Genetic Deletion of AT1a Receptor or Na+/H+ Exchanger 3 Selectively in the Proximal Tubules of the Kidney Attenuates Two-Kidney, One-Clip Goldblatt Hypertension in Mice

Abstract

The roles of angiotensin II (Ang II) AT1 (AT1a) receptors and its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of two-kidney, 1-clip (2K1C) Goldblatt hypertension have not been investigated previously. The present study tested the hypothesis that deletion of the AT1a receptor or NHE3 selectively in the proximal tubules of the kidney attenuates the development of 2K1C hypertension using novel mouse models with proximal tubule-specific deletion of AT1a receptors or NHE3. 2K1C Goldblatt hypertension was induced by placing a silver clip (0.12 mm) on the left renal artery for 4 weeks in adult male wild-type (WT), global Agtr1a−/−, proximal tubule (PT)-specific PT-Agtr1a−/− or PT-Nhe3−/− mice, respectively. As expected, telemetry blood pressure increased in a time-dependent manner in WT mice, reaching a maximal response by Week 3 (p < 0.01). 2K1C hypertension in WT mice was associated with increases in renin expression in the clipped kidney and decreases in the nonclipped kidney (p < 0.05). Plasma and kidney Ang II were significantly increased in WT mice with 2K1C hypertension (p < 0.05). Tubulointerstitial fibrotic responses were significantly increased in the clipped kidney (p < 0.01). Whole-body deletion of AT1a receptors completely blocked the development of 2K1C hypertension in Agtr1a−/− mice (p < 0.01 vs. WT). Likewise, proximal tubule-specific deletion of Agtr1a in PT-Agtr1a−/− mice or NHE3 in PT-Nhe3−/− mice also blocked the development of 2K1C hypertension (p < 0.01 vs. WT). Taken together, the present study provides new evidence for a critical role of proximal tubule Ang II/AT1 (AT1a)/NHE3 axis in the development of 2K1C Goldblatt hypertension.

Keywords: 1-clip Goldblatt hypertension; 2-kidney; AT1a receptor; NHE3; angiotensin II; proximal tubule.

Figure 1

Induction of 2K1C Goldblatt hypertension with 0.12 mm silver clip on the left kidney significantly decreases glomerular filtration rate (GFR), as measured using FITC-Sinistrin, equally by >40% in WT, global Agtr1a^−/−, proximal tubule-specific PT-Agtr1a^−/− or PT-Nhe3^−/− mice. N = 9–15 per strain, per group. ** p < 0.01 vs. Basal.

Figure 2

The development of 2K1C Goldblatt hypertension in WT mice with the activation of circulating and intratubular renin-angiotensin system. (A), the time-dependent increases in systolic blood pressure during the development of 2K1C hypertension. (B), increased intratubular expression of renin mRNAs in the clipped kidney. (C), no changes in intratubular expression of angiotensinogen mRNAs between control, clipped, and nonclipped kidneys. (D), no changes in intratubular expression of ACE mRNAs between control, clipped, and nonclipped kidneys. (E), Increased circulating Ang II level during the development of 2K1C hypertension. (F), increased intratubular Ang II in both clipped and contralateral nonclipped kidney during the development of 2K1C hypertension. Note that Control is defined as sham-operated control mice without clipping the left renal artery, LK as the left clipped kidney, and RK as the unclipped contralateral kidney, respectively. * p < 0.05 or ** p < 0.01 vs. control or baseline; ⁺⁺ p < 0.01 vs. the clipped kidney.

Figure 3

Deletion of the AT_1a receptor prevents the development of 2K1C Goldblatt hypertension in Agtr1a^−/− mice with whole-body AT_1a receptor knockout. (A), comparison of the maximal blood pressure responses between WT and Agtr1a^−/− mice during the development of 2K1C hypertension. (B), increased intratubular expression of renin mRNAs in both clipped and nonclipped kidneys. (C), no changes in intratubular expression of angiotensinogen mRNAs between control, clipped, and nonclipped kidneys. (D), no changes in intratubular expression of ACE mRNAs between control, clipped, and nonclipped kidneys. (E), Increased circulating Ang II during the development of 2K1C hypertension in Agtr1a^−/− mice. (F), increased intratubular Ang II in both clipped and contralateral nonclipped kidney during the development of 2K1C hypertension in Agtr1a^−/− mice. Note that Control is defined as sham-operated control mice without clipping the left renal artery, LK as the left clipped kidney, and RK as the unclipped contralateral kidney, respectively. ** p < 0.01 vs. control or baseline.

