bluuebunny/crossref_metadata_2025 huggingface dataset:
The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion
Authors: Czogalla Jan , Vohra Twinkle , Ribas David Penton , Kirschmann Moritz , Carrel Monique , Loffing Johannes
Published: November 2023

The mineralocorticoid receptor (MR) and its ligand aldosterone increase renal sodium reabsorption via regulation of the epithelial Na+ channel (ENaC) and the Na‐K‐ATPase in the renal collecting system. Previous studies suggested that aldosterone also regulates the thiazide‐sensitive cotransporter in the renal distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations such as changed plasma potassium, remained controversial. We have now generated a mouse model carrying a targeted deletion of MR in ~16% of cells randomly scattered along the renal tubule (MRko mice). In this model, MR‐positive and MR‐negative cells can be studied next to each other in the same physiological context via immunofluorescence. To evaluate microscope images objectively, we further developed an ImageJ script allowing radial quantification of optical density, thus allowing comparison of apical staining intensities. Although newborn MRko mice need NaCl supplementation to thrive, adult MRko do not show any differences to wildtype littermates in terms of general physiology, mRNA and protein expression of renal ion transport proteins and urine concentrating capacity, under both normal and low salt diet. MR‐negative cells in the renal collecting system of MRko mice do not show any detectable alphaENaC expression, while gammaENaC is diffusely distributed over the cytoplasm. In MR‐positive cells, these ENaC subunits are targeted to the apical cell surface. Although dietary Na+ restriction increases NCC abundance and phosphorylation to a similar extent in control and MRko mice, no differences in NCC regulation were observed between MR‐negative and MR‐positive DCT cells. In conclusion, MR is crucial for ENaC regulation but dispensable for the maintenance of NCC expression and phosphorylation under control conditions and for NCC upregulation in response to dietary Na+ restriction.

Support or Funding Information

This work was supported by a collaborative project grant from the Zurich Center for Integrative Human Physiology (ZIHP), research funds from the Swiss National Center for Competence in Research “Kidney.CH” and by a project grant (310030_143929/1) from the Swiss National Science Foundation.
γ subunit proteolytic fragment binds the epithelial Na+ channel at the finger‐thumb domain interface of the γ subunit
Authors: Kashlan Ossama B , Thompson Rebecca N
Published: November 2023

Epithelial Na+ channel (ENaC) maturation and activation entail proteolysis of both the α and γ subunits. Cleavage at multiple sites in the finger domain of each subunit liberates the subunit's autoinhibitory peptide. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by proteolysis. We previously showed that a peptide derived from the α‐subunit's autoinhibitory sequence binds at the α subunit's finger‐thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from γ subunit's autoinhibitory sequence inhibits the channel through an analogous mechanism. We tested our hypothesis using a crosslinking approach. We used bifunctional methane thiosulfonate (MTS) reagents to cross‐link experimentally‐introduced cysteines in the γ inhibitory peptide, and at select sites within the ENaC γ subunit. Our data show that the γ inhibitory peptide can be linked to both the finger and thumb domains of the γ subunit, consistent with our hypothesis. Our data also suggest that direct manipulation of the finger‐thumb interface using MTS reagents influences channel gating.

Support or Funding Information

This work is supported by NIH grant DK098204
Role of the NEDD4‐2 adaptor protein NDFIP2 in salt transport regulation
Authors: Vacle Sarah , Cheval Lydie , Doucet Alain , Fenton Robert A. , Staub Olivier
Published: November 2023

Proper Na+ and K+ homeostasis by the kidney is a key process in the prevention of hypertension, the most prevalent disease in Western societies, and a primary risk factor for cardiovascular disease. The distal part of the nephron, referred to as the Aldosterone Sensitive Distal Nephron (ASDN), plays an important role in this process. There are at least two main regulatory pathways that are involved in the regulation of ions transporters (NCC, ENaC and ROMK or BK channels). One involves the WNK/SPAK/OSR1 kinase cascade, and the other the concerted action of SGK1 kinase and the ubiquitin‐protein ligase NEDD4‐2. Both pathways have been extensively studied, but there remain numerous open questions with respect to their regulation and possible crosstalks. In this project, we investigate the role of a putative adaptor protein (NDFIP2), which may provide the link between NEDD4‐2 and NCC. We first assessed the expression of Ndfip2 mRNA in microdissected tubules, and found that it is predominantly expressed in the proximal tubule and the ASDN. In MDCK‐hNCC cells, expressing human NCC under the control of a tetracycline inducible promotor, as well as endogenous NDFIP2 and NEDD4‐2, we demonstrated by co‐immunoprecipitation that NCC, NEDD4‐2 and NDFIP2 form a complex together. In parallel, we generated inducible, nephron‐specific KO mice (Ndfip2Pax8/LC1). This model allows the doxycycline‐sensitive expression of Cre‐recombinase along the nephron and consequently the deletion of exon 3 of Ndfip2. As the deletion is induced at the age of 3 weeks, compensatory mechanisms that may occur during nephrogenesis are avoided. In whole kidney lysates, only weak Ndfip2 mRNA expression and nearly complete suppression of the NDFIP2 protein is observed, which persist at least up to 2 months after deletion. Our data suggest decreased circulating levels of aldosterone, but no observable changes in NCC expression. When animal are kept under a high Na+ diet (HSD), they demonstrate difficulties to handle Na+ excess and adapt their water consumption.

