Medulla Of Mice Research Articles

The immunomodulatory activity of the betulonic acid based compound.

The immunomodulatory activity of a betulonic acid-based compound with furocoumarin (BABCF; 2-azido, 9-N-methylpiperazinomethyl oreozelone) has been investigated. Male C57BL/6 mice (aged 3 months) treated with the cytostatic agent cyclophosphamide (CP) and intact individuals served as experimental models. The expression of genes was studied in bone marrow (IL-12, IL-10, IL-1β, TNF-α, TGF-β, M-CSF, GM-CSF) or in the suspension of peritoneal cells (IL-12, IL-10; as the injection site). The surface markers of T-lymphocytes (CD3, CD4, and CD8) in fractions of venous blood mononuclear cells (MNCs) were determined by means of flow cytometry using antibodies. Histological and morphometric studies were performed to assess the impact of CP and BABCF on the thymus. BABCF caused a pronounced (about 3-fold) increase in relative expression of the GM-KSF gene. BABCF caused a local increase in the expression of IL-12 in the peritoneal cavity cells and restored the relative content of T-lymphocytes in the blood of CP-treated mice treated affecting mainly CD3⁺CD4⁺ lymphocytes. This substance reduced the tissue density of the thymic cortex and thymic medulla in CP-treated mice. Thus, results of this study suggest that BABCF exhibits a stimulating effect on the cellular link of immunity and promotes maintenance of the number of T-lymphocytes in the blood due to their migration from the central organs of the immune system.

Alteration of monoaminergic systems in the caudal medulla and its possible link to diurnal increase of apnea in a mouse model of Rett syndrome.

Methyl-CpG binding protein 2 (MeCP2)-deficient (Mecp2-/y) mice exhibit apneas that resemble respiratory abnormalities observed in Rett syndrome (RTT) patients. The present study aimed to clarify whether Mecp2-/y mice show diurnal variations in apnea as seen in RTT and how the MeCP2 deficiency affects monoaminergic systems that control breathing. In 7-week-old Mecp2-/y mice, 24 h variation of apnea and effects of milnacipran, a serotonin/noradrenaline reuptake inhibitor, on the apnea were evaluated. The number of vesicular monoamine transporter 2 (VMAT2)-immunoreactive puncta in the caudal medulla was counted. Further, the effects of valproate (VPA) on the expression of tyrosine hydroxylase (TH) mRNA in the ventrolateral medulla of mice were assessed by RT-qPCR. Apnea occurred more frequently during the light phase under a 12:12 h light/dark environment in Mecp2-/y mice and milnacipran reduced apnea during the light phase but not during the dark phase. The number of VMAT2-immunoreactive puncta was reduced in Mecp2-/y mice. VPA treatment significantly increased TH mRNA expression in Mecp2-/y mice. Alteration of monoaminergic systems in the caudal medulla of Mecp2-/y mice is potentially relevant to the light-sensitive diurnal increase of apnea, and an improvement in monoaminergic neurotransmission can ameliorate the diurnal increase of apnea in Mecp2-/y mice.

Effects of recombinant human soluble thrombomodulin on neutrophil extracellular traps in the kidney of a mouse model of endotoxin shock

Sepsis is a life-threatening condition characterized by multi-organ dysfunction due to host immune system dysregulation in response to an infection. During sepsis, neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response. However, excessive NETs play a critical role in the development of organ failure during sepsis. Although recombinant human soluble thrombomodulin (rTM) can inhibit NET formation in the lungs and liver of a mouse model of endotoxin shock, its effects on the kidneys are unclear. The specific effects of NETs and rTM on the renal cortex and renal medulla were examined in a mouse model of endotoxin shock generated by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), followed by i.p. injection of rTM or an identical volume of saline 1h later. LPS injection increased serum creatinine, blood urea nitrogen, and histone H3 levels. However, rTM administration significantly decreased histone H3 and citrullinated histone H3 (citH3) levels. Immunohistochemical analysis revealed no significant changes in citH3 quantity in the renal cortex of any group. However, in the renal medulla, the increase in citH3 induced by LPS was abolished in the LPS+rTM group. Our findings demonstrate that rTM can suppress NETs in the renal medulla of mice with endotoxin-induced acute kidney injury.

