bluuebunny/crossref_metadata_2025_split huggingface dataset:
Renal epithelial electrophysiological response to cobalt chloride induced hypoxia (888.4)
Authors: Nag Subhra , Resnick Andrew 
Published: June 2021

Regulation of amiloride‐sensitive sodium current i.e., sodium channel (ENaC) current was found to be dependent on intact cilium in a kidney epithelial cell culture model. We hypothesize that cystic disease, for example Polycystic Kidney Disease (PKD), results in localized hypoxia due to creation of stagnant fluid‐filled cysts, interfering with normal salt and water transport.In the current study, we used a cortical collecting duct cell line (mCCD 1296 (d)) of an Immortomouse. We simulated hypoxic conditions with 100 µM Cobalt Chloride (CoCl2) and observed sodium transport under static and dynamic cell differentiation conditions. CoCl2 simulates hypoxia conditions by stabilizing the hypoxia inducible transcription factor 1α (HIF1α). We noticed a drop in the sodium current at time points of 24, 48, 72, and 96 hours in the presence of CoCl2. According to our hypothesis, the sodium current is expected to decrease in shorter time points of hypoxia whereas longer hypoxia induces cellular apoptosis totally nullifying the sodium current. We record sodium current in response to hypoxia at shorter time intervals within 48 hours as well as longer time periods upto 8 days. We use MTT cell viability assay to determine the time points at which CoCl2 induced hypoxia or cytotoxic CoCl2 start causing cell death. Correlating the MTT assay results with sodium current results will help us finding the direct correlation between hypoxia and sodium transport. This electrophysiological study will be complimented with immunocytochemistry to image cilium length alterations, and ciliary‐associated proteins, STAT‐6 and Phosphorylated‐STAT6 localizations. Thus, this study will provide better understanding of how hypoxia, sodium ion transport, effect of fluid flow and physiological and molecular cellular responses are interrelated.Grant Funding Source: Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716
KLHL3/CUL3 targets the ROMK endocytic protein ARH for degradation directly by ubiquitination (892.14)
Authors: Woodward Owen , Kim Boyoung , Fang Liang , Coleman Richard , Wade James , Welling Paul 
Published: June 2021

Mutations in KLHL3 and CUL3, components of an E3 ligase complex, cause a familial disease of hyperkalemia and hypertension (PHAII). Here, we explore how KLHL3/CUL3 normally regulates the ROMK channel for potassium balance. We previously reported that ROMK is targeted for endocytosis by a specific clathrin adaptor molecule, ARH (Fang et al, JCI ’09). Because KLHL3/CUL3 targets ARH for proteasomal degradation (Fang et al, EB ’13), we wondered if ARH is a direct substrate of KLHL3/CUL3 ubiquitination. In vitro protein‐protein interaction studies with purified ARH, KLHL3, CUL3 revealed that ARH directly interacts with KLHL3. We found separate interaction domains allow KLHL3 to simultaneously recruit CUL3 and the E3 ligase, ROC1, into a complex. As measured in ubiquitination assays in vitro with purified components, the direct interaction results in an increase in ARH ubiquitination, indicating that ARH is a direct substrate of KLHL3/CUL3. Importantly, KLHL3 localizes to the late DCT and CNT, overlapping with ROMK and ARH. Thus KLHL3/CUL3 are positioned to limit ARH and ROMK endocytosis to safeguard adequate potassium excretion.Grant Funding Source: NIDDK
Real time monitoring of NKCC2 endocytosis by total internal reflection fluorescence microscopy (892.19)
Authors: Jaykumar Ankita , Caceres Paulo , Sablaban Ibrahim 
Published: June 2021

