bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Epithelial Na+ Channel is Regulated by Biliary Components
Authors: Wang Xueping , Im Seohyun Janice , Ray Evan C. , Kashlan Ossama B. 
Published: June 2021

Cirrhosis of the liver often leads to edema and increased circulating and urinary levels of bile acids and bilirubin conjugates. We hypothesized that ENaC activation by increased concentrations of biliary metabolites in renal tubular ultrafiltrate could contribute to renal Na+ retention and edema in cirrhosis of the liver. The epithelial Na+ channel (ENaC) plays a key role in regulating Na+ reabsorption in the kidney, and is regulated by an array of factors, including extracellular ligands (e.g. H+, Na+, Cl−) and post‐translational modifications (e.g. cleavage and palmitoylation). To begin testing this, we expressed wild type and mutant ENaCs in Xenopus oocytes and measured effects on channel current by two‐electrode voltage clamp. We found that specific bile acids and conjugated‐bilirubin activate ENaC in a dose‐dependent manner, with deoxycholic acid having the strongest stimulatory effect. Notably, deoxycholic acid stimulated both wild type channels and ‘near silent’ channels in which the furin cleavage sites were mutated. However, fully activating channels by trypsin abolished deoxycholic acid stimulation in both wild type channels and channels lacking furin cleavage sites, consistent with an effect on open probability. We hypothesized that bile acids, conjugated bilirubin and Cys‐palmitoylation (palmitate addition to specific intracellular cysteines on the β and γ subunits) regulate ENaC currents through a common mechanism: by increasing the membrane association of specific intracellular structures, driving conformation changes in the channel's pore. To begin testing our hypothesis, we determined whether mutation of all 4 palmitoylation sites in the β and γ subunits (βC43, βC557, γC33 and γC41) to alanine would affect activation by deoxycholic acid. We found that deoxycholic acid stimulated currents of ENaC lacking of all 4 palmitoylation site cysteines by 7‐fold over wild type ENaC. When we tested the effect of mutating individual sites, we found that removal of the N‐terminal palmitoylation site cysteines resulted in the strongest stimulatory effects. We also found that deoxycholic acid had a stronger stimulatory effect when we removed both sites from the γ subunit as compared to the β subunit. Our results demonstrate that ENaC can be activated by biliary metabolites, including bile acids and bilirubin, and that these metabolites may activate ENaC through a mechanism in common with ENaC activation by palmitoylation.Support or Funding InformationDK098204 to O.B.K. and DK110332 to E.C.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The β‐adrenergic stimulation of the renal NaCl cotransporter is mediated via a cAMP‐dependent activation of protein phosphatase 1 inhibitor 1
Authors: Ribas David Penton , Moser Sandra , Wengi Agnieszka , Czogalla Jan , Rosenbaek Lena Lindtoft , Faresse Nourdine , Fenton Robert , Loffing‐Cueni Dominique , Loffing Johannes 
Published: June 2021

Sympathetic β‐adrenergic activation of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) was suggested to contribute to the development of salt‐sensitive hypertension. Previous studies demonstrated that β‐adrenergic agonists (e.g. norepinephrine and isoproterenol) increase cytoplasmic cAMP and the phosphorylation and hence the activity of NCC in the mouse DCT. Recently, we identified the endogenous inhibitor 1 (I1) of protein phosphatase 1 (PP1) as a DCT enriched gene product that controls NCC phosphorylation and blood pressure. As I1 is activated by cAMP‐dependent protein kinase A (PKA), we now tested the hypothesis that β‐adrenergic agonists inhibit PP1 via a cAMP/PKA‐dependent activation of I1 ultimately stimulating NCC phosphorylation. To test this hypothesis we performed experiments on MDCK cells stably transfected with NCC and on ex vivo preparations of kidneys from wildtype (WT) and I1‐knockout (I1‐KO) mice. The exposure of isolated DCTs to the cAMP‐elevating drugs forskolin and IBMX rapidly increased the phosphorylation of NCC via a protein kinase A (PKA)‐dependent pathway. The forskolin/IBMX‐induced phosphorylation of NCC was paralleled by I1 phosphorylation at its PKA‐consensus phosphorylation site (T35). Moreover, the forskolin/IBMX‐induced phosphorylation of NCC was diminished in kidney slices of from I1‐KO mice. The transgenic overexpression of a phosphomimetic I1 mutant (T35D) in kidneys of I1‐KO mice restored NCC phosphorylation, but made NCC resistant to forskolin/IBMX stimulation. Using an unbiased yeast‐two‐hybrid screen and co‐immunoprecipitation assays, we demonstrate that NCC and PP1 physically interact in transfected MDCK cells. Finally, the isoproterenol stimulated increase of NCC‐phosphorylation was impaired in I1‐KO mice as assessed in kidney slices and isolated‐perfused mouse kidneys. Remarkably, this occurred independent from any change in the phosphorylation of the kinases SPAK/OSR1. Thus, our data establish a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA‐dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway likely accounts for beta‐adrenergic NCC activation and hence may contribute to salt‐sensitive hypertension in patients with sympathetic hyperactivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Collecting‐duct‐principal‐cell‐select knockout (KO) of the mechanistic‐target‐of‐rapamycin (mTOR) alters sodium and acid/base homeostasis
Authors: Fluitt Maurice B. , Brown Aaron L. , Tiwari Swasti , Li Lijun , Ecelbarger Carolyn M. 
Published: June 2021