Renal lysophospholipase A1 contributes to Enterococcus faecalis-induced hypertension by enhancing sodium reabsorption.

Our recent study has found that gut bacteria Enterococcus faecalis contributes to hypertension and upregulates lysophospholipase A1 (LYPLA1) in the renal medulla of rats. This work aimed to investigate the role of LYPLA1 in the development of E.faecalis-induced hypertension. Compared to control, E.faecalis treatment increased blood pressure (BP), serum angiotensin II, sodium reabsorption, and expression of αENaC and LYPLA1 in the renal medulla of mice, and these effects were attenuated by knockdown of LYPLA1. Moreover, the intrarenal lypla1 overexpression increased sodium reabsorption and BP. Further studies showed that LYPLA1 promoted the accumulation of renal glycerophosphocholine (GPC), which directly elevated the expression of αENaC and sodium reabsorption. In addition, enriched abundance of LYPLA1 in the renal medulla and urine was also observed in other hypertensive animals. Overall, our results demonstrate that LYPLA1 contributes to E.faecalis-induced hypertension by accumulating GPC and activating ENaC in the renal medulla.

MicroRNA-195a-5p Regulates Blood Pressure by Inhibiting NKCC2A.

Previous studies showed that miR-195a-5p was among the most abundant microRNAs (miRNAs) expressed in the kidney. Lentivirus silencing of tumor necrosis factor-α (TNF) was performed in vivo and in vitro. Luciferase reporter assays confirmed that bumetanide-sensitive Na+-K+-2Cl- cotransporter isoform A (NKCC2A) mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure. TNF upregulates mmu-miR-195a-5p, and -203 and downregulates mmu-miR-30c and -100 in the medullary thick ascending limb of male mice. miR-195a-5p was >3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, whereas silencing TNF inhibited miR-195a-5p expression by ≈51%. Transient transfection of a miR-195a-5p mimic into medullary thick ascending limb cells suppressed NKCC2A mRNA by ≈83%, whereas transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in medullary thick ascending limb cells prevented increases in miR-195 induced by 400 mosmol/kg H2O medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of phosphorylated NKCC2 increased 1.5-fold in medullary thick ascending limb cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg H2O medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA, whereas injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and phosphorylated NKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl. The study identifies miR-195a-5p as a salt-sensitive and TNF-inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A.

Application of AP-MALDI Imaging Mass Microscope for the Rapid Mapping of Imipramine, Chloroquine, and Their Metabolites in the Kidney and Brain of Wild-Type Mice.

Mass spectrometry imaging (MSI) is well-known for the non-labeling visualization of analytes, including drugs and their metabolites in biological samples. In this study, we applied three different tools of MSI, desorption electrospray ionization (DESI)-MSI, matrix-assisted laser desorption ionization (MALDI)-MSI, and a newly developed atmospheric pressure (AP)-MALDI-MSI known as iMScopeTM QT for rapid mapping of imipramine, chloroquine, and their metabolites in C57BL/6 male wild-type mice. Among three MSI tools, better detection capability for targeted drugs at higher speed (up to 32 pixels/s) was observed in iMScope QT. It revealed that imipramine and its metabolites were significantly accumulated in the renal cortex of mice, but chloroquine and its metabolites were highly accumulated in the renal pelvis and renal medulla of mice. Additionally, a higher accumulation of imipramine was noted in the thalamus, hypothalamus, septum, and hindbrain of mice brains. However, chloroquine and its metabolites showed notable accumulation in the lateral ventricle, fourth ventricle, and fornix of the mice brains. These findings of our study can be helpful in understanding clinically relevant properties, efficacy, and potential side effects of these drugs. Our study also showed the potentiality of iMScope QT for rapid mapping of small drugs and their metabolites in biological samples.

Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice.

The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.

Endothelin system expression in the kidney following cisplatin-induced acute kidney injury in male and female mice.

The chemotherapeutic agent cisplatin accumulates in the kidney and induces acute kidney injury (AKI). Preclinical and clinical studies suggest that young female mice and women show greater recovery from cisplatin-AKI compared to young male mice and men. The endothelin (ET) and ET receptors are enriched in the kidney and may be dysfunctional in cisplatin-AKI; however, there is a gap in our knowledge about the putative effects of sex and cisplatin on the renal ET system. We hypothesized that cisplatin-AKI male and female mice will have increased expression of the renal ET system. As expected, all cisplatin-AKI mice had kidney damage and body weight loss greater than control mice. Cisplatin-AKI mice had greater cortical Edn1, Edn3, Ednra, and Ednrb, while outer medullary Ednra was significantly suppressed in both sexes. Of the ∼25 000 genes sequenced from the inner medulla, only 91 genes (comparing saline mice) and 134 genes (comparing cisplatin-AKI mice) were differentially expressed and they were unrelated to the ET system. However, Edn1 was significantly greater in the inner medulla of male and female cisplatin-AKI mice. Thus, RNA profiles of the ET system were significantly affected by cisplatin-AKI throughout the kidney regardless of sex and this may help determine the therapeutic potential of targeting the ET receptors in cisplatin-AKI.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Characterization and Ischemic Response of Telecytes in the Mouse Renal Medulla

Telocytes are currently defined as a novel type of interstitial (stromal) cells with very long and thin prolongations, and these cells can produce exosomes to transmit signals to different cells such as vascular smooth muscle cells, tubular cells and telocytes themselves. So far it remains unclear whether telocytes are present in the renal medulla and whether they are able to regulate renal medullary function. In the present study, we first characterized telocytes in the kidney, in particular, in the renal medulla, given that the renal medullary interstial cells (RMICs) are able to sense pressure and release bioactive lipids lowering arterial blood pressure. Using two selective markers of telocytes, CD34 and c‐kit for immunohistochemical analysis, we found that telocytes are present in the renal medullary interstitium between tubules, vessels or tubular‐blood vessels. Compared to the renal cortex, telocytes are more enriched in the renal medulla. Their long prolongations could be seen in the interstitium along the tubules or blood vessels. Using confocal microscopy, colocalization of CD34 and c‐kit are mainly detected around different tubules or vessels, but not in tubular or vascular cells. Electron microscopy found that many renal medullary telocytes had at least 3 prolongations, where a number of telopods were shown. In some interstitial space with enriched telopods, moreover, a large number of exosomes could be found. We also isolated and cultured telocytes, which were purified from renal medullary interstitial cells used in our laboratories by MiniMACS Cell Separator with anti‐c‐kit antibody beads‐loaded column. In culture, these cells showed typical telocyte features such as more than 3 prolongations, telopods and project connections between cells. Confocal microscopy showed that most cells through MiniMacs column could be double stained by antibodies against c‐kit and CD34, but the original renal medullary institial cells had much fewer cells stained, which was also confirmed by flow cytometry. In a group of mice with 2 kidney‐1 clip (2K1C) hypertensive model, we found that compared to the unclipped kidney, the clipped kidney had largely increased CD34 and c‐kit staining in the renal medullary interstitium, but not much in the renal cortex. These results suggest that telocytes are present in the renal medullary interstitium and in response to chronic ischemia produced by partially clipping renal artery, telocytes are increased. Given that exosomes are substantially enriched in the renal medulla of 2K1C mice, telocytes may be one of the important source for these exosomes that have antihypertensive action (Supported by NIH grants DK054927 and DK120491).

