New Findings What is the central question of this study?We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension. What is the main finding and its importance?The main finding is that PVN‐specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension in Sprague–Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. Abstract The central mechanisms underlying salt‐sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt‐resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein‐dependent, salt‐sensitive hypertension. Male Sprague–Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal‐salt (NS; 0.6% NaCl) or high‐salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co‐infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro‐inflammatory cytokines TNFα, IL‐1β and IL‐6 and the anti‐inflammatory cytokine IL‐10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt‐sensitive hypertension, increased plasma noradrenaline, PVN pro‐inflammatory cytokine mRNA upregulation, anti‐inflammatory cytokine mRNA downregulation and PVN‐specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co‐infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN‐infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial‐mediated PVN inflammation and the development of salt‐sensitive hypertension.

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α 1 - and β-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α 1 -adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine–evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt–sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α 1 -adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt–sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α 1 -adrenoceptor antagonism initiated before high–salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α 1 -adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (μeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

Aim: We hypothesize paraventricular nucleus (PVN)-specific blood brain barrier (BBB) disruption and neuroinflammation increase hypertension and sympathoexcitation with age, promoting cognitive impairment, that can be attenuated by an Angiotensin II Type 1 Receptor (AT 1 R)-dependent mechanism in the aging Sprague Dawley (SD) rat. Methods: In male and female SD rats aged 3, 8 and 16 months old (MO) (N=6/gp) and 16 MO male rats treated with losartan (21 days; sc 3 mg/kg/day), blood pressure (BP; femoral artery cannulation), sympathetic tone to the vasculature (iv hexamethonium) and plasma NE (ELISA) was measured. Memory function was assessed by the novel object recognition task. BBB disruption was assessed via FITC extravasation and IHC/IF was performed for microglia (CD11b/c), astrocytes (GFAP), IL-6 and TNF-α in the PVN. Results: Aged male, but not female, SD rats develop HTN, sympathoexcitation, and cognitive impairment. PVN neuroinflammation, proinflammatory cytokine production and BBB disruption increased in male, but not female, rats with age. Losartan significantly lowered BP, reduced sympathoexcitation, attenuated BBB disruption and neuroinflammation, and reversed cognitive impairment in aged male rats. Conclusions: Our data suggest there is a potential protective role of female sex steroids in preventing the development of age-dependent HTN which is associated with PVN neuroinflammation. Concurrent with recent clinical findings, we found lowering blood pressure improved cognitive function and, thus, an AT 1 R antagonism represents a new therapeutic modality to improve cognitive performance in hypertensive individuals.

Aim: Failure to upregulate hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins promotes salt sensitive hypertension (ssHTN) and central inflammation plays a role in HTN. We hypothesize that 1) PVN-specific inflammation contributes to Gαi 2 protein dependent ssHTN, and 2) GNAI2 SNPs correlate with HTN. Methods: 3-month-old male Sprague Dawley rats were implanted with I.C.V. cannulas fitted to osmotic minipumps and infused with Gαi 2 or control scrambled (SCR) oligodeoxynucleotides (ODN) (25μg/5μl/day), or ODN + minocycline (MINO) (120μg/5μl/day, HS only) and placed on a 7-day normal (NS; 0.6% NaCl) or high (HS; 4% NaCl) salt diet. Day 7 BP (femoral cannulation) and Plasma NE (ELISA) were measured. PVN and subfornical organ microglia and astrocyte activation (IHC), and PVN pro-inflammatory (PIC) and anti-inflammatory (AIC) cytokine mRNA and protein levels were analyzed. GNAI2 SNPs were examined in the UK BioBank for associations with HTN. Results: In contrast to SCR ODN infusion, Gαi 2 ODN infusion and HS evoked HTN, elevated plasma NE, PVN-specific microgliosis, upregulation of PVN PICs and downregulation of PVN IL-10. MINO co-infusion with Gαi 2 ODN and HS diet restored PIC and AIC mRNA and protein levels. Furthermore, MINO attenuated microgliosis and reduced the magnitude of ssHTN. GNAI2 SNPs rs2298952 (p=0.0411, MAF=0.103) and rs4547694 (p=0.0117, MAF=0.381) are both associated with essential HTN in BioBank subjects. Conclusion: PVN-specific microgliosis and inflammation may drive Gαi 2 protein dependent ssHTN and GNAI2 SNPs represent a potential biomarker of essential HTN.