The mechanistic target of rapamycin (mTOR), a serine‐threonine kinase downstream of insulin receptor signaling and existing in a complex (mTORC2) with ancillary proteins, has been demonstrated to activate the renal epithelial sodium channel (ENaC). To further elucidate mTOR's role in the kidney collecting duct (CD), we generated CD‐principal‐cell‐select mTOR KO mice by Cre‐lox recombination using aquaporin‐2 (AQP2) promoter to drive Crerecombinase. A single injection of benzamil (ENaC antagonist) to male mice (n = 6/genotype) showed significantly reduced natriuretic response in the KO (32%) supporting lower ENaC activity. Urine was collected in metabolic cages while mice (n = 13/genotype) were fed a normal chow (1% NaCl) or a very low sodium (Na+) diet (<0.05%, LSD) for one week. Urine sodium concentrations were significantly higher on day 1 of the LSD in the KO (16.3 ± 1.8 versus 27.4 ± 4.3 mM, p = 0.024); but not on remaining days. Urine aldosterone (ng/d) rose 3–4 fold by day 7 with LSD in both genotypes, but was ~15–25% lower in the KO on most days. Mice were then divided to receive control diet or the LSD for an additional week and euthanized. Western blotting of cortex homogenates revealed a 20–40% reduction in the protein abundances of α, β‐and γ‐ENaC and aquaporin‐2 in the KO, regardless of diet. Furthermore, Rhbg (renal ammonia transporter) was ~65% lower in the KO. Blood chemistry showed significantly lower Na+ and higher bicarbonate (HCO3) concentrations in the KO under normal diet, and these differences were eliminated by the LSD. To test responses when aldosterone levels were clamped high, in Study 2, male mice were infused with aldosterone (20 mg/30 g·bw/d) by osmotic minipump for 3 days, then ½ of each genotype were switched to a high NaCl diet (4%) for 4 days. Clamping aldosterone high resulted in some “reversals” of phenotype, i.e., KO mice had significantly higher blood Na+ and lower HCO3 concentrations (relative to WT). This was associated with a modest relative reduction in K+ excretion and urine volume in the KO in the “aldosterone escape” phase, leading to an elevation in plasma K+ at the completion of the study. This may suggest relative aldosterone insensitivity in the KO. In sum, these studies support a role for CD‐principal cell mTOR in electrolyte and acid/base homeostasis. They also support a central role for mTOR in integrating growth factor and aldosterone actions at this site.Support or Funding InformationGeorgetown Medical Center Pilot Award (CE)Post‐Doctoral TL‐2 Award (MF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of ZO‐1, ARHGEF11 and Actin Cytoskeleton at the Apical Junctional Complex
Authors: White Addison R. , Mudekunye Gabriella G. , King Jonathan M. 
Published: June 2021