Antihypertensive Action of Exosomes Released from the Renal Medulla after Unclipping of the Renal Artery of 2 Kidney‐1 Clip Hypertension Mice

The renal medulla has been long known to exert antihypertensive action by releasing different bioactive substances. However, so far it remains unknown what is the identity of such renal medullary antihypertensive substances. In the present study, we hypothesized that exosomes, but not a single molecule released from the renal medulla are importantly implicated in the renal medullary antihypertensive action. To test this hypothesis, we first produced the 2 kidney‐one clip (2K1C) hypertension mouse model by clipping the right renal artery (127.9 ± 1.40 mmHg vs. 94.8 ± 1.75 mmHg of sham mice). After 1 week, we unclipped the right renal artery and monitored the mean arterial blood pressure (MAP) for 1 hour and at the same time collected blood samples for analysis of exosomes. It was found that unclipping the right renal artery of 2K1C mice significanty reduced MAP by 21.2 ± 2.65 mmHg but increased the plasma exosome concentrations by more than 5 folds (before unclipping 2.9 ± 0.7 vs. 3.3±0.5, N.S., after unclipping 4±0.5 vs. 17±5× 108particles/mL, p<0.05). Immunohistochemical (IHC) assessments showed that the clipped kidneys had dramatic increase in exosome markers (CD63 and Annexin II) in the renal medulla of 2K1C mice compared to contralateral kidneys or sham mouse kidneys. These functional and IHC changes in 2K1C mice were almost completely blocked by pretreatment of mice with an exosome biogenesis inhibitor, GW‐4869 (1 mg/kg/day, i.p., n=8). We also found that rapamycin, a compound that increases autophagy and lysosome trafficking (6 mg/kg/day, i.p., n=8) substantially attenuated exosome release from the renal medulla and associated antihypertensive action in 2K1C with unclipping the right renal artery, even though it had some effects to reduce MAP and increase exosome release before the unclipping. In another series of studies using 2K1C hypertensive mice, we extracted and purified exosomes from the blood of mice with unclipping of the right renal artery and then infused these exosomes intravenously to another group of 2K1C mice. It was found that plasma exosomes from 2K1C mice with unlipping of the right renal artery signicantly reduced MAP when they are infused into recipient 2K1C mice (at a concentration of 3 x 108 exosomes) compard to exosomes from sham mice. Urinary flow and sodium excretion of recipient mice also increased slightly by infusion of exosomes from 2K1C mice. These results suggest that exosomes are enriched in the renal medulla of the clipped kidney in 2K1C mice and they can be released into the blood if the renal artery clip is released. These exosomes have strong effects to lower arterial blood pressure.

Functional and Molecular Studies of Renal Sphingosine 1 Phosphate Signaling Pathway in IUGR Benjamin Bhunu1 and Suttira Intapad1 Tulane University School of Medicine

