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.

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.

There are profound, yet incompletely understood, sex differences in the neurogenic regulation of blood pressure. Both corticotrophin signaling and glutamate receptor plasticity, which differ between males and females, are known to play important roles in the neural regulation of blood pressure. However, the relationship between hypertension and glutamate plasticity in corticotrophin-releasing hormone (CRF) receptive neurons in brain cardiovascular regulatory areas, including the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), is not understood. In the present study, we used dual label immunoelectron microscopy to analyze sex differences in slow-pressor angiotensin II (AngII) hypertension with respect to the subcellular distribution of the obligatory GluN1 subunit of the N-methyl-D-aspartate receptor (NMDAR) in the RVLM and PVN. Studies were conducted in mice expressing the enhanced green fluorescence protein (EGFP) under the control of the CRF type 1 receptor (CRF1) promoter (i.e., CRF1-EGFP reporter mice). By light microscopy, GluN1-immunoreactivity was found in CRF1-EGFP neurons of the RVLM and PVN. Moreover, in both regions tyrosine hydroxylase was found in CRF1-EGFP neurons. In response to AngII, male mice showed an elevation in blood pressure that was associated with an increase in the proportion of GluN1 on presumably functional areas of the plasma membrane in CRF1-EGFP dendritic profiles in the RVLM. In female mice, AngII was neither associated with an increase in blood pressure nor an increase in plasma membrane GluN1 in the RVLM. Unlike the RVLM, AngII-mediated hypertension had no effect on GluN1 localization in CRF1-EGFP dendrites in the PVN of either male or female mice. These studies provide an anatomical mechanism for sex-differences in the convergent modulation of RVLM catecholaminergic neurons by CRF and glutamate. Moreover, these results suggest that sexual dimorphism in AngII-induced hypertension is reflected by NMDA receptor trafficking in presumptive sympathoexcitatory neurons in the RVLM.

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 a (TNFa) has emerged as a critical modulator of blood pressure and neural plasticity; however, the mechanism by which TNFa signaling contributes to the development of hypertension is uncertain. We present evidence that following angiotensin II (AngII) infusion the TNFa 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.

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 b (ERb) 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 ERb 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 ERb agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERb neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERb in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERb 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.

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