Probes for INS

The brain renin-angiotensin system plays important roles in blood pressure and cardiovascular regulation. There are two isoforms of prorenin in the brain: the classic secreted form (prorenin/sREN) encoded by renin-a, and an intracellular form (icREN) encoded by renin-b. Emerging evidence indicates the importance of renin-b in cardiovascular and metabolic regulation. However, the role of endogenous brain prorenin in the development of salt-sensitive hypertension remains undefined. In this study, we test the hypothesis that renin-a produced locally in the brain contributes to the pathogenesis of hypertension. Using RNAscope, we report for the first time that renin mRNA is expressed in several regions of the brain, including the subfornical organ (SFO), the paraventricular nucleus of the hypothalamus (PVN), and the brainstem, where it is found in glutamatergic, GABAergic, cholinergic, and tyrosine hydroxylase-positive neurons. Notably, we found that renin mRNA was significantly elevated in the SFO and PVN in a mouse model of DOCA-salt-induced hypertension. To examine the functional importance of renin-a in the SFO, we selectively ablated renin-a in the SFO in renin-a-floxed mice using a Cre-lox strategy. Importantly, renin-a ablation in the SFO attenuated the maintenance of DOCA-salt-induced hypertension and improved autonomic function without affecting fluid or sodium intake. Molecularly, ablation of renin-a prevented the DOCA-salt-induced elevation in NADPH oxidase 2 (NOX2) in the SFO without affecting NOX4 or angiotensin II type 1 and 2 receptors. Collectively, our findings demonstrate that endogenous renin-a within the SFO is important for the pathogenesis of salt-sensitive hypertension.

Epidermal keratinocytes express various purinergic 2 receptors that play an essential role in cell growth, differentiation, and proliferation. In the conditions of injury, concentrations of extracellular adenosine triphosphate (ATP) may dramatically increase due to cell damage and inflammatory processes. In this situation activation of purinergic signaling in keratinocytes could act as a double-edged sword contributing to skin regeneration or cell apoptosis. As the role of keratinocytes in transducing and modulating nociceptive stimuli has been increasingly appreciated in recent years, the aim of the present study was to evaluate whether peripheral nerve injury affects purinergic signaling in keratinocytes. Spared nerve injury (SNI), a classical model of peripheral neuropathic pain, was induced in mice. The injury was induced by sparing of the tibial nerve, and ligation and cut of the sural and common peroneal nerves. Keratinocytes were isolated and cultured on Days 2-4 post-injury and ATP-mediated calcium responses in keratinocytes were examined by confocal imaging. On average, the number of keratinocytes that responded to ATP with an increase in intracellular calcium gradient as well as the magnitude of the peak response was not significantly different between sham and SNI groups. However, significantly less delay in ATP-induced increase in intracellular calcium concentration was observed in keratinocytes in SNI group compared to sham. Selective pharmacological inhibition of keratinocyte response to ATP indicated a major role of P2 × 4 receptors in the modulation of calcium homeostasis in SNI. Our results indicate that epidermal purinergic signaling undergoes dramatic changes following peripheral nerve injury that may contribute to injury-induced mechanical hypersensitivity.

Objectives: We recently observed that Transient Receptor Potential V2 knockout (TRPV2 KO) mice show late-onset fetal growth restriction and perinatal lethality, which are most severe on a C57BL6 compared to 129Sv background. In the placenta of both strains, TRPV2 expression is confined to the labyrinth zone (Lz). Here, we investigated whether there were strain-specific alterations in placental morphology and nutrient transport that may underlie the difference in fetal outcomes due to TRPV2 KO. Methods: The cellular expression of TRPV2 was assessed in wildtype placentas using RNAscope. Placental clearance of glucose and amino acid (AA) was assessed using 3H-methyl-D glucose and 14C-aminoisobutyric acid in vivo on E18.5 (term=20 days). In representative placentas, mRNA levels of glucose (SLC2A1,3) and AA transporters (SLC38A1,2,4) were quantified by q-RT-PCR in the Lz and placental structure determined using stereology. Data were compared between wildtype and TRPV2 KO littermates on a 129Sv and C57Bl6 background. Results: In the labyrinth, TRPV2 was highly expressed by syncytial trophoblast and absent from fetal endothelial cells. The placental transfer of glucose and AA was adaptively increased in TRPV2129Sv KO compared to WT littermates (+15% and +130%, respectively). This was not related to a change in the expression of glucose or amino acid transporters in TRPV2129Sv KOs. Placental AA transport was also increased in TRPV2C57 KO, albeit to a lesser extent (+35%), while glucose transport and expression of SLC2A1 and SLC2A3 were decreased (-20%, -35% and -52%, respectively). Lz volume was similarly decreased in TRPV2129Sv and TRPV2C57 KOs (-18% and -28%, respectively). Conclusion: Thus, there are strain-specific adaptations in placental transport function that seem to optimise fetal outcomes in response to TRPV2 deficiency. The less extensive upregulation of placental AA transport and failure to upregulate glucose transport in TRPV2C57 KOs likely explains poorer offspring growth and survival compared to TRPV2129Sv KOs.