Fetal Growth Restriction (FGR) is now regarded as a risk factor for adult renal diseases, however, the precise underlying mechanism that is responsible for this observation remains vague. Sphingosine 1 Phosphate (S1P) is a lysophospholipid with important functions in the regulation of renal physiology and the cardiovascular system. Previously, we demonstrated that in FGR but not control animals, acute activation of Sphingosine 1 Phosphate Receptor 1 (S1PR1) transiently reduces Blood Pressure (BP) in an endothelial nitric oxide synthase dependent manner. We have also shown that both male and female FGR mice have lower nephron number in the kidneys, however, unlike females, male FGR offspring have reduced renal function suggesting sex specific differences in fetal programing of kidney diseases. We have also shown that within 5 minutes of pharmacologically activating S1PR1 with SEW2871, GFR significantly reduces in male control but not IUGR animals. The objective of this study was to test the effect of longer stimulation of S1PR1 agonist on renal function and to determine the distribution of renal S1P signaling molecules. On their 13th day of gestation, pregnant C57BL mice went through a Reduced Uterine Pressure Perfusion (RUPP) or sham surgery to generate FGR and control offspring respectively. At 6 months, measurement of Glomerular Filtration Rate (GFR) and expression of proteins that are involved in S1P signaling was performed using plasma FITC‐Anulin decay, PCR, and immunohistochemistry. For immunohistochemistry, the Integrated Optical Densities (IDO) values from (Diaminobenzidine) DAB‐stained kidney sections were used to statistically quantify protein expression. GFR was lower in male FGR offspring than control (8.85 ±0.55 vs 13.86±1.13 µL/min/kg BW, P˂0.05, FGR vs control males) while in females there was no statistical difference (12.33±0.87 vs 15.92 ±1.94 L/min/kg BW, P˃ 0.05 control females vs FGR). Administration of SEW2871 and measurement of GFR after one hour confirmed that GFR decreases only in control males but not IUGR while in females both FGR and control groups had similar reduction response (6.00±0.46 vs 8.41±0.79 µL/min/kg BW, P˃ 0.05 control males vs FGR) (5. 97±0.48 vs 5.72 ±1.01 µL/min/kg BW, P˃ 0.05 control females vs FGR). Whole kidney transcriptomics showed no statistical differences in the expression profile of S1PR 1‐3 and Sphingosine 1 Phosphate Kinases 1 and 2 (SPHK1‐2) between FGR and control male and female animals. Immunohistochemistry analysis of the kidney medulla and cortex of male animals revealed no difference in the expression profile of S1PR1 in both FGR and control and increased expression of S1PR2 in both the medulla and cortex of FGR animals. S1PR3 was decreased in both the medulla and cortex of FGR mice. SPHK 1 and 2 were not differentially expressed in the medulla of control and FGR animal whereas in the cortex both the two receptors were downregulated in FGR. Our results indicate that acute activation of S1PR1 differentially regulates GFR between male and female FGR. Consequences of the upregulation of S1PR2 and the down regulation of S1PR 3 and SPHK 1‐2 proteins in the kidney of male FGR warrants further investigations.

Genetic Deletion of the Prostaglandin EP4 Receptor in the Kidney Tubule of Adult Male Mice Has Mild Impact on Kidney Water Handling

Prostaglandin E2 (PGE2) is an essential lipid mediator modulating several aspects of renal function. PGE2 acts via four PGE2 receptors, EP1‐EP4. EP4 is abundant in the kidney glomerulus, where it plays a role in renal hemodynamics. EP4 is also expressed in the collecting duct, and constitutive deletion of EP4 from this segment in mice suggests it plays a role in urine concentration. To assess the tubular role of EP4 in adult mice we generated a mouse model with doxycycline‐dependent deletion of EP4 along the renal tubule (Pax8Cre system) and assessed their renal phenotype regarding water handling. Two weeks after treatment with doxycycline, EP4 mRNA expression was reduced by >80% in the renal medulla (RT‐q‐PCR) of knockout mice compared to control mice. Under basal conditions, there were no significant differences between genotypes in food and water intake, bodyweight or plasma biochemistries. EP4 knockout males showed a small increase in basal urinary output and a decreased osmolality, concomitant with lower urinary Na+, K+, Cl‐, urea and creatinine concentrations. There were no differences between genotypes in their renal handling of water during water deprivation for 14 hours, or in their response to the vasopressin V2 receptor agonist dDAVP. In a water overloading test only the EP4 knockout females showed an increased urinary output after 1 hour of the water overload. In conclusion, EP4 in the renal tubule has a mild role in renal water handling. This new model provides a novel tool for assessing the role of EP4 in other aspects of kidney function or disease independently of potential developmental abnormalities or systemic effects.

Bromodeoxyuridine Methodology for Labeling Renal Stem Cells During Kidney Development of Mice.