Figure 4

Proximal tubule-specific deletion of AT_1a receptors in the kidney significantly attenuates the development of 2K1C Goldblatt hypertension in PT-Agtr1a^−/− mice. (A), the lack of the time-dependent systolic blood pressure responses for 4 weeks during the development of 2K1C hypertension. (B), no change in intratubular expression of renin mRNAs in both clipped and nonclipped kidneys. (C), no changes in intratubular expression of angiotensinogen mRNAs between control, clipped, and nonclipped kidneys. (D), no changes in intratubular expression of ACE mRNAs between control, clipped, and nonclipped kidneys. (E), circulating Ang II slightly but not statistically increased during the development of 2K1C hypertension. (F), increased intratubular Ang II in both clipped and contralateral nonclipped kidney during the development of 2K1C hypertension likely due to the decreases in AT₁ (AT_1a) receptor occupancy in the proximal tubules. ** p < 0.01 vs. control kidney.

Figure 5

Proximal tubule-specific deletion of the Na⁺/H⁺ exchanger 3 (NHE3), a major downstream target of AT_1a receptor activation by intratubular Ang II, in the kidney significantly attenuates the development of 2K1C Goldblatt hypertension in PT-Nhe3^−/− mice. (A), the lack of the time-dependent systolic blood pressure responses for 4 weeks during the development of 2K1C hypertension, a phenotypic response similar to PT-Agtr1a^−/− mice. (B), comparisons of maximal systolic blood pressure responses at Week 3 between wild-type and PT-Nhe3^−/− mice during the development of 2K1C hypertension. (C), comparisons of maximal systolic blood pressure responses to 2-week systemic infusion of a pressor dose of Ang II, 1.5 mg/kg/day, i.p., between wild-type and PT-Nhe3^−/− mice. ** p < 0.01 vs. control or baseline in the same mouse strain. ⁺⁺ p < 0.01 vs. wild-type control or during the development of 2K1C Goldblatt hypertension (B) or Ang II-induced hypertension (C).

Figure 6

Deletion of AT_1a receptors or NHE3 selectively in the proximal tubules of the kidney promotes the natriuretic responses to the induction of 2K1C Goldblatt hypertension in proximal tubule-specific PT-Agtr1a^−/− or PT-Nhe3^−/− mice. N = 9–15 per strain, per group. * p< 0.05 or ** p < 0.01 vs. WT + 2K1C.

Figure 7

Deletion of AT_1a receptors selectively in the proximal tubules of PT-Agtr1a^−/− mice significantly attenuates the development of tubulointerstitial injury in the renal cortex induced by 2K1C Goldblatt hypertension, as revealed by Masson’s Trichome staining. (A), the nonclipped contralateral kidney in a representative WT mouse kidney. (B), the development of marked tubulointerstitial injury in a representative WT mouse kidney in response to the development of 2K1C hypertension with blue color staining in the peri-glomerular and tubulointertsitial areas. (C), the development of marked tubulointerstitial injury in a representative WT mouse kidney in response to the development of Ang II-induced hypertension with blue color staining in the peri-glomerular and tubulointertsitial areas. (D), the nonclipped kidney of a representative PT-Agtr1a^−/− mouse. (E), marked tubulointerstitial injury as developed in WT mice in response to the development of 2K1C hypertension was attenuated in a representative PT-Agtr1a^−/− mouse kidney. (F), marked tubulointerstitial injury as developed in WT mice in response to the development of Ang II-induced hypertension was also markedly attenuated in a representative PT-Agtr1a^−/− mouse kidney. G, glomerulus; PT, proximal tubule. Insets represent the cortical tubulointerstitial areas.

Figure 8

The schematic representation showing the iL-Sglt2-Cre/LoxP approach to generate mutant mouse models with proximal tubule-specific deletion of the AT_1a receptor or its downstream target protein NHE3 [32,33,34,35] (A) and the working hypothesis that deletion of the AT_1a receptor or NHE3 selectively in the proximal tubules attenuates or blocks the development of 2K1C Goldblatt hypertension (B).