(Pro)renin receptor knockdown in the paraventricular nucleus of the hypothalamus attenuates hypertension development and AT1 receptor-mediated calcium events

Abstract

Activation of the brain renin-angiotensin system (RAS) is a pivotal step in the pathogenesis of hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a critical part of the angiotensinergic sympatho-excitatory neuronal network involved in neural control of blood pressure and hypertension. However, the importance of the PVN (pro)renin receptor (PVN-PRR)-a key component of the brain RAS-in hypertension development has not been examined. In this study, we investigated the involvement and mechanisms of the PVN-PRR in DOCA-salt-induced hypertension, a mouse model of hypertension. Using nanoinjection of adeno-associated virus-mediated Cre recombinase expression to knock down the PRR specifically in the PVN, we report here that PVN-PRR knockdown attenuated the enhanced blood pressure and sympathetic tone associated with hypertension. Mechanistically, we found that PVN-PRR knockdown was associated with reduced activation of ERK (extracellular signal-regulated kinase)-1/2 in the PVN and rostral ventrolateral medulla during hypertension. In addition, using the genetically encoded Ca²⁺ biosensor GCaMP6 to monitor Ca²⁺-signaling events in the neurons of PVN brain slices, we identified a reduction in angiotensin II type 1 receptor-mediated Ca²⁺ activity as part of the mechanism by which PVN-PRR knockdown attenuates hypertension. Our study demonstrates an essential role of the PRR in PVN neurons in hypertension through regulation of ERK1/2 activation and angiotensin II type 1 receptor-mediated Ca²⁺ activity. NEW & NOTEWORTHY PRR knockdown in PVN neurons attenuates the development of DOCA-salt hypertension and autonomic dysfunction through a decrease in ERK1/2 activation in the PVN and RVLM during hypertension. In addition, PRR knockdown reduced AT_1aR expression and AT₁R-mediated calcium activity during hypertension. Furthermore, we characterized the neuronal targeting specificity of AAV serotype 2 in the mouse PVN and validated the advantages of the genetically encoded calcium biosensor GCaMP6 in visualizing neuronal calcium activity in the PVN.

Keywords: (pro)renin receptor; GCaMP6; calcium; central nervous system; hypertension; paraventricular nucleus of the hypothalamus; renin-angiotensin system.

Fig. 1.

Expression and cellular localization of the PRR in the PVN of the mouse hypothalamus. The PVNs of WT C57Bl/6J mice were immunofluorescently labeled for the PRR (red), GFAP (green), and NeuN (blue) (A). The PVNs of CX3CR1-GFP reporter mice were immunofluorescently labeled for the PRR (red) and counterstained with DAPI (blue); white arrows indicate colocalization of the PRR with GFP (B). No primary antibody controls using secondary antibodies conjugated with Alexa 594 (red), Alexa 488 (green), or Alexa 647 (blue) (C). Validating the specificity of the PRR antibody by Western blot analysis of whole cell extracts of Neuro2A cells infected with scrambled (SC) or PRR shRNA (D). PVNs are shown in a dashed outline; the green dashed boxes are areas that appear in higher magnification on the right. 3V, third ventricle; AAV, adeno-associated virus; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; PRR, (pro)renin receptor; PVN, paraventricular nucleus; WT, wild-type.

Fig. 2.

Characterization of AAV2 targeting in the PVN of the hypothalamus. AAV2-eGFP was bilaterally injected into the PVN of PRR-floxed mice; 7 days later, mouse brains were collected for imaging. A and B: schematic showing the bilateral PVN injection site and stereotaxic injection coordinates. C: representative images of eGFP fluorescence following bilateral injection of AAV2-eGFP into the PVN. D: representative images showing the specificity of PVN injection in slices from the SFO and RVLM. E: representative images of immunolabeling for the neuronal cell marker NeuN (red), astrocyte marker GFAP (red), and microglial marker IBA1 (red), and their colocalization with eGFP (green). White arrows indicate colocalization of eGFP with NeuN, and arrowheads indicate no colocalization of eGFP with either GFAP or Iba1. 3V, third ventricle; AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; PRR, (pro)renin receptor; PVN, paraventricular nucleus; RVLM, rostral ventrolateral medulla; SFO, subfornical organ.

Fig. 3.

PRR knockdown in the PVN by AAV2-delivered Cre recombinase. A: representative images of mouse PRR (red) immunostaining and eGFP fluorescence (green) in the PVN. White dashed circles in higher magnification images (10 μm) indicate representative cells expressing both PRR and eGFP. B: semiquantitative analysis of the relative intensity of PRR fluorescence signals in eGFP-positive cells. C: number of eGFP-positive cells in each PVN (n = 7 PVNs from 3 mice/group). D: PRR mRNA levels in the PVN, cortex, and brain stem (BS), determined by RT-qPCR (*P < 0.05 vs. AAV-Con; n = 3 mice/group, 2 PVNs from each mouse were combined). 3V, third ventricle; AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; PaPo, paraventricular hypothalamic nucleus, posterior part; PaMP, paraventricular hypothalamic nucleus, medial parvicellular part; PRR, (pro)renin receptor; PVN, paraventricular nucleus; RFU, relative fluorescence units; RT-qPCR, reverse-transcription quantitative PCR.

Fig. 4.