Support or Funding Information

This work was supported by the Swiss National Science Foundation Grant # 310030_159735 (to OS), the Swiss Kidney.ch NCCR.
Vasopressin‐dependent regulation of potassium channel distribution in thick ascending limb of Henle's loop
Authors: Smorodchenko Alina , Sharkovska Yuliya , Böhlick Alexandra , Dittmayer Carsten , Cornejo‐Müller Alejandro , Mutig Kerim , Bachmann Sebastian
Published: November 2023

Introduction

NaCl reabsorption via the bumetanide‐sensitive Na‐K‐2Cl‐cotransporter (NKCC2) in the thick ascending limb (TAL) of Henle's loop is crucial for kidney salt and water regulation. The inward‐rectifying potassium channels, luminal renal outer medullary potassium channel (ROMK) and basolateral KCNJ10 (Kir4.1) provide outward movement of potassium; both are essential for the action of NKCC2. A discontinuous pattern of ROMK‐positive and ‐negative cells along TAL has been shown earlier, whereas less is known about TAL Kir4.1 expression. Vasopressin (AVP) may increase TAL function significantly. In this study we investigated how ROMK and Kir4.1 cellular abundances adapt to the activation of TAL via AVP.

Methods

Control Long Evans (LE; n=5) and AVP‐deficient Brattleboro rat kidneys (BB) were analyzed. BB rats were treated with V2 vasopressin receptor agonist (dDAVP) (n=10) or saline (n=10) for 72h. Kidneys were perfusion‐fixed or prepared for biochemical analysis. Immunohistochemistry, in situ hybridization, and EM analysis were used for localization and quantification of NKCC2, ROMK, and Kir4.1. Expression was further studied by Western blot.

Results

In LE, NKCC2 was expressed in all TAL cells throughout the medullary and cortical portions, whereas ROMK and Kir4.1 showed selective expression patterns. Between 60% (medulla) and 40% (cortex) of TAL cells were devoid of ROMK, and respective 35% and 50% were Kir4.1‐negative at steady state as revealed by IHC. Expression of ROMK and Kir4.1 was often mutually exclusive. Administration of dDAVP to BB for 72h led to significant upregulation of both channels and induced an overlapping expression in most cells along the TAL. Changes were confirmed by Western blot, with increases in protein abundance by 220% for ROMK and 25% for Kir4.1, which were restricted to the medulla (p<0.01). ISH revealed no changes at all in mRNA abundances.

Conclusion

Our results demonstrate expressional cell heterogeneity with respect to ROMK and Kir4.1 signals in TAL. Transepithelial transport activation by AVP coincides with an augmentation of positive cell numbers, presumably via altered protein stability. Changes likely reflect an adaptive mechanism in potassium handling by the TAL to maintain solute homeostasis during changes in transport.
Comparative characterization of the ɛ‐ and α‐subunit of the epithelial sodium channel in Xenopus laevis
Authors: Wichmann Lukas , Perniss Alexander , Althaus Mike
Published: November 2023

The vectorial transport of sodium ions across most vertebrate epithelia is tightly linked to the activity of the epithelial sodium channel (ENaC) which allows for entry of sodium ions across the apical membrane of epithelial cells. The canonical ENaC is composed of the pore‐forming α‐subunit which assembles with the accessory β‐ and γ‐subunits to form a heterotrimeric ion channel. While α‐ENaC is crucial for proper channel functionality it may be substituted by α‐like subunits such as the delta‐subunit (δ) in humans or the epsilon‐subunit (ɛ) in Xenopus laevis. Although the inherent role of α‐containing ENaC in epithelial sodium absorption is well established, the physiological function of the ɛ‐ENaC subunit remains elusive. This study aimed to examine the expression of ɛ‐ and α‐ENaC subunits in various Xenopus laevis tissues and to comparatively characterize heterologously expressed ɛβγ‐ and αβγ‐ENaCs.

Screening of ɛ‐ and α‐ENaC expression in tissue homogenates was performed by reverse transcription PCR. Transcripts for ɛ‐ENaC were predominantly detected in tissues of the urogenital tract (kidneys, urinary bladder, testis, ovary, cloaca). With the exception of the ovary these tissues also expressed α‐ENaC. Functional characterization of heterologously expressed ɛβγ‐and αβγ‐ENaC in Xenopus laevis oocytes was carried out using the two‐electrode voltage‐clamp technique. In comparison with αβγ‐ENaCs, ɛ‐containing channels produced an increased transmembrane current which was more sensitive to inhibition by extra‐ and intracellular sodium ions. Furthermore, activity of ɛβγ‐ENaC was enhanced by acidic extracellular pH but decreased under alkaline conditions, whereas α‐ENaC containing channels were activated in both cases. Additionally, ɛβγ‐ENaC was less sensitive to the ENaC blocker amiloride. A decreased amiloride‐sensitivity of transepithelial sodium transport was also detected in Ussing‐chamber recordings of urinary bladder preparations compared to lung epithelia which did not express ɛ‐ENaC. Since ɛ‐ENaC was the only difference in the ENaC‐subunit transcriptome of both tissues, its presence likely accounts for the distinct amiloride‐sensitivities.

These results indicate a functional role for the ɛ‐ENaC subunit in urogenital tissues of Xenopus laevis. The presence of ɛ‐ENaC might facilitate a distinct responsiveness of these tissues to sodium and extracellular pH.
Glycosylated Asparagines Are Important Constituents For The Shear Force Dependent Activation Of The Epithelial Sodium Channel (ENaC)
Authors: Barth Daniel , Knoepp Fenja , Fronius Martin
Published: November 2023