Erythropoietin (EPO) is regulated by hypoxia-inducible factor (HIF)-2. In the kidney, it is produced by cortico-medullary perivascular interstitial cells, which transdifferentiate into collagen-producing myofibroblasts in response to injury. Inhibitors of prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-2 and stimulate kidney and liver EPO synthesis in patients with anemia of chronic kidney disease (CKD). We examined whether HIF-PHIs can reactivate EPO synthesis in interstitial cells that have undergone myofibroblast transdifferentiation in established kidney fibrosis.We investigated Epo transcription in myofibroblasts and characterized the histological distribution of kidney Epo transcripts by RNA in situ hybridization combined with immunofluorescence in mice with adenine nephropathy (AN) treated with HIF-PHI molidustat.  Lectin absorption chromatography was used to assess liver-derived EPO.  In addition, we examined kidney Epo transcription in Phd2 knockout mice with obstructive nephropathy.In AN, molidustat-induced Epo transcripts were not found in areas of fibrosis and did not colocalize with interstitial cells that expressed α-smooth muscle actin, a marker of myofibroblast transdifferentiation. Epo transcription was associated with megalin-expressing, kidney injury molecule 1-negative nephron segments and contingent on residual renal function. Liver-derived EPO did not contribute to serum EPO in molidustat-treated mice. Epo transcription was not associated with myofibroblasts in Phd2 knockout mice with obstructive nephropathy.Our studies suggest that HIF-PHIs do not reactivate Epo transcription in interstitial myofibroblasts and that their efficacy in inducing kidney EPO in CKD is dependent on the degree of myofibroblast formation, the preservation of renal parenchyma and the level of residual renal function.

The fate of neurons in the developing brain is largely determined by the combination of transcription factors they express. In particular, stem cells must follow different transcriptional cascades during differentiation in order to generate neurons with different neurotransmitter properties, such as glutamatergic and GABAergic neurons. In the mouse cerebral cortex, it has been shown that large Maf family proteins, MafA, MafB and c-Maf, regulate the development of specific types of GABAergic interneurons but are not expressed in glutamatergic neurons. In this study, we examined the expression of large Maf family proteins in the developing mouse olfactory bulb by immunohistochemistry and found that the cell populations expressing MafA and MafB are almost identical, and most of them express Tbr2. Since Tbr2 is expressed in glutamatergic neurons in the olfactory bulb, we further examined the expression of glutamatergic and GABAergic neuronal markers in MafA and MafB positive cells. The results showed that in the olfactory bulb, MafA and MafB are expressed exclusively in glutamatergic neurons, but not in GABAergic neurons. We also found that few cells express c-Maf in the olfactory bulb. These results indicate that, unlike the cerebral cortex, MafA and/or MafB may regulate the development of glutamatergic neurons in the developing olfactory bulb. This study advances our knowledge about the development of glutamatergic neurons in the olfactory bub, and also provides insight into the mechanism by which the cortex and olfactory bulb, although both generated from the telencephalon, generate projection and interneurons with different properties. This article is protected by

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96 hours post-inoculation for serotonergic, GABAergic, and enkephalinergic expressions using In situ Hybridization and/or Immunohistochemistry techniques. The results revealed that >50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker TpH2. Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated GABA transporter VGAT with a concomitant increase in TpH2 expression in bladder-linked RVM neurons suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity. This article is protected by

