values are indicated.

Figure 2:. Vascular Function.

Vascular function was assessed in three different vascular beds in Ren-b^Null and wildtype (WT) mice: aorta (left), carotid artery (center), and basilar artery (right) in response to acetylcholine (A), and sodium nitroprusside (SNP, B). Data was analyzed by repeated measure two-way ANOVA with Sidak’s multiple comparisons procedure and significances are noted in each panel.

Figure 3:. Pressor Response to Ang-II.

A) Systolic blood pressure measured by tail-cuff plethysmography at baseline and weekly after angiotensin II (Ang-II) minipump implantation. B) The same data is expressed as dot-plot graph format to transparently reveal the distribution of the individual animal data. Data are plotted as individual points, the bar height reflects the mean, and the error bars reflect SEM. Data were analyzed with repeated measures two-way ANOVA. *p<0.05 WT+Ang-II vs WT+veh and ^#p<0.05 Ren-b^Null+Ang-II vs Ren-b^Null+veh by Bonferroni’s multiple comparisons procedure.

Figure 4:. Cardiac End-Organ Damage.

Cardiac tissues were collected and weighed at the end of the fourth week. A) Heart weight/body weight (HW/BW) ratio. B) Collagen 1A2 (Col1A2), C) transforming growth factor beta (TGF-β), and D) tumor necrosis factor-alpha (TNF-α) mRNA were measured in cardiac homogenates by quantitative RT-PCR. The ROUT method (with Q set to 5%) was used to identify outliers (1 outlier in the WT+veh in TGF-β and TNF-α measurements; and 3 outliers, 1 each in WT+veh; Ren-b^Null+veh; WT+Ang-II in Col1A2 determination). Data are plotted as individual points, the bar height reflects the mean, and the error bars reflect SEM. Data were analyzed with repeated measures two-way ANOVA with Bonferroni’s multiple comparisons procedure and significances are noted in each panel.

Figure 5:. Plasma Renin and Renal Cortical Renin.

A) The levels of renin were measured by enzyme immunoassay in plasma samples from vehicle or Ang-II-infused Ren-b^Null and wildtype (WT) mice. ROUT method (with Q set to 5%) was used to identify outliers (1 outlier in Ren-b^Null+veh and 2 in Ren-b^Null+Ang-II group). B) Renin mRNA expression was measured by quantitative RT-PCR in kidney cortex. C) Renin protein expression was measured by western blotting in kidney cortex. D) Using in situ hybridization was used to assess the distribution of renin expression within the renal cortex. Renin-positive glomerular and tubular cells were quantified. Representative in situ hybridization are shown in Figure S2. Data are plotted as individual points, the bar height reflects the mean, and the error bars reflect SEM. *p<0.05 WT+Ang-II vs WT+veh and ^#p<0.05 Ren-b^Null+Ang-II vs Ren-b^Null+veh by Bonferroni’s multiple comparisons procedure.

Figure 6:. Water Intake

During the fourth week on vehicle or Ang-II infusion, Ren-b^Null and wildtype (WT) mice were housed in metabolic cages for the assessment of water intake. Data were collected in three separate groups of mice and then combined. Twenty four-hour water intake (A), urinary volume (B), Urine osmolarity, sodium excretion (UNaV), and potassium excretion (UKV) (C) are shown. D) The relationship between blood pressure and water intake is shown. E) Aldosterone and copeptin, a surrogate marker of vasopressin, levels were measured in urine samples by enzyme immunoassay. Data are plotted as individual points, the bar height reflects the mean, and the error bars reflect SEM. Data were analyzed with repeated measures two-way ANOVA and significances are noted in each panel.