Tumor Necrosis Factor α Receptor Type 1 Activation in the Hypothalamic Paraventricular Nucleus Contributes to Glutamate Signaling and Angiotensin II-Dependent Hypertension.

There are significant neurogenic and inflammatory influences on blood pressure, yet the role played by each of these processes in the development of hypertension is unclear. Tumor necrosis factor α (TNFα) has emerged as a critical modulator of blood pressure and neural plasticity; however, the mechanism by which TNFα signaling contributes to the development of hypertension is uncertain. We present evidence that following angiotensin II (AngII) infusion the TNFα type 1 receptor (TNFR1) plays a key role in heightened glutamate signaling in the hypothalamic paraventricular nucleus (PVN), a key central coordinator of blood pressure control. Fourteen day administration of a slow-pressor dose of AngII in male mice was associated with transcriptional and post-transcriptional (increased plasma membrane affiliation) regulation of TNFR1 in the PVN. Further, TNFR1 was shown to be critical for elevated NMDA-mediated excitatory currents in sympathoexcitatory PVN neurons following AngII infusion. Finally, silencing PVN TNFR1 prevented the increase in systolic blood pressure induced by AngII. These findings indicate that TNFR1 modulates a cellular pathway involving an increase in NMDA-mediated currents in the PVN following AngII infusion, suggesting a mechanism whereby TNFR1 activation contributes to hypertension via heightened hypothalamic glutamate-dependent signaling.SIGNIFICANCE STATEMENT Inflammation is critical for the emergence of hypertension, yet the mechanisms by which inflammatory mediators contribute to this dysfunction are not clearly defined. We show that tumor necrosis factor α receptor 1 (TNFR1) in the paraventricular hypothalamic nucleus (PVN), a critical neuroregulator of cardiovascular function, plays an important role in the development of hypertension in mice. In the PVN, TNFR1 expression and plasma membrane localization are upregulated during hypertension induced by angiotensin II (AngII). Further, TNFR1 activation was essential for NMDA signaling and the heightening NMDA currents during hypertension. Finally, TNFR1 silencing in the PVN inhibits elevated blood pressure induced by AngII. These results point to a critical role for hypothalamic TNFR1 signaling in hypertension.

Estrogen Receptor ß Contributes to Both Hypertension and Hypothalamic Plasticity in a Mouse Model of Peri-Menopause.

Hypertension susceptibility in women increases at the transition to menopause, termed perimenopause, a state characterized by erratic estrogen fluctuation and extended hormone cycles. Elucidating the role of estrogen signaling in the emergence of hypertension during perimenopause has been hindered by animal models that are confounded by abrupt estrogen cessation or effects of aging. In the present study, accelerated ovarian failure (AOF) in estrogen receptor ß (ERß) reporter mice was induced by 4-vinylcyclohexene diepoxide in young mice to model early-stage ovarian failure (peri-AOF) characteristic of peri-menopause. It was found that administering ERß agonists suppressed elevated blood pressure in a model of neurogenic hypertension induced by angiotensin II (AngII) in peri-AOF, but not in age-matched male mice. It was also found that ERß agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERß neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERß in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERß signaling in the PVN plays an important role in blood pressure regulation in female mice and contributes to hypertension susceptibility in females at an early stage of ovarian failure comparable to human perimenopause.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Tumor necrosis factor alpha induces NOX2-dependent reactive oxygen species production in hypothalamic paraventricular nucleus neurons following angiotensin II infusion.