The epithelial sodium channel (ENaC) contributes to electrolyte/fluid‐homeostasis and blood pressure regulation and its activity is regulated by shear force (SF). Previous studies have shown that SF sensation of α‐ENaC depends on an interaction with the extracellular matrix (ECM) via N‐linked glycans of two glycosylated asparagines (N312 and N511). To determine whether the asparagines themselves or the attached N‐glycans are required for SF sensation, site‐directed mutagenesis combined with two‐electrode voltage clamp (TEVC) experiments were performed. α‐ENaC subunits were stripped of their N‐glycans by individually disrupting the glycosylation consensus sequence (NXS/T) N312NS to N312NA and N511YT to N511YA. When co‐expressed with β and γ ENaC, a reduced SF‐response was observed compared with wildtype α ENaC. Furthermore, the role of glycosylated asparagines for SF sensation was confirmed in experiments with the δ‐subunit. While functionally related to the α‐subunit (35% of sequence identity), δ ENaC (when co‐expressed with β and γ) is less SF sensitive and lacking the glycosylated asparagines responsible for proper SF sensation in α‐ENaC. Insertion of asparagines corresponding to α N312 and N511, including their glycosylation consensus sequence, at the corresponding positions in the δ‐subunit, generated two δ‐subunit mutants that were more sensitive to SF.

These experiments further confirm that glycosylated asparagines and the attached N‐glycans in particular are crucial for SF sensation of ENaC.

Support or Funding Information

The work was supported by University of Otago and Justus‐Liebig‐University Giessen PhD Scholarships and research grants.
COMMD10: A Novel Regulator Of ENaC Trafficking?
Authors: Ware Adam , Burstein Ezra , McDonald Fiona
Published: November 2023

The Epithelial Sodium channel (ENaC) is a heterotrimeric (consisting of one α, one β and one γ subunit) Na+ channel and is located at the apical membrane of polarised epithelia, such as the distal kidney, lung and colon, where it has an important role in Na+ reabsorption for maintenance of total body Na+, blood pressure and blood volume. ENaC's trafficking to and from the plasma membrane is a highly regulated but not completely understood process. One protein that has been identified as playing a role in ENaC trafficking is COMMD1, a member of the COMMD (Copper Metabolism Murr1 domain containing) family of proteins. The other family members however have not been as extensively studied, including COMMD10, the focus of our study. Preliminary data has suggested an interaction between COMMD10 and Arf1 (a small GTP binding protein with various functions throughout the trafficking pathway) which suggests a role for COMMD10 in protein trafficking. Our work aims to investigate whether COMMD10 is a novel regulator of the protein trafficking pathway, and thus, whether it is involved in ENaC trafficking to and/or from the plasma membrane. A stable COMMD10 knockdown reduces amiloride‐sensitive short circuit current in αβγ‐ENaC transfected FRT epithelia. A COMMD10 knockdown reduces protein expression of some proteins (Vps35 and COMMD1) involved in the retromer complex (a complex implicated in the recycling of ENaC back to the plasma membrane). These results suggest that when COMMD10 is knocked down ENaC trafficking is disrupted. We have also identified that COMMD10 and Arf1, both in its wild‐type form as well as constitutively active and constitutively inactive forms, co‐localise. However, no co‐localisation was observed between COMMD10 and other Arf family members (Arf4 and Arf5) suggesting an Arf1 specific function for COMMD10. Using a cell‐line in which COMMD10 is stabily knocked down we have identified a change in localisation of the active form of Arf1, that when taken with the other results suggests a role for COMMD10 in the protein trafficking pathway.

Support or Funding Information

University of Otago PhD Scholarship
L‐type Ca2+ Current Increase by Rotenone, Stimulant of Mitochondrial Superoxide Release, in A7r5 Arterial Smooth Muscle Cells. Effect of Dithiothreitol, Hydrogen Peroxide, Tempol and Staurosporine
Authors: Ochi Rikuo , Dhagia Vidhi , Patel Dhara , Wolin Michael S. , Gupte Sachin A.
Published: November 2023

L‐type Ca current (ICaL) is primary Ca2+ current to initiate contraction in arterial smooth muscle cells. Mitochondrial respiratory chain (MRC) generates and releases superoxide (O2−) in energy metabolism. O2− is converted to H2O2 by superoxide dismutase (SOD). Rotenone (Rot), phytogenic toxin, inhibits complex I of MRC and stimulates the release of O2−. We recently found that Rot increases ICaL in A7r5 cultured aortic smooth muscle cells. Here, we performed further pharmacological study of the increase by applying several redox‐related agents. Rot (0.01 mM) and other drugs were introduced during repetitive depolarization at 1/20s or recording I–V relationship with 2 min interval. Rot increased ICaL by 25% in 1–2 minute. It increased macroscopic conductance and shifted V0.5 of activation curve to the hyperpolarizing direction. Dithiothreitol (DTT, 2 mM), thiol‐reducing agent, increased ICaL by 20% without producing the negative shift and, in the presence of DTT, Rot increased ICaL with the negative shift. H2O2 (0.3 mM) inhibited ICaL by 13% in 5 min without shifting I–V and, in the presence of H2O2, Rot induced 17% increase of ICaL with negative shift. Pretreatment by Tempol, SOD mimetic scavenger of O2−, markedly depressed Rot‐induced increase of ICaL. Staurosporine (100 nM), broad‐spectrum protein‐kinase inhibitor, inhibited ICaL with slow time course and completely suppressed Rot‐induced increase of ICaL. Rotenone increases ICaL by increasing the release of O2− from mitochondrial complex I and the increase is independent of thiol‐oxidation but is mediated by phosphorylation of proteins that regulates ICaL.
Increased Function of Large‐Conductance of Potassium Channels following Acute Ischemic Stroke
Authors: Rottgen Trey , Dick Gregory , Chantler Paul , Frisbee Jefferson
Published: November 2023