A continuous Bromodeoxyuridine (BrdU) labeling approach was used during the whole process of the mice kidney development to explore the best BrdU-labeling time, the distribution of BrdU-retaining cells, and to probe into the niche of stem cells in adult kidney. BrdU was injected intraperitoneally to the mice once daily for 3 consecutive days from day 11.5 of embryonic period (E11.5) to the postnatal day 21.5 (P21.5). The kidneys were harvested 24 h after the last BrdU injection and 6 months of age. A renal injury model of subtotal nephrectomy (Nx) in adult mice treated with BrdU was used to observe the response of BrdU-retaining cells to renal injury. When BrdU labeled at E11.5-13.5, the BrdU-retaining cells were mainly detected in the papilla and inner medulla in adult mice. When BrdU labeled at P0.5-11.5, the BrdU-retaining cells were mainly detected in the inner medulla and outer medulla. When BrdU labeled at P12.5-17.5, the BrdU-retaining cells were mainly detected in the outer medulla. When BrdU labeled at P18.5-21.5, almost no BrdU-positive cells could be found, except the cortex. Seventy-two hours after Nx operation in adult mice by BrdU-labeling at P0.5-2.5 or P15.5-17.5, a significant increase of BrdU-retaining cells was found in many cortical proximal tubules, while a dramatic decrease was detected in medulla near the incision edge. Moreover, most of BrdU-positive cells were not costained with proliferating cell nuclear antigen. The distributions of label-retaining cells in the mice kidney were different if BrdU was administered in different periods of kidney development. Most of BrdU-retaining cells were quiescent, the proximal tubules were the only segments that always contained BrdU positive cells, which may have the niche of stem cells in adult kidney.

EIF2A-knockout mice reveal decreased life span and metabolic syndrome.

Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)‐containing mRNAs and/or mRNAs harboring upstream near cognate/non‐AUG start codons. These non‐canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A‐knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A‐KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex‐specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A‐KO and wild‐type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.

HIF2\u03b1 regulates the synthesis and release of epinephrine in the adrenal medulla

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia.Key messagesHIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells.The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia.Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

Abstract P219: Losartan Prevents The Recruitment Of Renal M1-like Macrophages And Dendritic Cells In Medulla Of Angiotensin Ii Hypertensive Mice

Immune cells play a major role in the development and progression of hypertension. Previous studies have shown that antigen presenting cells (APCs), such as macrophages (Mø) and dendritic cells (DCs) are particularly abundant in kidney. However, the relevance of these renal APCs on hypertension and whether their distribution change during the anti-hypertensive treatment remain unknow. We evaluated whether losartan (Los) treatment changes the abundance of APCs in the renal cortex and medulla in Angiotensin (Ang) II-infused mice.Male C57BL/6 mice (8-12wo) were treated with AngII (490ng/Kg/min), AngII+Los (20mg/Kg/day) or Vehicle for 14 days (n=4-6). Systolic blood pressure (SBP) was measured by the tail cuff method, and renal cortex/medulla were isolated for the measurements of: APCs (MHC-II + :CD11c + ), DCs (APCs:F4/80 - :CD64 - /CD103 + for type-1 DCs, or APCs:F4/80 - :CD64 - :CD11b + for type-2 DCs), and M1-like Mø (APCs:F4/80 - :CD64 + :CD11b + ), by flow cytometry.Los treatment prevented the increased SBP (AngII+Los=118.8±6.4 mmHg vs. AngII=158.0±21.1 mmHg; p<0.001), and the APCs recruitment in renal cortex (AngII+Los=23.2±2.7 vs. AngII=36.0±5.9%; p<0.01) and in renal medulla (Veh=16.3±7.7; AngII=26.3±4,7; AngII+Los=14.9±3.3%; p<0.05) induced by AngII. In addition, we observed an increase of DC2 and M1-like Mø recruitments in renal medulla of AngII mice (DC2 Veh =29.0±5.0 vs. DC2 AngII =45.5±7.3%; p<0.05; M1 Veh =44.8±7.5 vs. M1 AngII =58.3±5.3%; p<0.05), which were prevented by Los treatment (DC2 AngII+Los =27.1±6.8%; p<0.05; M1 AngII+Los =47.0±3.5%; p<0.05). Interestingly, we did not observe differences between groups on M1-like Mø, and DC2 populations in renal cortex. However, Los treatment prevented the increase of DC1 on renal cortex (Veh=2.1±1.4; AngII=5.2±2.4; AngII+Los=2.1±0.8%; p<0.05), without differences between groups at medullar level.Our results show that Los treatment has a differential effect on the APCs populations in renal cortex and medulla, suggesting that renal APCs have different participations on hypertension according their microenvironment.Supported by Fondecyt #1201251 and #3201016