There is evidence that tumor necrosis factor alpha (TNFα) influences autonomic processes coordinated within the hypothalamic paraventricular nucleus (PVN), however, the signaling mechanisms subserving TNFα's actions in this brain area are unclear. In non-neuronal cell types, TNFα has been shown to play an important role in canonical NADPH oxidase (NOX2)-mediated production of reactive oxygen species (ROS), molecules also known to be critically involved in hypertension. However, little is known about the role of TNFα in NOX2-dependent ROS production in the PVN within the context of hypertension. Using dual labeling immunoelectron microscopy and dihydroethidium (DHE) microfluorography, we provide structural and functional evidence for interactions between TNFα and NOX2 in the PVN. The TNFα type 1 receptor (TNFR1), the major mediator of TNFα signaling in the PVN, was commonly co-localized with the catalytic gp91phox subunit of NOX2 in postsynaptic sites of PVN neurons. Additionally, there was an increase in dual labeled dendritic profiles following fourteen-day slow-pressor angiotensin II (AngII) infusion. Using DHE microfluorography, it was also shown that TNFα application resulted in a NOX2-dependent increase in ROS in isolated PVN neurons projecting to the spinal cord. Further, TNFα-mediated ROS production was heightened after AngII infusion. The finding that TNFR1 and gp91phox are positioned for rapid interactions, particularly in PVN-spinal cord projection neurons, provides a molecular substrate by which inflammatory signaling and oxidative stress may jointly contribute to AngII hypertension.

Angiotensin II Infusion Results in Both Hypertension and Increased AMPA GluA1 Signaling in Hypothalamic Paraventricular Nucleus of Male but not Female Mice.

The hypothalamic paraventricular nucleus (PVN) plays a key role in hypertension, however the signaling pathways that contribute to the adaptability of the PVN during hypertension are uncertain. We present evidence that signaling at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluA1 receptor contributes to increased blood pressure in a model of neurogenic hypertension induced by 14-day slow-pressor angiotensin II (AngII) infusion in male mice. It was found that AngII hypertension was associated with an increase in plasma membrane affiliation of GluA1, but decreased GluA2, in dendritic profiles of PVN neurons expressing the TNFα type 1 receptor, a modulator of AMPA receptor trafficking. The increased plasma membrane GluA1 was paralleled by heightened AMPA currents in PVN-spinal cord projection neurons from AngII-infused male mice. Significantly, elevated AMPA currents in AngII-treated mice were blocked by 1-Naphthyl acetyl spermine trihydrochloride, pointing to the involvement of GluA2-lacking GluA1 receptors in the heightened AMPA signaling in PVN neurons. A further functional role for GluA1 in the PVN was demonstrated by the attenuated hypertensive response following silencing of GluA1 in the PVN of AngII-infused male mice. In female mice, AngII-infusion did not impact blood pressure or plasma membrane localization of GluA1 . Post-translational modifications that increase the plasma membrane localization of AMPA GluA1 and heighten the rapid excitatory signaling actions of glutamate in PVN neurons may serve as a molecular substrate underlying sex differences in hypertension.

Estrogen receptor beta activity contributes to both tumor necrosis factor alpha expression in the hypothalamic paraventricular nucleus and the resistance to hypertension following angiotensin II in female mice.

Sex differences in the sensitivity to hypertension and inflammatory processes are well characterized but insufficiently understood. In male mice, tumor necrosis factor alpha (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension following slow-pressor angiotensin II (AngII) infusion. However, the role of PVN TNFα in the response to AngII in female mice is unknown. Using a combination of in situ hybridization, high-resolution electron microscopic immunohistochemistry, spatial-temporal gene silencing, and dihydroethidium microfluorography we investigated the influence of AngII on both blood pressure and PVN TNFα signaling in female mice. We found that chronic (14-day) infusion of AngII in female mice did not impact blood pressure, TNFα levels, the expression of the TNFα type 1 receptor (TNFR1), or the subcellular distribution of TNFR1 in the PVN. However, it was shown that blockade of estrogen receptor ß (ERß), a major hypothalamic estrogen receptor, was accompanied by both elevated PVN TNFα and hypertension following AngII. Further, AngII hypertension following ERß blockade was attenuated by inhibiting PVN TNFα signaling by local TNFR1 silencing. It was also shown that ERß blockade in isolated PVN-spinal cord projection neurons (i.e. sympathoexcitatory) heightened TNFα-induced production of NADPH oxidase (NOX2)-mediated reactive oxygen species, molecules that may play a key role in mediating the effect of TNFα in hypertension. These results indicate that ERß contributes to the reduced sensitivity of female mice to hypothalamic inflammatory cytokine signaling and hypertension in response to AngII.