The apical Na‐K‐2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TALs is determined by dynamic endocytosis from the apical membrane mediated by clathrin and lipid rafts. However, the time resolution of previous methods used to measure endocytosis was low (5 min), and internalization of NKCC2 containing vesicles not measurable. We hypothesize that TIRF microscopy imaging of fluorescently labeled apical NKCC2 would allow us to monitor its endocytosis in real‐time. We transduced polarized MDCK cells (in Transwell filters) with an N‐terminus GFP‐tagged NKCC2 using adenoviruses. Similar to native TALs, GFP‐NKCC2 was located in the apical membrane, at and above tight junctions. Surface biotinylation confirmed targeting of mature GFP‐NKCC2 (≍190 kDa) to the apical membrane. For TIRF imaging, cells were placed in a chamber with the apical membrane facing the TIRF illumination field. To selectively label NKCC2 at the apical surface we generated NKCC2 containing a biotin acceptor domain of 16 aa between one of the extracellular loops. Once expressed in MDCK cells, we specifically biotinylated surface NKCC2 with recombinant biotin ligase. Labeling apical NKCC2 with streptavidin‐Qdot, or streptavidin‐AlexaFluor allowed us to monitor single endocytic events and apical surface NKCC2 distribution in real‐time. In the absence of biotin ligase we did not observe labeling, indicating specificity for NKCC2 labeling. The overall rate of constitutive NKCC2 endocytosis was 1.2±0.1 % per minute (n=6). In MDCK and TAL cells most apical NKCC2 was located within small discrete domains that exhibited little lateral movement. We conclude that TIRF microscopy of labeled NKCC2 allows the imaging of individual endocytic events at the apical membrane.Grant Funding Source: NIH‐AHA
NKCC2 activity is inhibited upon the expression of the gain‐of‐function CASR mutant A843E in renal cells (892.23)
Authors: Carmosino Monica , Hendy Geoffrey , Torretta Silvia , Rizzo Federica , Gerbino Andrea , Procino Giuseppe , Svelto Maria 
Published: June 2021

The mutation gain‐of‐function A843E of the Calcium Sensing Receptor (CASR) causes a Bartter syndrome type V. Patients carrying this CASR variant showed a remarkable reduced renal NaCl reabsorption in Thick Ascendent Limb (TAL) resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl‐ ion transporters. The molecular mechanisms underlying this clinical phenotype are still unknown. Indeed, we investigated, in LLC‐PK1 epithelial renal cells, the possible functional cross‐talk of CASR‐A843E mutant with NKCC2, the main transporter involved in the NaCl reabsorption in the TAL. The steady‐state NKCC2 phosphorylation and activity were inhibited by about 60% in cells transfected with CASR‐A843E mutant compared to CASR WT‐transfected cells, used as control. Of note, the low‐chloride‐dependent NKCC2 activation was also strongly inhibited upon the expression of CASR‐A843E mutant. Interestingly the maximal activation of CASR WT mimicked the effect of CASR‐A843E on NKCC2 regulation. The expression of the CASR mutant did not alter the apical localization of NKCC2 in LLC‐PK1 cells, suggesting that the CASR‐A843E affected intracellular pathway/s modulating NKCC2 activity rather than NKCC2 intracellular trafficking. Our findings opened new avenues not only to gain more insight in the role of CASR in the physiopathology of the kidney, but also to conceive novel therapeutic strategies for the treatment of the Bartter syndrome type V.Grant Funding Source: Supported by PRIN 2009 n# 2009J53ALK to MS
Cytoskeletal regulation of ENaC: the role of ankyrin G (892.28)
Authors: Klemens Christine , Carananti Maria , Liu Xiaoning , Butterworth Michael 
Published: June 2021

The epithelial sodium channel (ENaC) is a major determinant of sodium (Na+) and water balance in the kidney. The hormone aldosterone increases ENaC subunit translation and modifies expression of key regulators that alter ENaC function and membrane density. Using a microarray screen we identified the scaffolding protein, ankyrin G (Ank3), as an aldosterone‐induced protein. We hypothesize that Ank3 regulates Na+ transport by increasing ENaC density at the apical membrane. Ank3 overexpression in a mouse kidney collecting duct epithelial cell line (mCCD‐cl1) increased ENaC‐mediated Na+ transport by 67%±11% (n=12), whereas knockdown reduced ENaC activity by 69%±2% (n=15). Ank3 depletion blunted the ENaC response to aldosterone stimulation in this cell line, from a 70.4±7% increase in control cells to 19.8±5% (n=5) increase in Ank3 knock‐down cells. Membrane permeabilization experiments confirmed the reduction in Na+ transport was due to reduced ENaC, and not Na/K‐ATPase activity. Future studies will identify critical domains governing Ank3’s association with ENaC and the regulation of this interaction. These findings introduce Ank3 as a key regulator in kidney Na+ transport.Grant Funding Source: Supported by DK078917, DK079307
Rosiglitazone promotes AQP‐2 translocation in renal cells via a Ca2+ dependent/cAMP independent mechanism (892.9)
Authors: Gerbino Andrea , Procino Giuseppe , Milano Serena , Nicoletti Maria , Mastrofrancesco Lisa , Carmosino Monica , Svelto Maria 
Published: June 2021