Effects of leptin on proliferation of astrocyte- and tanycyte-like neural stem cells in the adult mouse medulla oblongata

Astrocyte- and tanycyte-like neural stem cells (NSCs) were recently detected in the area postrema (AP) and central canal (CC) of the adult medulla oblongata, respectively. The present study aimed to examine dynamical behaviors of the astrocyte- and tanycyte-like NSCs of the mouse medulla oblongata to leptin. The neurosphere assay identified astrocytes in the AP and tanycytes in the CC as NSCs based on their self-renewing neurospherogenic potential. Both NSCs in neurosphere cultures were multipotent cells that generate astrocytes, oligodendrocytes, and neurons. Astrocyte-like NSCs actively proliferated and tanycyte-like NSCs were quiescent under physiologically-relevant in vivo conditions. Chronic leptin treatment promoted proliferation of astrocyte-like NSCs in the AP both in vitro and in vivo. Leptin receptors were expressed in astrocyte-like, but not tanycyte-like NSCs. Food deprivation significantly diminished proliferation of astrocyte-like NSCs. Therefore, the present study indicates that proliferation of astrocyte-like, but not tanycyte-like NSCs is regulated by nutritional conditions.

Neuronal Mediators of the Frequency Increase Evoked by Activation of P2Y 1 Receptors in the preBötzinger Complex Inspiratory Rhythm Generating Network in Vitro

The ventilatory response to acute hypoxia (HVR) comprises an initial, carotid body-mediated increase in breathing followed by a centrally-mediated secondary respiratory depression when ventilation falls to a new steady state. Recent evidence suggests that ATP is released from astrocytes in the preBötzinger Complex (preBötC; site of inspiratory rhythm generation) during the secondary phase where it acts via P2Y1 receptors (Rs) to excite preBötC neurons, increase breathing frequency and attenuate the secondary respiratory depression. The preBötC network comprises a heterogenous population of excitatory and inhibitory inspiratory neurons that can be further classified based on expression of transcription factors, receptors and transmitters, discharge pattern and membrane properties. An important goal is to establish the neuronal phenotype(s) that underlie the P2Y1R frequency increase. Perhaps surprisingly, preBötC frequency increases in response to optogenetic stimulation of excitatory OR inhibitory preBötC neurons. Thus, the objective of this study was to determine if the P2Y1R-mediated frequency increase results from activation of excitatory (glutamatergic) or inhibitory (GABA/glycinergic) preBötC inspiratory neurons. Analysis of P2Y1R and somatostatin (SST) immunolabeling in the medulla of neonatal vGLUT2TdTomato and GLYT2EGFP reporter mice showed that P2Y1R immunolabeled neurons are concentrated in a discrete region of the ventral respiratory column (VRC) that features strong SST immunolabeling (a preBötC marker). P2Y1R+ neurons were sparse in all other regions of the VRC. In addition, 95% of P2Y1R+ neurons were glutamatergic (VGLUT2+), but <10% of glutamatergic neurons were P2Y1R+. None of the GLYT2+ (glycinergic) preBötC neurons were P2Y1R+. Whole-cell analyses of neurons in rhythmic slices from vGLUT2 mice showed that all VGLUT2+ inspiratory preBötC neurons excited by the P2Y1R agonist MRS2365 (100 µM) were glutamatergic, but only1/3 of VGLUT2+ neurons were P2Y1R sensitive. In contrast, calcium imaging of rhythmic slices identified subpopulations (4/16) of glutamatergic and non glutamatergic inspiratory neurons that in TTX were P2Y1R sensitive. Finally, we demonstrated that the frequency increase evoked by MRS2365 (100 µM) in the preBötC was unaffected by block of inhibitory synaptic transmission via GABAA (bicuculline, 20 µM) and glycine (strychnine, 20 µM) receptor antagonists. In summary, we found that P2Y1R-expressing neurons are concentrated in the preBötC and are primarily glutamatergic. P2Y1R-sensitive neurons are primarily glutamatergic, but a subpopulation is nonglutamatergic. These data, combined with the observation that the P2Y1R-evoked excitation of the preBötC does not depend on synaptic inhibition, suggest that the P2Y1R-mediated excitation of the preBötC is primarily achieved through activation of excitatory, glutamatergic neurons.