BackgroundZonula occludens (ZO‐1) is a tight junction cytosolic scaffolding protein known to bind the actin cytoskeleton in epithelial cells. ARHGEF11 is a Rho guanine nucleotide exchange factor known to activate the Rho family of small GTPases which are regulators of the actin cytoskeleton of the cell. ARHGEF11 associates with ZO‐1 by binding to the ZU5 domain in the C‐terminal tail. We are interested in understanding the interplay between ZO‐1, ARHGEF11 and the actin cytoskeleton in confluent MDCK II cells.MethodsConfluent monolayers stably transfected with EGFP‐ZO‐1 or EGFP‐ZO‐1Δ ZU5 were treated with latrunculin A to observe the actin depolymerization protection capacity of ZO‐1 with and without the ZU5 domain. Additionally, studies were performed to investigate the localization of EGFP‐ZO‐1 or EGFP‐ZO‐1ΔZU5 proteins following recovery from latrunculin A exposure. Colocalization studies were performed in mCherry‐ARHGEF11/EGFP‐ZO‐1 co‐transfected cells. Pharmacological inhibition of Rho‐kinase was used to target the ARHGEF11 pathway. Electrophysiological studies were conducted to evaluate junctional permeability.ResultsFull‐length EGFP‐ZO‐1 protects against latrunculin A induced actin depolymerization more robustly than EGFP‐ZO‐1ΔZU5. In the actin polymerization recovery paradigm EGFP‐ZO‐1ΔZU5 exhibited enhanced recovery rate when compared to full‐length EGFP‐ZO‐1 following latrunculin A exposure. Enhanced actin networks are observed in mCherry‐ARHGEF11 positive cells in both wildtype and ZO‐1 knockdown MDCK II cells.ConclusionsThe increased recovery rate exhibited by EGFP‐ZO‐1ΔZU5 in the latrunculin A recovery assay coupled with the greater actin protection capacity of full‐length ZO‐1 suggests that truncated ZO‐1 is more mobile. ARHGEF11 elicits actin cytoskeleton reorganization in both wildtype and ZO‐1 knockdown cells that was partially reversed by inhibition of Rho kinase. There was limited accumulation of ARHGEF11 at the apical junctional complex in confluent monolayers.Support or Funding InformationSan Antonio Medical Foundation, Trinity University Biology Department, Mach Research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purinergic Receptors Profile in the ARPKD Cystic Epithelia
Authors: Palygin Oleg , Ilatovskaya Daria V. , Levchenko Vladislav , Pavlov Tengis , Staruschenko Alexander 
Published: June 2021

Polycystic kidney diseases (PKD) are a group of inherited nephropathies marked with the formation of fluid‐filled cysts along the nephron. Growing evidence suggests that autocrine and paracrine effects of purinergic signaling via P2 receptors could detrimentally contribute to cyst expansion. However, little is known about purinergic signaling in renal cyst epithelium, which is characterized by loss of polarity, dedifferentiation and other abnormalities which can lead to purinergic signaling remodeling. We have proposed that ATP via associated intracellular signaling can contribute to cystogenesis by modulation of calcium influx. PCK/CrljCrl‐Pkhd1pck/CRL (PCK) rat, an established model of ARPKD, was used here to test this hypothesis. The cystic fluid of PCK rats and their cortical tissues exhibited significantly higher levels of ATP compared to Sprague Dawley (SD) rat kidney cortical interstitium as assessed by highly sensitive for ATP enzymatic biosensors (211±55 vs 1082±147 nM for SD and PCK cortex correspondingly, and 2,078±391 nM for cystic fluid). Confocal calcium imaging of the freshly isolated cystic monolayers revealed a stronger response to ATP in a higher range of concentrations (above 100 μM). The removal of extracellular calcium results in the absence of ATP evoked transient, which pointed towards the extracellular (ionotropic) calcium entry in cyst‐lining cells rather than the metabotropic P2Y‐mediated internal depot. Application of iso‐PPADS (a non‐selective P2X antagonist) resulted in partial blockade of ATP response (calcium release after ATP was 30.1 ± 0.9, 22.2 ± 1.8 and 19.5 ± 3.2 a.u. in control, after incubation with iso‐PPADS, and after washout, respectively) indicating the contribution of P2X4 purinoreceptor in the cystic monolayer. Next, to specifically assess the role of P2X7 in the ATP‐mediated calcium influx, we employed AZ10606120, a potent P2X7 receptor antagonist. Application of AZ10606120 (5 μM) resulted in a bunted calcium response to ATP (32.4 ± 2.2, 13.5 ± 6.4, and 11.1 ± 3.1 a.u. of total calcium release in control, after incubation with AZ10606120, and after washout, respectively), which corroborates the commonly hypothesized role of P2X7 in cyst development. Further use of pharmacological agents (α,β‐methylene‐ATP, 5‐BDBD, and NF449) allowed to narrow down potential candidate receptors and suggested a significant involvement of the P2X4 and/or P2X7 signaling axis in the regulation of cytosolic calcium level in the cystic epithelia. In conclusion, our ex vivo study provides direct evidence that the profile of P2 receptors is altered in the ARPKD cystic epithelia towards the prevalence of P2X4 and/or P2X7 receptors, which opens new avenues for the treatment of this disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Annexin II light chain p11 interacts with ENaC to increase functional activity at the membrane
Authors: McDonald Cowles Fiona J. , Cheung Tanya T. , Ismail Noor AS. , Moir Rachel , Arora Nikhil , Condliffe Steven 
Published: June 2021

The epithelial Na+ channel (ENaC) provides for Na+ absorption in various types of epithelia including the kidney, lung and colon where ENaC is localized to the apical membrane to enable Na+ entry into the cell. The degree of Na+ entry via ENaC largely depends on the number of active channels localized to the cell membrane. While regulation of ENaC endocytosis has been well studied, relatively little is understood of the proteins that govern ENaC exocytosis. We hypothesized that the annexin II light chain, p11 could participate in the transport of ENaC along the exocytic pathway. Our results demonstrate that all three ENaC channel subunits interacted with p11 in an in vitro binding assay. Quantitative mass spectrometry of affinity‐purified ENaC‐p11 complexes recovered several other trafficking proteins including HSP‐90, COMMD10 and annexin A6. We also found that p11 exhibits a robust protein expression in cortical collecting duct epithelial cells. However, the expression of p11 in these cells was not influenced by either short‐term or long‐term exposure to aldosterone. To determine whether the p11 interaction affected ENaC function, we measured amiloride sensitive Na+ currents in Xenopus oocytes or mammalian epithelia coexpressing ENaC and p11, or a siRNA to p11. Results from these experiments showed that p11 significantly augmented amiloride‐sensitive current, whereas knockdown of p11 decreased current. Further, knockdown of p11 reduced the ENaC cell surface population suggesting p11 promotes membrane insertion of ENaC. Overall, our findings reveal a novel protein‐protein interaction of p11 with ENaC that controls the number of ENaC channels inserted at the plasma membrane via the exocytic pathway.Support or Funding InformationThis work was supported by a University of Otago Research Grant, and an AIM grant from the Department of Physiology, University of Otago.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Learning to Breathe: Cholinergic modulation of plasticity associated with the gating of pulmonary stretch receptor input in the nucleus of the solitary tract.
Authors: Furuya Werner Issao , Dhingra Rishi , Dick Thomas , Colombari Debora , Dutschmann Mathias 
Published: June 2021

The lateral subnucleus of nucleus of the solitary tract (l‐NTS) is a primary synaptic site of visceral afferents, including pulmonary stretch receptors (PSRs). The PSR‐mediated inspiratory off‐switch (IOS), which is a component of the Hering‐Breuer reflex is a major determinant of the sensory modulation of breathing. Previously, we demonstrated that rhythmic vagal stimulation or lung inflations entrain phrenic nerve activity. Repetitive vagal stimulation or lung inflation induces a progressive shift from sensory (PSR) to anticipatory (learned) centrally mediated IOS (Dutschmann et al., 2009). In the present study, we investigated whether cholingeric neurotransmission within l‐NTS modulates PSR associated plasticity.Juvenile Sprague‐Dawley rats of either gender weighing 40–80 g (P21–28) were used in the decerebrated arterially‐perfused in situ preparations to record the phrenic nerve activity (PNA). The Hering‐Breuer reflex was evoked by inflating the lungs (1–2 mL tidal volume) at 40 inflations min−1 for 2 min. Lung inflation trials were repeated 10 times, at For the experimental groups, either atropine (ATR, muscarinic antagonist, 5 mM) or mecamylamine (MEC, nicotinic antagonist, 5 mM) were microinjected bilaterally in the l‐NTS after one lung inflation trial. Following a 3‐min interval, the lung inflation trials were repeated 10 times with a 3‐min recovery‐period between each trial. The control group received only 10 lung inflation trials. The 1st, 3rd, 5th and 7th trials were analyzed.The injection of atropine, a muscarinic receptor antagonist, in the l‐NTS promoted the plasticity switching the IOS from being initiated only by sensory activation to being initiated centrally, before the sensory input. In contrast, the nicotinic antagonist mecamylamine prevented the shift from sensory to central of IOS. In conclusion, we suggest that the muscarinic and nicotinic receptors in the l‐NTS have opposite roles in modulating the Hering‐Breuer‐induced plasticity that affects the IOS.Support or Funding InformationThe study was funded by the Australian Research Council and a fellowship for WF from FAPESP, Brazil.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gestational intermittent hypoxia induces neuroinflammation and impairs compensatory respiratory plasticity in adult offspring
Authors: Meza Armand , Gumnit Maia , Ewald Andrea , Braegelmann Kendra , Kiernan Elizabeth A. , Ouellette Jonathan N. , Johnson Stephen M. , Watters Jyoti J. , Baker Tracy 
Published: June 2021