We tested the hypothesis that acute ischemic stroke is associated with increased function of K+ channels in vascular smooth muscle. Basilar smooth muscle from male rats was usedas a model. Animals underwent carotid surgery where a thin‐filament was inserted and guided to the bifurcation of the middle cerebral artery (MCA) from the internal carotid. The filament was then wedged to provide a transient occlusion of the MCA (MCAO) of sixtyminutes. Animals were then euthanized thirty days following MCAO. Whole‐cell patch clamp experiments indicated that K+ current density was elevated in the stroke animals. Outward current density was 2.5 ± 0.2 fold greater in stroke animals. Ca2+‐activated (BK) K+ currents dominated membrane conductance at the more depolarized potentials. Paxilline (1 uM) a selective antagonist of BK channels was used to determine the contribution of these channels to the whole‐cell current. In control rats, paxilline inhibited 64 ± 2% of current at +80 mV. In stroke rats, paxilline inhibited 61 ± 3% of current at +80mV. The paxilline‐sensitive BK current was 2.4 ± 0.3 fold greater in stroke rats. These data indicate that acute ischemic stroke increases the expression or function of K channels invascular smooth muscle cells, especially BK channels. We suggest this change in K+ channel function may represent a compensatory response aimed at preserving cerebral blood flow after ischemic stroke.
Functional Characterization of the First Known Mutation of the Human SLC12A2 (NKCC1) Gene
Authors: Flores Bianca , Schornak Cara , Wolfe Lynne , Adams David , Delpire Eric
Published: November 2023

An 8 year old girl with multiple health problems that began at the age of 6 months visited the NIH Undiagnosed Disease Program in 2010. At the time of her visit, she suffered from obstructive apnea, episodes of vomiting and dehydration; decrease energy and fatigue, exercise intolerance, dilated cardiomyopathy (left ventricle dilation) and seizure‐like episodes. In early 2015, she was found to have a de novo mutation in SLC12A2, the gene encoding the Na‐K‐2Cl cotransporter, NKCC1. Using genomic DNA isolated from the patient, we confirmed an 11‐bp deletion in exon 22 leading to protein truncation. We created the mutant cDNA for expression in Xenopus laevis oocytes. In contrast to wild‐type cotransporter which demonstrated significant bumetanide‐sensitive and hypertonic‐stimulated K+ uptake, the mutant cotransporter was non‐functional. Western blot analysis of oocytes expressing of a c‐myc tagged mutant cotransporter revealed a molecular size consistent with a dimer, in contrast to oocytes expressing wild‐type cotransporters that showed all three forms of the cotransporter: core, glycosylated, and dimer. Co‐expression of mutant NKCC1 with wild‐type NKCC1 did not result in dominant‐negative effect of mutant on wild‐type transporter. K+ influx measurements in fibroblasts isolated from the patient revealed that 50% of the K+ influx was ouabain‐sensitive or mediated by the Na+/K+ pump, whereas 41% of the flux was bumetanide‐sensitive or mediated by NKCC1. Addition of ouabain and bumetanide resulted in 87% reduction in K+ influx. These data indicate that the mutation leads to a non‐functional allele without deleterious effect on the function of the cotransporter derived from the wild‐type allele. Whether the mutant protein exerts deleterious effects on other cellular functions still need to be explored.

Support or Funding Information

Supported by NIH grant DK093501
KCC3 activity contributes to the elongated spindle‐shaped morphology of invasive gliomas
Authors: Gagnon Kenneth Bradley
Published: November 2023

Salt and obligatory water efflux across the cell membrane contributes to the elongated spindle‐shaped morphology commonly observed with invasive glioma cells. Immunofluorescent localization of the K‐Cl cotransporter isoform 3 (KCC3) to migrating extensions of rat glioblastoma (C6) cells suggests that its activity may be responsible for this salt and water efflux and thus play a significant role in the motility of these tumor cells through the tight extracellular spaces in the brain. Unfortunately, the ubiquitous expression of KCC3 in other tissues makes it an unlikely target for novel anti‐cancer therapeutics. K‐Cl cotransporters are activated by dephosphorylation of specific threonine residues located in their intracellular carboxy‐terminal domain. Western blot analysis of protein lysates from several different immortalized tumor cell lines demonstrates an abundant expression of protein phosphatase 1 (PP1). In a series of wound healing experiments, Calyculin A inhibition of PP1 activity prevented C6 cells from moving into the wound area without varying the viability of cells outside the wound area. Furthermore, C6 cells incubated with Calyculin A no longer exhibited an elongated spindle‐shape, but rather a truncated rounder form. Interestingly, this same truncated form was observed in C6 cells where KCC3 was stably knocked down with short hairpin RNA, confirming that KCC3 activity plays a role in maintaining the elongated spindle‐shape associated with invasive motility. These results suggest that targeting the regulatory pathway of K‐Cl cotransport in glioma cells might be an effective way of slowing the spread of these malignant brain tumors.

Support or Funding Information

Supported by a Special Projects Grant from the University of Saskatchewan College of Medicine to Kenneth B. Gagnon.
Pathway Analyses of Kidney Transcriptome from inv/inv Mice Reveal Altered Cellular Processes and Sodium Ion Transport
Authors: Kulkarni Nalini H , Smith Rosamund C , Wei Tao , Blazer‐Yost Bonnie L
Published: November 2023