Receptor activator of NF-κB (RANK) expression is increased in podocytes of patients with diabetic nephropathy. However, the relevance of RANK to diabetic nephropathy pathobiology remains unclear. Here, to evaluate the role of podocyte RANK in the development of diabetic nephropathy, we generated a mouse model of podocyte-specific RANK depletion (RANK-/-Cre T), and a model of podocyte-specific RANK overexpression (RANK TG), and induced diabetes in these mice with streptozotocin. We found that podocyte RANK depletion alleviated albuminuria, mesangial matrix expansion, and basement membrane thickening, while RANK overexpression aggravated these indices in streptozotocin-treated mice. Moreover, streptozotocin-triggered oxidative stress was increased in RANK overexpression, but decreased in the RANK depleted mice. Particularly, the expression of NADPH oxidase 4, and its obligate partner, P22phox, were enhanced in RANK overexpression, but reduced in RANK depleted mice. In parallel, the transcription factor p65 was increased in the podocyte nuclei of RANK overexpressing mice but decreased in the RANK depleted mice. The relevant findings were largely replicated with high glucose-treated podocytes in vitro. Mechanistically, p65 could bind to the promoter regions of NADPH oxidase 4 and P22phox, and increased their respective gene promoter activity in podocytes, dependent on the levels of RANK. Taken together, these findings suggested that high glucose induced RANK in podocytes and caused the increase of NADPH oxidase 4 and P22phox via p65, possibly together with the cytokines TNF- α, MAC-2 and IL-1 β, resulting in podocyte injury. Thus, we found that podocyte RANK was induced in the diabetic milieu and RANK mediated the development of diabetic nephropathy, likely by promoting glomerular oxidative stress and proinflammatory cytokine production.

Background Sexual dimorphism in depression is well documented. Women and men differ in the prevalence, symptom presentation, and responses to antidepressant treatment. Methods Chronic social defeat stress protocol was used to induce depression-like behavior in mice. Multiplex cytokine assay was used to assess peripheral inflammation. RNA-seq was conducted to assess for gene expression regulation in the PFC. RNAscope combined with immunohistochemistry was used to identify cell-specific expression of CCL5 receptors in the brain. Results Characterization of peripheral inflammation in female mice revealed positive correlation between susceptibility and plasma levels of CCL5, but not with IL-6 which was associated with stress susceptibility in male mice. RNA-seq analysis of PFC revealed that CCR5, one of the major receptors for CCL5 was significantly higher in defeat stressed female mice compared to the control mice. However, this increase was not seen in stressed male mice. We found that the expression of CCR5 is mainly in the microglia. Treatment with CCR5 antagonist significantly attenuated CSDS-induced depression-like behavior in female mice. Conclusions Higher level of CCL5 was also reported in human subjects with MDD and higher levels of peripheral CCL5 in women compared to men. Cross-examination with human MDD RNA-seq data showed that in female MDD subjects, the level of CCR5 in the ventromedial PFC was 2.8 fold higher compared to the control subjects and this increase was not seen in male MDD subjects. The human data supports our finding, strongly implicating sexual dimorphic interactions between CCL5/CCR5 expression and depression. CCL5/CCR5 signaling may be potential target for treating female depression.

Significance Molecules regulated by neuronal activity are necessary for circuits to adapt to changing inputs. Specific classical major histocompatibility class I (MHCI) molecules play roles in circuit and synaptic plasticity, but the function of most members of this family remains unexplored in brain. Here, we show that a nonclassical MHCI molecule, Qa-1 (H2-T23), is expressed in a subset of excitatory neurons and regulated by visually driven activity in the cerebral cortex. Moreover, CD94/NKG2 heterodimers, cognate receptors for Qa-1, are expressed in microglia. A functional interaction between Qa-1 and CD94/NKG2 is necessary for regulating the magnitude of ocular dominance plasticity during the critical period in the visual cortex, implying an interaction in which activity-dependent changes in neurons may be monitored by microglia.

Obesity and high-fat diet (HFD) consumption result in hypothalamic inflammation and metabolic dysfunction. While the TLR4 activation by dietary fats is a well-characterized pathway involved in the neuronal and glial inflammation, the role of its accessory proteins in diet-induced hypothalamic inflammation remains unknown. Here, we demonstrate that the knockdown of TLR4-interactor with leucine-rich repeats (Tril), a functional component of TLR4, resulted in reduced hypothalamic inflammation, increased whole-body energy expenditure, improved the systemic glucose tolerance and protection from diet-induced obesity. The POMC-specific knockdown of Tril resulted in decreased body fat, decreased white adipose tissue inflammation and a trend toward increased leptin signaling in POMC neurons. Thus, Tril was identified as a new component of the complex mechanisms that promote hypothalamic dysfunction in experimental obesity and its inhibition in the hypothalamus may represent a novel target for obesity treatment.

The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor-expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain.