Knockdown of mineralocorticoid or angiotensin II type 1 receptor gene expression in the paraventricular nucleus prevents angiotensin II hypertension in rats

Abstract

Circulating Ang II activates an aldosterone-mineralocorticoid receptor (MR) - angiotensin II (Ang II) - angiotensin type 1 receptor (AT1R) pathway in the hypothalamus. To obtain insights into the actual neuronal projections involved, adeno-associated virus carrying small interfering RNA against either AT1aR (AAV-AT1aR-siRNA) or MR (AAV-MR-siRNA) were infused into the paraventricular nucleus (PVN) in Wistar rats. Intra-PVN infusion of AAV-AT1aR-siRNA or AAV-MR-siRNA decreased AT1R or MR expression in the PVN but not in the subfornical organ (SFO) or supraoptic nucleus (SON). Subcutaneous infusion of Ang II at 500 ng kg(-1) min(-1) for 2 weeks increased mean arterial pressure by 60-70 mmHg, and increased AT1R and MR expression in the SFO, SON and PVN. Intra-PVN AT1aR-siRNA prevented the Ang II-induced increase in AT1R but not MR expression in the PVN, and MR-siRNA prevented MR but not AT1R expression in the PVN. The increases in AT1R and MR expression in both the SFO and the SON were not changed by the two AAV-siRNAs. Specific knockdown of AT1R or MR in the PVN by AAV-siRNA each prevented most of the Ang II-induced hypertension. Prevention of the subcutaneous Ang II-induced increase in MR but not the increase in AT1R by knockdown of MR and vice versa suggests an independent regulation of MR and AT1R expression in the PVN. Both AT1R and MR activation in the PVN play a critical role in Ang II-induced hypertension in rats.

Figure 1. MR mRNA and protein expression and AT₁R mRNA in the PVN, SON and SFO

MR mRNA and protein expression (A, B) and AT₁R mRNA (C) in the PVN, SON and SFO 2 weeks after intra-PVN infusion of AAV-SCM-siRNA (n = 4), AAV-AT₁R-siRNA (n = 5), 2.5 or 5 × 10⁷ genomic particles or AAV-MR-siRNA (n = 5), 2.5×10⁷ genomic particles. Values are means ± SEM, normalized with PGK 1 (mRNA) or β-actin (protein). Values for MR/β-actin in the SCM-siRNA + saline group were normalized to 1. *P < 0.05 versus other(s); a, P < 0.05 versus 2.5×10⁷ genomic particles.

Figure 2. Representative images of eGFP fluorescence in the PVN

eGFP expression was evaluated in brain sections of a rat infused in the PVN with AAV-AT_1aR-siRNA at 25 × 10⁷ genomic particles. Red arrows indicate eGFP fluorescence in the PVN, and the white arrows point to the SFO or SON. The higher magnification image shows eGFP expression in both neurons and glia in the PVN. Control image was obtained from a rat without any AAV infusion.

Figure 3. MAP and HR after intra-PVN infusion of Ang II

Maximal increases in MAP and HR after intra-PVN infusion of Ang II at 100, 300 or 600 ng min⁻¹ for 5 min at 2 weeks after intra-PVN infusion of AAV-SCM-siRNA or AAV-AT₁R-siRNA at 2.5 or 5 × 10⁷ genomic particles. Values are means ± SEM (n = 4 per group). *P < 0.05 versus resting values; a, P < 0.05 versus others.

Figure 4. AT₁R and MR protein expression in the PVN

AT₁R and MR protein expression in the PVN after intra-PVN infusion of AAV-SCM-siRNA, AAV-AT₁R-siRNA or AAV-MR-siRNA at 5 × 10⁷ genomic particles, and sc infusion of Ang II at 500 ng kg⁻¹ min⁻¹ or saline for 2 weeks. Values are means ± SEM, normalized with β-actin (n = 5 per group). Values for AT₁R or MR/β-actin in the SCM-siRNA + saline group were normalized to 1. *P < 0.05 versus others; a, P < 0.05 versus SCM-siRNA.

Figure 5. AT₁R and MR mRNA expression in the PVN, SFO and SON

AT₁R and MR mRNA expression in the PVN, SFO and SON after intra-PVN infusion of AAV-SCM-siRNA at 5 × 10⁷ genomic particles, AAV-AT₁R-siRNA at 2.5 (low) or 25 (high)  × 10⁷ genomic particles or AAV-MR-siRNA at 5 × 10⁷ genomic particles, followed by sc infusion of Ang II at 500 ng kg⁻¹ min⁻¹ for 2 weeks. Values are means ± SEM, normalized with PGK (n = 4–5 per group). *P < 0.05 versus others.

Figure 6. Responses of MAP to sc infusion of Ang II

Responses of MAP to sc infusion of Ang II at 150 or 500 ng kg⁻¹ min⁻¹ for 2 weeks after intra-PVN infusion of AAV-SCM-siRNA, AAV-AT₁R-siRNA at 2.5 (low) or 25 (high) × 10⁷ genomic particles, or AAV-MR-siRNA at 5 × 10⁷ genomic particles. Values are means ± SEM (n = 4–6). *P < 0.05 versus AAV-AT₁R-siRNA at 25 × 10⁷ or AAV-MR-siRNA; a, P < 0.05 versus baseline.