Type II Nephronophthisis (NPHP2) is an auto somalrecessive disorder caused by mutations in the NPHP2 gene that encodes inversin protein. Homozygous inv/inv transgenic mice exhibit situsinversus, enlarged cystic kidneys and die within a week of birth. We hypothesized that increased kidney size in NPHP2 may involve altered cell growth, apoptosis, electrolyte transport and fluidaccumulation in the cysts. To test this hypothesis, histology and transcriptome analyses were performed on 1 day old kidneys of wild type and inv/inv mice. Histology of inv/inv kidneys showed dilated tubules and expansion of Bowman's space around the glomeruli compared to wild type. Transcriptome analyses revealed a total of over 700 genes that were differentially regulated (P<0.05, fold change 1.5, fdr < 0.05) in inv/inv compared to wild type kidneys. Differentially expressed genes were subjected to pathway analysis (KEGG, GO) to help elucidate mechanisms involved in cyst formation and growth. Analysis of the top 30 significant GO biological processes showed an overrepresentation of genes involved in cell proliferation, apoptosis, response to stress, metabolic processes and ion transport. Interestingly, neural precursor cell expressed developmentally downregulated 4‐like (Nedd4l), an ubiquitin proteinligase and a negative regulator of the amiloride‐sensitive sodium channel (ASSC)was upregulated (x1.7) in inv/inv kidneys. Nedd4l mRNA levels were also increased in an inversin‐depleted (using shRNA) mouse renal epithelial cell line (mCCD). In the cultured cells, the increase in Nedd4l, a negative regulator of ASSC, is consistent with an observed decrease in ASSC activity measured using an electrophysiological technique. Thus the transcriptional change in the inv/inv mice is manifested as a functional alteration intransepithelial Na+ transport. In summary, these results suggest that inversin exerts its effect on kidneys, at least in part through transcriptional regulation of genes involved in cellular pathways and in modulating ion transport.
Hydronephrosis and Urinary Bladder Hypertrophy in ROMK Bartter's Mouse
Authors: Kim Jun‐Mo , Guo Xiaoyun , Hu Haiyan , Xu Shuhua , Wang Tong
Published: November 2023

The renal outer medullary potassium channel (ROMK; Kir1.1) plays an important role in Na+ and K+ homeostasis. ROMK knockout mice (KO) show a similar phenotype to Bartter's syndrome of salt wasting and dehydration due to reduced Na‐2Cl‐K‐cotransporter activity. ROMK KO mice also show hydronephrosis; however, the mechanism of this phenotype has not been understood. We investigated ROMK expression and regulation by high and low K intake in mouse bladder, by Western blot and immunofluorescence (IF) staining. We also compared the bladder weight, bladder capacity, and thickness of urothelium and smooth muscle layer between WT and ROMK KO mice. Western blotting detected ROMK expression at 45~48 kDA, and IF staining showed that the luminal membrane of umbrella cells were strongly stained in WT compared to KO mice. The expression of ROMK in the luminal membrane disappeared and signal intensity of the detrusor muscle was decreased by low potassium diet (LK) in WT mice. In contrast, there was no significant change in ROMK intensity, but the thickness of urothelium was increased after mice were treated with HK diet. The thickness of urothelium was 168.1±25.8 μm in normal K (NK) and was 300.7±83.6 μm (p<0.001) in the HK treated group compared with NK in WT mice at the same age. The bladder weight, bladder capacity and the thickness of urothelium in ROMK KO showed average increases 2~4 fold greater than WT at both 2 months and 12 months of age. The thickness of the urothelium was 648.8±33.2 μm vs 302.7±16.5 μm (p<0.001) and the detrusor muscle 1940.7±98.9 μm vs 1308.2±102.1 μm (p=0.013) respectively in 12‐month ROMK KO mice. In conclusion, ROMK expressed in the urinary bladder can be regulated by K+ intake; significant enlargement and hypertrophy of the bladder may contribute to hydronephrosis in ROMK KO mice.

Support or Funding Information

NIH RO1 DK099284 and PO1 DK 17433
Chromogranin B forms the chloride channel required for regulated secretion
Authors: Jiang Qiu‐Xing , Yadav Gaya , Zheng Hui , Yang Qing
Published: November 2023

Endocrine cells use regulated secretory pathway to process and package small proteins and organic molecules into granular secretory vesicles that are released in a finely controlled manner (1–3). Acidification of secretory granules is essential for cargo maturation inside them. Vesicular H+‐ATPase pumps protons into secretory granules(4). But identity of the accompanying anion channel to neutralize charge accumulation in granules remains uncertain (5–7). We discover that chromogranin B (CHGB) is both sufficient and necessary to serve the highly selective anion conductance required for acidification and cargo maturation in insulin‐secreting cells. We find that CHGB uses different domains for channel formation and granular biogenesis, allowing the separation of these two essential functions. Our results establish that CHGBs constitute a new type of pH and calcium‐regulated chloride channel specific to regulated secretion.

Support or Funding Information

NIH R01GM093271Welch Foundation I‐1684CPRIT RP120474AHA 12IRG9400019NIH R01GM088745
An Investigational Anti‐Cancer Agent Synergizes with VX‐809 in F508del Correction
Authors: Ambrosetti Adam , Fan Yifei , Kirby Emily F. , Riggs Robert M. , Wang X. Robert
Published: November 2023

