Brain-targeted (pro)renin receptor knockdown attenuates angiotensin II-dependent hypertension

Abstract

The (pro)renin receptor is a newly discovered member of the brain renin-angiotensin system. To investigate the role of brain (pro)renin receptor in hypertension, adeno-associated virus-mediated (pro)renin receptor short hairpin RNA was used to knockdown (pro)renin receptor expression in the brain of nontransgenic normotensive and human renin-angiotensinogen double-transgenic hypertensive mice. Blood pressure was monitored using implanted telemetric probes in conscious animals. Real-time PCR and immunostaining were performed to determine (pro)renin receptor, angiotensin II type 1 receptor, and vasopressin mRNA levels. Plasma vasopressin levels were determined by ELISA. Double-transgenic mice exhibited higher blood pressure, elevated cardiac and vascular sympathetic tone, and impaired spontaneous baroreflex sensitivity. Intracerebroventricular delivery of (pro)renin receptor short-hairpin RNA significantly reduced blood pressure, cardiac and vasomotor sympathetic tone, and improved baroreflex sensitivity compared with the control virus treatment in double-transgenic mice. (Pro)renin receptor knockdown significantly reduced angiotensin II type 1 receptor and vasopressin levels in double-transgenic mice. These data indicate that (pro)renin receptor knockdown in the brain attenuates angiotensin II-dependent hypertension and is associated with a decrease in sympathetic tone and an improvement of the baroreflex sensitivity. In addition, brain-targeted (pro)renin receptor knockdown is associated with downregulation of angiotensin II type 1 receptor and vasopressin levels. We conclude that central (pro)renin receptor contributes to the pathogenesis of hypertension in human renin-angiotensinogen transgenic mice.

Figure 1. The PRR is highly expressed in the brain and primarily located in neurons

Green staining represents the neuron marker NeuN (A) or astroglial cell marker GFAP (D), red staining represents the PRR (B and E) and merged pictures (C and F) show that the PRR is mainly expressed in neurons. Double staining pictures show PRR expression in the SFO (G), PVN (H), NPR (I),NTS (J), RVLM (K), and brain cortex (L).

Figure 2. The PRR is up-regulated in the SFO and PVN of RA mice

Immunotstaining shows PRR expression in the SFO and PVN of NT mice (A and E) and RA mice (B and F). C and G represent quantitative data for PRR immunostaining in the SFO and PVN. D and H represent PRR mRNA levels from micro-punched SFO and PVN (n=4/group).* P<0.01 vs. NT mice (Student t test).

Figure 3. AAV-PRR-shRNA knocks down PRR expression in Neuro-2A cells and ICV administration infects the SFO

PRR levels from Neuro-2A cells infected with AAV-PRR-shRNA or AAV-eGFP were detected by real-time PCR (A) and western blotting (B) (n=4/group). Representative pictures show Neuro-2A cells 3 days after AAV-PRR-shRNA infection (C) and SFO thress days after ICV administration of AAV-PRR-shRNA (D). * P<0.01 vs. AAV-eGFP treatment (Student t test).

Figure 4. ICV administration of AAV-PRR-shRNA mediates PRR knockdown in SFO

AAV-PRR-shRNA significantly decreases PRR mRNA levels in the SFO of both NT and RA mice (A, n=4/group), but not in the PVN (B, n=8/group).* P<0.01 vs. NT mice with AAV-eGFP, # P<0.01 vs. RA mice with AAV-eGFP (Two-way ANOVA).

Figure 5. Brain-targeted PRR knockdown attenuates hypertension in RA mice

Continuously telemetric BP was recorded in conscious NT and RA mice for baseline and after ICV injection (A). SBRS was calculated using the sequence method (B). Cardiac sympathetic tone (C), vasomotor sympathetic tone (D), parasympathetic tone (E), and intrinsic heart rate (F) were determined following administration of propranolol, chlorisondamine, and methylatropine. n=4/group, * P<0.05 vs. NT mice with AAV-eGFP, # P<0.05 vs. RA mice with AAV-eGFP (Two-way ANOVA).

Figure 6. AT1 receptor and AVP levels are decreased following PRR knockdown in the RA mice

AT1 receptor mRNA levels (A, n=4/group) were detected by real time PCR and protein levels (B, n=10/group) were detected by western blotting in NT and RA mice with ICV delivery of AAV-PRR-shRNA or control virus. C and D (n=5/group) represent AVP mRNA level in the PVN and plasma AVP level of NT and RA mice after ICV delivery of AAV-PRR-shRNA or control virus.* P<0.01 in Figure A and B, P<0.05 in Figure C and D vs. NT mice with AAV-eGFP, # P<0.01 in Figure A and B, P<0.05 in Figure C and D vs. RA mice with AAV-eGFP (Two-way ANOVA).