Sleep disordered breathing during pregnancy is increasing at an alarming rate, but virtually nothing is known about long‐lasting effects on adult offspring physiology. Preliminary data suggest that microglia isolated from adult rat offspring exposed to in utero intermittent hypoxia exhibit enhanced inflammatory gene expression and primed responses to inflammatory stimuli. Since neuroinflammation abrogates some forms of respiratory plasticity, we tested the hypothesis that gestational intermittent hypoxia (GIH) impairs respiratory plasticity in adult offspring. Pregnant rat dams were exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or intermittent normoxia from gestation days 10–21 (GIH and GNX, respectively). Respiratory plasticity was assessed by measuring the response to recurrent neural apnea in urethane‐anesthetized, mechanically ventilated male and female GNX and GIH offspring. Recurrent neural apnea was induced by briefly hyperventilating below the CO2 apneic threshold for breathing (5, ~1 min neural apnea episodes, separated by 5 min). In GNX offspring, recurrent neural apnea triggered a significant increase in phrenic burst amplitude (45±3 %baseline, n=6, p<0.05), a form of plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). By contrast, intermittent neural apnea did not trigger increased phrenic burst amplitude in GIH offspring (7±8 %baseline, n=10, p>0.05), suggesting that in utero intermittent hypoxia impairs the capacity to elicit iPMF. Further, the phrenic response to intermittent neural apnea was significantly different in GIH and GNX rats (p<0.05). A critical role for microglial neuroinflammation is suggested by preliminary data indicating that the capacity to elicit iPMF can be restored in adult GIH offspring in which microglia are depleted with Pexidartinib (PLX3397) or following administration of the non‐steroidal anti‐inflammatory drug ketoprofen. Since iPMF is hypothesized to be a compensatory form of respiratory plasticity that prevents recurrent apneas, the impact of impaired iPMF on breathing stability in adult GIH offspring during sleep is currently being investigated.Support or Funding InformationHL105511, NS085226This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Participation in a High Fidelity Patient Simulation Prevents Knowledge Loss after One Week in Undergraduate Students
Authors: Harris David , Fragapane Lauren , Cheng Zixi 
Published: June 2021

High fidelity patient simulations (HFPS) have been used successfully in hospital training, medical education, and nursing to improve clinical, communication and procedural skills. Some reports have indicated that HFPS is underutilized for teaching basic science (Heyler R, Adv in Physio Educ, 2016). There are limited studies elucidating the benefits/challenges of using HFPS to learn physiology in undergraduate learners. One potential benefit is that HFPS allow students to apply physiology through recall and retrieval. The purpose of this study is to determine if HFPS enhances long‐term learning (1 week) more than re‐reading the material.METHODSSeventy‐six biomedical undergraduate students participated in the study and were assigned to one of two groups. The schematic for the study is included as an image. Both groups took a pre‐simulation quiz before reading the assigned text on pulmonary fibrosis and asthma (4 pages). All quizzes consisted of 10 true/false questions based on the text. The BLACK group (n=35) was given 15 mins to read the text and then another 15 mins to re‐read. The GOLD group (n=41) was given 15 mins to read the text and then participate in an asthma HFPS (intervention) for 15 mins. Afterwards, both groups took a post‐simulation quiz 1. One week later, both groups took another post‐simulation quiz 2. The pre‐simulation and post‐simulation quizzes 1 and 2 were all different, but the same between groups.RESULTSThere was a significant increase in the average number of questions correct between the pre‐simulation and post‐simulation quiz 1 in both groups. However, there were no differences between the two groups at either point. One week later, the BLACK group had a significant decrease in the number of correct answers on post‐simulation quiz 2 compared to post‐simulation quiz 1. There was no significant decrease in the number of correct questions on post‐simulation quiz 2 compared to post‐simulation quiz 1 for the GOLD group.CONCLUSIONSThe data suggest that HFPS may provide a platform to decrease the amount of knowledge loss after one week in undergraduate students. This may be due to HFPS acting as a method to promote recall and retrieval in students.Support or Funding InformationN/A