Cystic Fibrosis (CF) is caused by functional deficiency of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel located on the plasma membrane of the epithelia lining the lungs, pancreatic ductules, sweat glands, and other organs. Over 2,000 CFTR mutations have been identified to cause CF with the majority of patients carrying at least one copy of the CFTR gene that has the deletion of a phenylalanine at residue 508 (F508del). This mutation causes improper folding of the nascent CFTR protein and its impaired trafficking to the cell surface. While there are a number of ways to treat the symptoms, current evidence posits that small‐molecule combination therapy is an effective method to correct the underlying defects that cause CF. B6, an investigational anti‐cancer agent, was found to alter the level of an endoplasmic reticulum (ER) chaperone. The objective of the current study is to evaluate the impact of B6 on the steady‐state level of F508del CFTR both in the ER and at the cell periphery. HEK293 cells stably expressing F508del CFTR were incubated with increasing concentrations of B6, and the steady‐state level of CFTR in both the ER core‐glycosylated (band B) and Golgi complex‐glycosylated (band C) forms was determined using quantitative immunoblotting. B6 increases the level of F508del band B in a dose‐dependent manner with proportional increase in band C. To further explore the potential usefulness of B6 in F508del rescue, B6 was combined with F508del investigational corrector VX‐809. Combination of 1 μM VX‐809 with B6 at 10 and 40 μM increases the level of F508del band C by 57% and 171%, respectively, as compared with VX‐809 alone. Similar experiments were performed on CFBE‐ΔF, a CF airway epithelial cell line stably expressing F508del CFTR. Addition of 40 μM B6 to 5 μM VX‐809 increased the level of F508del band C by 119%. Our data suggest a synergistic enhancement of F508del correction by the combination of B6 and VX‐809. These data suggest B6 operates synergistically with VX‐809 to promote maturation of F508del CFTR. A better understanding of the underlying mechanism might lead to the development of novel small‐molecule compounds that dramatically enhance the efficacy of existing F508del correctors.

Support or Funding Information

This work was supported by NIDDK/NIH grant P30DK072482 and the Cystic Fibrosis Foundation grant WANG12P0. A.A. and E.F.K. were supported by Samford University McWhorter School of Pharmacy Pharmaceutical Sciences Summer Internship Program.
Sarcolipin Deletion in mdx Mice Worsens Dystrophic Pathology by Impairing Calcineurin Signaling and Reducing Utrophin Expression
Authors: Fajardo Val Andrew , Gamu Daniel , Rietze Bradley A , Kwon Frenk , Chambers Paige J , Quadrilatero Joe , Tupling A. Russell
Published: November 2023

Duchenne's muscular dystrophy (DMD) is the most severe form of muscular dystrophy affecting 1 in 3500 live male births. There is no cure for DMD and males typically succumb to early mortality induced by respiratory and cardiac insufficiency. Thus, finding viable therapeutic targets is of utmost importance. Sarcolipin (SLN), a sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump inhibitor, is highly upregulated in dystrophic muscles from mdx mice, a mouse model for DMD. To date, the role of SLN in muscular dystrophy remains unknown, and therefore, in this study we crossed SlnKO mice with mdx mice to generate the mdx/SlnKO double knockout mutant mouse. We hypothesized that Sln deletion would worsen dystrophic myopathy based on unpublished results from our laboratory and previous studies showing that SLN can stimulate calcineurin – a Ca2+‐dependent serine/threonine phosphatase shown to counteract muscular dystrophy. Our findings show that mdx/SlnKO myofibers from soleus (P = 0.0001) and diaphragm (P < 0.0001) were significantly smaller than those from mdx. Furthermore, mdx/SlnKO soleus and diaphragm muscles displayed a slow‐to‐fast fiber type shift, which was associated with a significant reduction in utrophin (P = 0.004). Utrophin, a dystrophin homolog that confers compensatory membrane stability, is largely controlled by calcineurin, suggesting that SLN in mdx mice stimulates calcineurin signaling. In addition, the significant reductions in utrophin expression in the mdx/SlnKO mice can account for their worsened dystrophic pathology revealed by greater serum creatine kinase (P = 0.01) and greater variability in myofiber size (minimal Feret's diameter, P = 0.01) compared with mdx. Finally, lower central nuclei counts in muscles from mdx/SlnKO mice compared with mdx (P = 0.04) suggests that genetic deletion of Sln also potentially impairs the muscles’ regenerative capacity. Taken together, these results, for the first time clearly demonstrate SLN's vital role in countering muscular dystrophy.

Support or Funding Information

This work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).
Fluorescence Spectroscopy Reveals a 2:1 Stoichiometry for Dimeric Cardiac Calcium Pump (SERCA2a) Bound to a Phospholamban Monomer
Authors: Blackwell Daniel , Robia Seth
Published: November 2023

Previous in vitro studies have suggested that the cardiac calcium ATPase (SERCA) can form functional homo‐dimers. To determine whether SERCA dimerizes in live cells, we fused Cerulean (Cer) or yellow fluorescent protein (YFP) to the N‐terminus of canine SERCA2a. We measured 18% maximum fluorescence resonance energy transfer (FRETmax) from Cer‐SERCA to YFP‐SERCA, suggesting an interaction between SERCA protomers. To determine whether the measured FRET was due to a specific interaction, we co‐transfected cells with unlabeled SERCA to compete for binding with Cer‐SERCA and YFP‐SERCA. FRETmax progressively decreased with increasing expression of unlabeled SERCA to a minimum of 5%. Competition with increasing molar ratios of unlabeled phospholamban (PLB) did not reduce the measured FRET or the apparent affinity of the SERCA dimer. In order to determine whether PLB phosphorylation status alters SERCA dimerization, we induced phosphorylation of PLB at serine 16 with PKA activation by Forskolin. PLB phosphorylation or phosphomimetic mutation (S16E) did not alter the maximum SERCA‐SERCA FRET or the apparent dissociation constant (Kd) of the SERCA dimer. The apparent affinity of the dimer did not vary with the presence or absence of calcium, ATP, or with the addition of the SERCA inhibitor, thapsigargin. The SERCA‐SERCA interaction was further supported by co‐immunoprecipitation experiments in cells co‐transfected with YFP‐SERCA and myc‐tagged SERCA. Immunoprecipitation with an anti‐mycTag antibody co‐immunoprecipitated YFP‐SERCA, as detected by western blot. Rabbit left ventricular myocytes were isolated and permeabilized in the presence of a benzophenone‐4‐maleimide crosslinker. Western blot analysis revealed two bands at approximately 110 and 200 kD, consistent with the expected molecular weights of a monomer and dimer, respectively. PLB was also detected in these higher molecular weight bands. We also quantified FRET from GFP‐SERCA to mCherry‐PLB using time correlated single photon counting to determine the GFP excited state lifetime. We observed a biexponential fluorescence decay consistent with two populations of donors with distinct lifetimes. The long lifetime was similar to that of a GFP‐alone control. The shorter lifetime was consistent with a high FRET population. Interestingly, the FRET efficiency of this species was 45%, approximately double the FRETmax value obtained from steady‐state FRET measurements. The population of the high FRET species approached, but did not exceed, 50% of the total, even at PLB to SERCA ratios as high as 40:1. This observation is consistent with a complex of two SERCA protomers in which only one of the pumps engages in FRET with a bound PLB.
Transforming Growth Factor‐beta Promotes Glioblastoma Multiforme to Establish Radiation Resistance by Mesenchymal Differentiation
Authors: Liu Chan‐Chuan , Yeh I‐Chun , Wu Sheng‐Nan , Sze Chun‐I
Published: November 2023