Knockdown of the PRR in the PVN attenuates the development of DOCA-salt-induced hypertension and dysautonomia. A: schematic of the experimental protocol. B and C: mean arterial pressure (MAP) and HR at baseline and during 21 days of DOCA-salt treatment. D: vasomotor sympathetic tone, measured as changes in MAP in response to intraperitoneal injection of 6 mg/kg chlorisondamine. E: cardiac sympathetic tone, measured as changes in HR in response to intraperitoneal injection of 5 mg/kg propranolol. F: cardiac parasympathetic tone, measured as changes in HR in response to intraperitoneal injection of 1 mg/kg methylatropine. n = 4–8 mice/group; *P < 0.05 vs. AAV-Con sham; **P < 0.01 vs. AAV-Con Sham; $P < 0.05 vs. AAV-Con DOCA-salt. AAV, adeno-associated virus; HR, heart rate; PRR, (pro)renin receptor; PVN, paraventricular nucleus.

Fig. 5.

PVN-targeted knockdown of the PRR reduces ERK1/2 activation in the PVN and RVLM. Schematics illustrating the activation of ERK1/2 by the PRR and effect of PVN-PRR knockdown on ERK1/2 activation (A). Representative images (B) and summary data (C) quantifying p-ERK1/2 immunostaining intensity in the SFO, PVN, and RVLM of AAV-Con sham, AAV-Con DOCA-Salt, and AAV-Cre DOCA-Salt groups following 2 wk of DOCA-salt or sham treatment. *P < 0.05 vs. AAV-Con Sham; **P < 0.01 vs. AAV-Con sham; ***P < 0.001 vs. AAV-Con Sham; $$$P < 0.001 vs. AAV-Con DOCA-Salt (n = 2–5 brain sections from each mouse, 3–5 mice/group). AAV, adeno-associated virus; PRR, (pro)renin receptor; PVN, paraventricular nucleus; RFU, relative fluorescence units; RVLM, rostral ventrolateral medulla; SFO, subfornical organ.

Fig. 6.

Effects of ICV-infused losartan on ERK1/2 activation in the SFO, PVN, and RVLM. Schematics illustrating activation of ERK1/2 by the PRR and the pathways by which chronically ICV-infused losartan inhibits ERK1/2 activation (A). Representative images (B) and summary data (C) quantifying p-ERK1/2 immunostaining in the SFO, PVN, and RVLM following 2 wk of chronic ICV infusion of aCSF or losartan (3 mg·kg⁻¹·day⁻¹) (n = 3–5 brain sections per mouse, 3 mice/group). Data normalized and replotted from Fig. 5C (dotted gray squares) and Fig. 6C (D). *P < 0.05 vs. ICV aCSF; **P < 0.01 vs. ICV aCSF; ****P < 0.001 vs. ICV aCSF. $$$P < 0.001 vs. AAV-Con DOCA-Salt; ^##P < 0.01 vs. ICV aCSF; ###P < 0.001 vs. PVN AAV-Cre DOCA-Salt. aCSF, artificial cerebrospinal fluid; ICV, intracerebroventricular; PRR, (pro)renin receptor; PVN, paraventricular nucleus; RFU, relative fluorescence units; RVLM, rostral ventrolateral medulla; SFO, subfornical organ.

Fig. 7.

PVN-PRR knockdown regulates angiotensin receptor expression. AT_1aR (A), AT₂R (B), and MasR (C) mRNA levels. **P < 0.01 vs. AAV-Con Sham; ***P < 0.001 vs. AAV-Con sham; $$P < 0.01 vs. AAV-Con DOCA-Salt; $$$P < 0.001 vs. AAV-Con DOCA-Salt (n = 3–4 mice/group). AAV, adeno-associated virus; AT_1aR, angiotensin II type 1a receptor; AT₂R, angiotensin II type 2 receptor; PRR, (pro)renin receptor; PVN, paraventricular nucleus.

Fig. 8.

PVN-PRR knockdown regulates AT₁R-mediated Ca²⁺-signaling events in the PVN. Representative snapshot images (A) and 3-D images from 3-D FOV reconstructions (B) of Ca²⁺ activity within PVN slices of AAV-Con Sham, AAV-Con DOCA-Salt, and AAV-Cre DOCA-Salt mice under control conditions and following treatment with ANG II (100 nM) or KCl (100 mM). Summary data for live-cell Ca²⁺ imaging showing amplitude (C), sites/FOV (D), and frequency (E) in response to ANG II, detected using the Ca²⁺ biosensor GCaMP6, in slices containing the PVN from AAV-Con sham, AAV-Con DOCA-Salt, and AAV-Cre DOCA-Salt mice. *P < 0.05 vs. AAV-Con sham in the same treatment group (vehicle or ANG II); ****P < 0.0001 vs. AAV-Con sham in the same treatment group (vehicle or ANG II); $$$$P < 0.0001 vs. AAV-Con DOCA-salt in the ANG II group; #P < 0.05 ANG II vs. vehicle or ANG II vs. ANG II + losartan within the same treatment group; ##P < 0.01 ANG II vs. Vehicle or ANG II vs. ANG II + losartan within the same treatment group; ####P < 0.0001 ANG II vs. vehicle or ANG II vs. ANG II + losartan within the same treatment group (AAV-Con sham, AAV-Con DOCA-salt, AAV-Cre DOCA-Salt) (n = 4 mice/group). 3-D, three-dimensional; AAV, adeno-associated virus; ANG II, angiotensin II; AT₁R, angiotensin II type 1 receptor; AT₂R, angiotensin II type 2 receptor; FOV, field of view; PRR, (pro)renin receptor; PVN, paraventricular nucleus.