Preliminary results by our group showed that exposure to Rosiglitazone (RGZ) induces phosphorylation and apical translocation of AQP2 in mouse collecting duct clone 4 (MCD4) cells. Here we studied the effect of short‐term exposure to 50 µM RGZ on cAMP‐ and Ca2+‐mediated signaling pathways, both key players for AQP2‐mediated water reabsorption in the collecting duct. Cytosolic Ca2+ and cAMP levels were imaged in real‐time in MCD4 cells loaded with Fura‐2 or transiently transfected with the EPAC‐based fluorescent probe H90, respectively.Physiologically, AQP2 phosphorylation/translocation depends on cytosolic cAMP levels. Nonetheless, cAMP measurements showed that RGZ did not induce significant changes in cAMP levels. Conversely, 20 minutes RGZ stimulation of Fura‐2 loaded MCD4 cells induced a large, transient cytosolic Ca2+ peak that was not the result of direct blockade of the SERCA pump since the rate of store empting elicited by CPA in the absence of external Ca2+ was not significantly different in the presence of RGZ. Importantly, removal of external Ca2+ and inhibition of Ca2+ channels with ruthenium red prevented the RGZ‐induced increase in cytosolic Ca2+ indicating a prominent role for Ca2+ entry at the plasma membrane.In conclusion, RGZ‐induced AQP2 phosphorylation/translocation process is likely initiated by a fast, large extracellular Ca2+ influx most likely via Ca2+‐dependent transient receptor potential channels. Further studies to ascertain which cascade of kinases is involved in this scenario are in progress.Grant Funding Source: Supported by Telethon #GGP1204/AIFA MRAR08P011
Deletion of KCNJ10 eliminates potassium conductance in the basolateral membrane of the early distal convoluted tubule (DCT1). (893.17)
Authors: Wang Wen‐Hui , Zhang Chengbiao , Wang Lijun , Scholl Ute , Zhang Junhui , Lifton Richard , Lin Dao‐Hong 
Published: June 2021

The basolateral K channels in the DCT1 are composed of KCNJ10 and KCNJ16. Moreover, a 20 pS K channel was detected in the basolateral membrane of DCT of KCNJ16 knockout mice, suggesting that KCNJ10 alone is able to form homotetramers in the absence of KCNJ16. The aim of the present study is to test whether deletion of KCNJ10 would eliminate the basolateral K channel activity in the DCT1. Global deletion of KCNJ10 significantly impaired the animal growth, and the body weight of KCNJ10‐/‐ mice was significantly lower than that of their +/+ and +/‐ littermates. Moreover, KCNJ10‐/‐ mice did not survive beyond postnatal day 9‐12, while KCNJ10+/‐ mice had a life span similar to that of wt. To study the role of KCNJ10 in constructing the basolateral K channel, we performed patch‐clamp experiments in the isolated DCT1 of 8‐10 days old KCNJ10‐/‐, +/‐ and +/+ mice. The patch‐clamp study detected a 40 pS K channel in the basolateral membrane of the DCT1 of wt and KCNJ10+/‐, but not in KCNJ10 ‐/‐ mice. We also measured the Ba2+‐sensitive whole cell K currents which represent the basolateral K channel activity in the DCT1 since it has no apical K conductance. The mean Ba2+ ‐sensitive K currents at ‐60 mV were 2950 pA (wt), 1400 pA (+/‐) and 250 pA (‐/‐), respectively. Thus, deletion of KCNJ10 largely eliminated the basolateral K conductance in DCT1. We conclude that KCNJ10 is a major type of K channel expressed in the basolateral membrane of the DCT1.Grant Funding Source: NIH
Characterization of SLC4A11 as a novel ammonia: 2H+ transporter (893.29)
Authors: Zhang Wenlin , Ogando Diego , Bonanno Joseph , Obukhov Alexander 
Published: June 2021

SLC4A11 mutations cause corneal endothelial dystrophies. This degenerative condition leads to corneal haze, edema, and vision loss. SLC4A11 is a recently identified membrane transporter, of which multiple substrates have been suggested. Intracellular pH measurements in SLC4A11‐expressing PS120 cells revealed that SLC4A11 is NH3/NH4+ and H+ permeable. To further characterize SLC4A11 substrates and transport stoichiometry, we performed patch‐clamp recordings in SLC4A11‐PS120 cells. We found that extracellular 10mM NH3/NH4+ induced inward current, which was inhibited by bath acidification and potentiated by bath alkalinization. Current density (pA/pF) at pHo 6.5, 7.5 and 8.5 were ‐0.22±0.19, ‐6.18±1.09, ‐18.43±3.43, respectively (mean±SE, p<0.001). Reversal potential shift in response to [NH3]o and pHo was consistent with an NH3/H+ coupled transport model. 10mM methylammonium induced smaller current (p<0.05) with similar pH sensitivity. Removal of K+, Na+, Cl‐ did not affect SLC4A11 NH3/H+ transport (K+ p=0.90, Na+ p=0.06, Cl‐ p=0.65), suggesting neither Na+, K+, nor Cl‐ is substrate. Stoichiometry of SLC4A11 NH3:H+ transport was estimated by measuring Erev at different [NH3]o. A plot of Erev against log[NH3]o was linear, and the slope was consistent with a 1:2 stoichiometry. We here identify SLC4A11 as a new member of ammonia channels/transporters, such as AQP8, Rh, MEP, and Amt.Grant Funding Source: Supported by 5R01EY008834(JAB); 5R01HL115140(AGO).
Cloning, localization and characterization of a Zn transporter (Zip10) from fly, dog, and human (893.39)
Authors: Williams Paige , Strohmaier Käri , Hirata Taku , Pandey Mukesh , Jiang Huailei , Aditya Bansal , Cabrero Pablo , Dow Julian , DeGrado Timothy , Furrow Eva , Romero Michael 
Published: June 2021