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Current standard therapy is surgery combined with radio‐therapy and/or chemo‐therapy. Due to the existence of resistance, GBM is one of the most difficult‐to‐treat tumor. It has been shown radio‐therapy may induce mesenchymal differentiation (MD). Transforming Growth Factor‐beta (TGF‐β) is a modifier of radiation response, and plays an important role in cell transformation. Therefore, we hypothesized that TGF‐β pathway might be involved in the development of radiation resistance via regulating MD.

Thus, we explored whether TGF‐β regulates MD in radiation resistance in GBM. We first examined the effect of radiation doses on cell viability by Trypan blue assay, and evaluated cell viability and morphology. The cells showed morphology change were defined as transformed cells. We used the radiation dose of 50% cell viability to establish consecutive‐irradiation‐induced resistance. The radiation dose of the cell viability greater than 50% was established high‐dose‐irradiation‐induced resistance positive control. When viability of cells recovered from consecutive‐irradiation exposures which were higher than positive control, the cells were defined as consecutive‐irradiation‐induced resistance. To develop radiation resistance, 3.5Gy exposure for 6 times were required for 1306MG, and 2Gy exposure for 4 times were needed for U87MG. The numbers of transformed cells increased in radiation resistant cells population. Moreover, immunocytochemistry staining showed expression of certain MD markers on transformed cells were different from on non‐transformed cells. Immunoblotting results showed increased mesenchymal marker N‐cadherin and decreased epithelial marker beta‐catenin in radiation resistance. Finally, we used TGF‐β and TGF‐β receptor inhibitor LY364947 to determine if TGF‐β/Smad pathway regulates radiation‐induced resistance. The immunoblotting results revealed that inhibition of TGF‐β pathway reversed MD in consecutive‐irradiation‐induced resistant cell lines via increasing the expression of β‐catenin and reducing the expression of N‐cadherin and Fibronectin. We concluded that TGF‐β promotes GBM to establish radiation resistance by MD.
The effect of dehydration on the metabolic rate and locomotor performance of two species of tree frogs found in Southern California (Pseudacris regilla and P. cadaverina)
Authors: Alvarez Chelsea , Contreras Heidy L.
Published: November 2023

Pacific tree frogs (Psedacris regilla) and California tree frogs (P. cadaverina), native to Southern California, have adapted to frequent periods of drought. These tree frogs show both behavioral and physiological adaptations that help diminish water loss to their environment. However, despite these adaptations, tree frogs may still experience periods of dehydration in their natural habitat. Dehydration significantly decreases aerobic scope in amphibians; frogs being more susceptible than toads. When dehydrated, semi‐aquatic frogs rely heavily on anaerobic metabolism, which has been shown to induce hyperglycemia and decreased organ ATP levels. In this study we are interested in evaluating the effects of dehydration on the aerobic scope of P. regilla and P. cadaverina, two highly terrestrial treefrogs. Furthermore we will investigate the effects of dehydration on the respiratory quotient of these frogs. Evaporative water loss in frogs can lead to a loss of 6–9% of initial body mass per day on land. In this study 8 frogs from each species were dehydrated to 5, 10, 15% of their initial body weight and then placed in a respiratory chamber so that standard metabolic rates were measured. After metabolic rates were recorded, hoping speed and the time it took for animals to right themselves were measured. After an exercise bout, the metabolic rate of all frogs was once again measured in all animals to determine the effect of dehydration on aerobic scope. Previously we have reported that P. regilla drinks water opportunistically whereas P. cadaverina must reach at least 18% dehydration before water absorption behaviors are consistently employed. Since there are significant differences in the levels of dehydration that each frog can endure before rehydration is normally established we expect to see differences in both aerobic scope, locomotion performance and the time it takes for frogs to switch to carbohydrates as the primary fuel for metabolism.

Support or Funding Information

Title V STEM Grant
Sex differences in the effects of environmental enrichment after maternal separation
Authors: Doreste‐Mendez Raura J. , Morales‐Bernecer Dayanira , Rivera‐Lopez Leslie , Torres‐Reveron Annelyn
Published: November 2023

It is well known that early stressful experience in life affect neurochemical, cognitive and behavioral development. Maternal separation in early life is a stressor which can cause depression and anxiety behaviors later in life. On the other hand, environmental enrichment can improve behavioral response and neurochemical markers. The aim in this study was compare the effect of early environmental enrichment in anxiety and depressive‐like behaviors and assess synaptic plasticity markers after maternal separation in both males and females. Our hypothesis is that there will be a sex‐specific response to environmental enrichment in animals that have undergone an early life stressor. Male and female Sprague‐Dawley rat pups (n=46) were separated from their dam for 3 hours daily during postnatal days (PND) 1 to 21. At PND 22 rats were divided into enriched (toys, larger cage, larger social group) and standard environment until PND 77. Open field test was used to measure anxiety and forced swim test to measure depressive‐like behaviors. After this, rats were sacrificed and their brains removed and processed for later immunohistochemical studies of the synaptic vesicle protein, synaptophysin in CA3 and dentate gyrus (DG) of the hippocampus. We found that female rats showed higher anxiety and depression compared to males. Male rats from the environmental enrichment group have reduced labeling of synaptophysin in the DG than males from the standard housing. This difference was not observed in the females. The increase in synaptophysin in control males can be a compensatory change in response to an impoverished environment. Environmental enrichment seems to have more effect in improving behavioral disorders in male rats than females after an early life stressor. For future studies, why this compensation is occurring in males but not in females remains to be elucidated.

