Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension

Abstract

The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

Keywords: (pro)renin receptor; angiotensin II; central nervous system; hypertension.

Figure 1. Localization of the PRR in mouse brain neurons

Mice brain sections were immune-stained with PRR (Red) and DAPI (Blue) with or without Triton. (A, B) Non-immune serum antibody control. (C, D) Representative images of PRR immunostaining without Triton permeabilization. (E, F) Representative images of PRR immunostaining with 0.2% Triton permeabilization. (G, H) Semi-quantitative analysis of PRR raw fluorescence intensity and membrane/cytoplasm ratio respectively using Image J software.

Figure 2. Reduction of PRR expression in the Nefh-PRRKO mice

(A) PRR mRNA levels in WT and Nefh-PRRKO mice detected by real-time PCR. (B, C) Crerecombinase (green) and PRR (red) expression in WT mice. (D, E) Cre-recombinase and PRR expression in Nefh-PRRKO mice. (F–H) Cre-recombinase and PRR in the SFO (F), PVN (G), and RVLM (H) of WT mice. (I–K) Cre-recombinase and PRR expression in the SFO (I), PVN (J), and RVLM (K) of Nefh-PRRKO mice.

Figure 3. PRR knockout in neurons yields normal cardiovascular phenotypes

(A) BP, (B) HR and (C) locomotor activity were continuously recorded in WT and Nefh-PRRKO mice. (D) Body weight was measured in adult (14-wk-old) mice. N=5 mice/group.

Figure 4. Prorenin mediates Ang II-dependent pressor responses via binding to the PRR

(A) The BP and HR responses to ICV infusion of carbachol (300ng; 0.3 μl/min) and artificial CSF in the Nefh-PRRKO and WT mice. (B) BP and HR responses to Ang II (300 ng) and Ang II + losartan (30 μg). (C) BP and HR responses to ICV infusion of mouse prorenin (300 ng), mouse prorenin + losartan (30 μg), and mouse prorenin + captopril (30 μg). *P<0.05 vs. WT + mProrenin; n=5 mice/group. (D) The BP and HR responses to ICV infusion of mouse renin (300 ng), mouse renin + losartan (30 μg), and mouse renin + captopril (30 μg). (E) Ang II levels in the brain cortex, hypothalamus (Hypo), and brainstem (BS) at baseline and after prorenin infusion in Nefh-PRRKO and WT mice. * P<0.05 vs. WT; # P<0.05 vs. WT +mProrenin; n=5 mice/group.

Figure 5. PRR, (pro)renin, and Ang II levels in DOCA-salt-induced hypertensive mice

(A) PRR mRNA levels in various brain regions including the SFO, PVN, RVLM, NTS, and brain cortex. (B) Ang II levels in the brain cortex, hypothalamus (Hypo), and brainstem (BS) in WT and Nefh-PRRKO mice before and after DOCA-salt treatment. (C, D) Ang II in the kidney and plasma. (E) (Pro)renin levels in the brain cortex, Hypo, and BS in WT and Nefh-PRRKO mice before and after DOCA-salt treatment measured by ELISA. (F) Renin and prorenin levels in the Hypo in WT and Nefh-PRRKO mice before and after DOCA-salt treatment, measured by EKA. * P<0.05 vs. WT; # P<0.05 vs. WT + DOCA-salt; n=4–5 mice/group.

Figure 6. PRR knockout in neurons prevents the development of DOCA-salt hypertension

BP (A) and HR (B) traces in WT mice (n=7), Nefh-PRRKO mice (n=9), and WT mice receiving ICV losartan (n=6) at baseline and following 21-d DOCA-salt treatment. (C) HR response to propranolol (IP, 5 mg/kg). (D) BP response to chlorisondamine (IP, 6 mg/kg), (E) HR response to methylatropine (IP, 1 mg/kg). (F) SBRS in WT and Nefh-PRRKO mice before and after DOCA-salt treatment. * P<0.05 vs. WT; #P<0.05 vs. WT + DOCA-salt.