Serotonergic Modulation of Persistent Inward Currents in Serotonergic Neurons of Medulla in ePet-EYFP Mice.

Serotonergic (5-HT) neurons in the medulla play multiple functional roles associated with many symptoms and motor activities. The descending serotonergic pathway from medulla is essential for initiating locomotion. However, the ionic properties of 5-HT neurons in the medulla remain unclear. Using whole-cell patch-clamp technique, we studied the biophysical and modulatory properties of persistent inward currents (PICs) in 5-HT neurons of medulla in ePet-EYFP transgenic mice (P3–P6). PICs were recorded by a family of voltage bi-ramps (10-s duration, 40-mV peak step), and the ascending and descending PICs were mirrored to analyze the PIC hysteresis. PICs were found in 77% of 5-HT neurons (198/258) with no significant difference between parapyramidal region (n = 107) and midline raphe nuclei (MRN) (n = 91) in either PIC onset (−47.4 ± 10 mV and −48.7 ± 7 mV; P = 0.44) or PIC amplitude (226.9 ± 138 pA and 259.2 ± 141 pA; P = 0.29). Ninety-six percentage (191/198) of the 5-HT neurons displayed counterclockwise hysteresis and four percentage (7/198) exhibited the clockwise hysteresis. The composite PICs could be differentiated as calcium component (Ca_PIC) by bath application of nimodipine (25 μM), sodium component (Na_PIC) by tetrodotoxin (TTX, 2 μM), and TTX- and dihydropyridine-resistance component (TDR_PIC) by TTX and nimodipine. Ca_PIC, Na_PIC and TDR_PIC all contributed to upregulation of excitability of 5-HT neurons. 5-HT (15 μM) enhanced the PICs, including a 26% increase in amplitude of the compound currents of Ca_PIC and TDR_PIC (P < 0.001, n = 9), 3.6 ± 5 mV hyperpolarization of Na_PIC and TDR_PIC onset (P < 0.05, n = 12), 30% increase in amplitude of TDR_PIC (P < 0.01), and 2.0 ± 3 mV hyperpolarization of TDR_PIC onset (P < 0.05, n = 18). 5-HT also facilitated repetitive firing of 5-HT neurons through modulation of composite PIC, Na_PIC and TDR_PIC, and Ca_PIC and TDR_PIC, respectively. In particular, the high voltage-activated TDR_PIC facilitated the repetitive firing in higher membrane potential, and this facilitation could be amplified by 5-HT. Morphological data analysis indicated that the dendrites of 5-HT neurons possessed dense spherical varicosities intensively crossing 5-HT neurons in medulla. We characterized the PICs in 5-HT neurons and unveiled the mechanism underlying upregulation of excitability of 5-HT neurons through serotonergic modulation of PICs. This study provided insight into channel mechanisms responsible for the serotonergic modulation of serotonergic neurons in brainstem.