Cell‐Type Specific Angiotensin II‐Driven Nrf2 Impairment within Central Cardioregulatory Nuclei of Spontaneously Hypertensive Rats

Abstract

Angiotensin II (AngII) plays a primary role in the pathophysiology of hypertension, directly influencing glia and neurons through its type I receptor (AT1R) within central cardioregulatory nuclei. Yet, the exact cell type‐specific signaling pathway(s) by which AngII contributes to a hypertensive state are unclear. We hypothesized that AngII impairs cellular antioxidant defense systems via Nrf2 (nuclear factor erythroid 2‐related factor 2) dysregulation, resulting in astrogliosis and contributing to neuroinflammation. 12–13 week‐old male spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs) were used to examine protein (immunofluorescence [IF]) and mRNA (RT‐PCR) in the hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM). High‐magnification confocal imaging of IF assays (primary antibodies: interleukin [IL]‐6, tumor necrosis factor [TNF]‐α, Nrf2, GFAP [astrocyte marker], and NeuN [neuron marker]) were analyzed with ImageJ to determine single‐cell and subcellular localization of Nrf2 colocalized with NeuN or GFAP in the whole cell, the nucleus, and the non‐nuclear (cytosolic) cell regions. IL‐6 and TNF‐α IF densities were elevated in SHR PVN (IL‐6: +19.9±5.2; TNF‐α: +147.2±22.5% vs. WKY) and RVLM (IL‐6: +52.2±7.4; TNF‐α: +119.6±19.9% vs. WKY), indicating a pro‐inflammatory cytokine profile in hypertensive animals that is consistent with astrocyte reactivity. Relative to WKYs, Nrf2 IF density was reduced in SHR neurons of the PVN (cell: −43.3±4.0, nucleus: −33.3±6.7, cytosol: − 63.9±6.8% vs. WKY) and the RVLM (cell: −31.0±2.9, nucleus: −55.2±6.8, cytosol: −46.7±5.9% vs. WKY). Similarly, Nrf2 protein density was diminished in SHR astrocytes within the PVN (cell: −55.2±5.0, nucleus: −71.9±4.0, cytosol: −54.1±5.7% vs. WKY) and RVLM (cell: −50.5±6.4, nucleus: −72.9±3.7, cytosol: −61.8±4.5% vs. WKY). In addition, PVN punches from SHRs showed increased mRNA levels of the Nrf2 repressor Keap1 (2.1±0.2), alongside reductions in Nrf2‐regulated antioxidant genes, Gpx1 (0.7±0.1) and NQO1 (0.5±0.1 fold change), compared to WKYs. To determine the relative contribution of AngII, SHRs were treated with Losartan (AT1R inhibitor; 20mg/kg/day; gavage; SHRLos). After four weeks, mean arterial pressure (tail‐cuff volume‐pressure system [CODA‐6, Kent Scientific]) was normalized in SHRLos (SHR: 154.9±1.7; SHRLos: 103.6±2.4mmHg), and the alterations in Nrf2 protein density were reversed in neurons (PVN: cell: −2.8±6.8, nucleus: −5.8±6.5, cytosol: +4.6±13.9; RVLM: cell: +1.3±6.8, nucleus: +2.8±13.6, cytosol: −2.8±13.9% vs. WKY) and astrocytes (PVN: cell: −3.9±9.0, nucleus: +1.2±11.9, cytosol: −4.1±10.2; RVLM: cell: +5.1±18.5; nucleus: −5.6±15.6; cytosol: −7.1±19.2% vs. WKY). Furthermore, AT1R inhibition abolished the increased levels of GFAP IF density observed within the PVN of SHRs (WKY: 15.9±0.6, SHR: 26.4±0.7, SHRLos: 15.3±0.6% area). Taken together, these findings suggest that AngII impairs Nrf2‐based antioxidant defense systems and promotes astrocyte reactivity towards a pro‐inflammatory phenotype within the PVN and RVLM of hypertensive animals.Support or Funding InformationThis work was funded by AHDR, Auburn University to VCB.