Support or Funding Information

Funding: 1K07AT008027 and MD007579.
GABA Transporters (GATs) within the nucleus Tractus Solitarii (nTS) Modulate GABAergic Signaling, Neuronal Activity and Cardiorespiratory Function
Authors: Matott Michael P , Hasser Eileen M , Kline David D
Published: November 2023

GABAergic neurons and their signaling are the major inhibitory influence within the nTS and provide both tonic and activity‐dependent modulation of nTS circuits, cardiorespiratory function and visceral reflexes. Altered GABAergic function is implicated in cardiorespiratory pathologies such as hypertension. Thus, maintaining GABA concentration within the synaptic and extrasynaptic space is critical to maintaining the efficacy of the nTS synapse and the system as a whole. GABA is removed from the extracellular milieu by GATs found on neurons and astrocytes. Despite the importance of GABAergic signaling in the nTS, the role of nTS GATs on synaptic and neuronal modulation and cardiorespiratory function is unknown. We hypothesized GABA transporters tonically restrain inhibitory synaptic activity to 2nd order nTS neurons to influence cardiovascular function. RT‐PCR of nTS tissue demonstrated GAT3 mRNA was expressed nearly 2‐fold greater than GAT1. Immunohistochemistry revealed robust GAT3 immunoreactivity (‐IR) whereas GAT1‐IR was weak. GAT3‐IR surrounded GAD67‐IR synaptic terminals and colabeled with the astrocyte marker S‐100B. Given this expression pattern, we focused primarily on the functional role of GAT3. Blockade of GAT3 in nTS slices with (S)‐SNAP 5114 (SNAP) produced a significant hyperpolarizing current in nTS neurons. In the majority of neurons, GAT3 blockade significantly decreased spontaneous inhibitory postsynaptic current (IPSC) frequency but did not affect sIPSC amplitude. Stimulation of the medial nTS (0.5 Hz) evoked nTS‐IPSCs which were not altered by GAT3 blockade. Bilateral microinjection of SNAP into the caudal nTS of anesthetized rats significantly increased mean arterial pressure (MAP), heart rate (HR), sympathetic nerve activity (SNA), and minute phrenic (Phr) nerve activity. The latter was due primarily to augmentation of Phr amplitude. These responses mimicked the cardiorespiratory effects of exogenous GABA microinjection. Furthermore, SNAP pretreatment enhanced the effects of GABA microinjection on MAP, HR, and SNA. Together these results demonstrate that inhibitory tone under basal conditions in nTS is significantly affected by GAT activity and modulates basal cardiorespiratory function. GATs therefore are likely to be important in cardiorespiratory reflex modulation under physiological and pathophysiological conditions.

Support or Funding Information

15GRNT25710159 (AHA) and HL098602 (NIH)
Hypothalamic Activated Microglia Augment Blood Pressure Elevation during Early Developing Phase of Hypertension in Stroke‐prone Spontaneously Hypertensive Rats
Authors: Takesue Ko , Kishi Takuya , Hirooka Yoshitaka
Published: November 2023

Background

Inflammation within the paraventricular nucleus of the hypothalamus (PVN) mediated by microglia with microglial morphological change is an important pathology of sympathoexcitation. Although previous reports showed activation of microglia within the PVN of angiotensin II‐induced hypertensive or spontaneously hypertensive rats (SHR), we did not confirmed that deactivation of microglia within the PVN could decrease blood pressure in hypertensive rats. Our previous data indicated that PVN microglia were much more activated at earlier stage of hypertension in stroke‐prone SHR (SHRSP) compared to normotensive Wistar‐Kyoto (WKY) rats. Therefore, We examined whether deactivation of microglia within the PVN could attenuate blood pressure elevation during early developing phase of hypertension in genetically hypertensive rats.

Methods and results

Six week‐old SHRSP and age‐matched normotensive WKY were intracerebroventriculary (ICV) administered minocycline (5 μg/h), microglial deactivator, for 4 weeks. ICV infusion of minocycline significantly attenuated systolic blood pressure elevation in SHRSP over 4 weeks (at the end of experiments; 203.2±2.2 mm Hg vs. 215.9±2.7 mm Hg, n=8–9, P<0.05) but not in WKY. 24‐h urinary norepinephrine excretion tended to be lowered by minocycline infusion in SHRSP. Activation of microglia within the PVN, identified by microglia specific ionized calcium‐binding adaptor molecule 1 (Iba‐1) immunoreactivity, was evaluated by the roundness and perimeter of microglia. Minocycline significantly decreased roundness and increased perimeter of microglia, indicating deactivation of microglia, within the PVN of SHRSP.

Conclusion

Hypothalamic activated microglia with morphological alteration accelerate blood pressure elevation probably by sympathoexcitation during early developing phase of hypertension in SHRSP.

Support or Funding Information

This study was supported by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24390198 to Dr. Hirooka and 22790709 to Dr. Kishi).