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63Zn, with varying pHs and ion concentrations. 63Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping).Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl‐ (with HCO3舉) or all Na+ (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral).Conclusions: Our results indicated that all three Zip10 clones transport Zn2+. Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption.Grant Funding Source: Supported by DK83007, DK92408
Molecular determinants of the interaction between E3 ubiquitin ligase Nedd4‐2 and ENaC (893.5)
Authors: Labarca Mariana , Goens Donald , Dong Wuxing , Bhalla Vivek 
Published: June 2021

Phenotypic characterization of Nedd4‐2 knockout mice demonstrates the critical importance of this E3 ubiquitin ligase to activity of the epithelial sodium channel (ENaC) in several tissues including lung and kidney. However, determinants of the interaction between Nedd4‐2 and ENaC are not well understood and have implications for the treatment of several diseases including cystic fibrosis and hypertension.We characterized several novel determinants of this interaction using cell‐based biochemical assays to analyze exogenously or endogenously expressed Nedd4‐2 and ENaC.Nedd4‐2 preferentially binds to proteolytically cleaved ENaC in transfected HEK293T cells (10.9:1 +/‐ 0.009, p < 0.05). The preference for Nedd4‐2 to interact with cleaved channels is independent of Nedd4‐2 species and Nedd4‐2 ligase activity. Additionally, we demonstrate that neither an ENaC channel inhibitor, amiloride, nor ENaC channel mutants with increased or decreased activity alter the preference of Nedd4‐2 for cleaved channels. However, the calcium‐binding C2 domain of Nedd4‐2 decreases the binding of Nedd4‐2 to cleaved and uncleaved ENaC channels by approximately 5‐fold (p < 0.01). Surprisingly, active serum‐and‐glucocorticoid‐induced protein kinase 1 (SGK1) increased interaction between Nedd4‐2 and ENaC. Ongoing studies of the subcellular localization of Nedd4‐2 and cleaved or uncleaved ENaC channels is critical to understanding the determinants of this interaction.Proteolytic cleavage, but not activity, of ENaC; regulatory domains on Nedd4‐2; and unexpectedly, SGK1 kinase activity enhance Nedd4‐2 and ENaC interaction and can inform our understanding of how to target these pathways for treating lung and kidney disease.Grant Funding Source: NIH
14‐3‐3 ‐β and ‐ε Contribute to nuclear factor of activated T‐cells 5 (NFAT5) activity by regulating NFAT5 protein expression (895.3)
Authors: Izumi Yuichiro , Burg Maurice , Ferraris Joan 
Published: June 2021

NFAT5 is a transcription factor that induces transcription of osmoprotective genes in response to high NaCl. 14‐3‐3s are regulatory proteins that have numerous roles in protein interaction and enzymatic activity. There are multiple 14‐3‐3 isoforms that form homo‐ and hetero‐dimers and play isoform‐specific regulatory roles.In the present study, the effects of small interfering RNAs against 14‐3‐3 ‐β, ‐ε, ‐γ,‐η,‐θ, and ‐ζ on NFAT5 function were examined in HEK293 cells incubated at 300 and 500 mosmol/kg (NaCl added). We used Luciferase reporters to measure NFAT5 transcriptional (ORE‐X) and transactivating (TAD) activity. We quantified mRNA abundance of the NFAT5 target genes, aldose reductase (AR) and betaine/GABA transporter (BGT1) by real time PCR. We measured nuclear localization of NFAT5 by quantitative Western analysis of nuclear and cytoplasmic proteins. We also measured protein abundance of NFAT5.Knock down of 14‐3‐3 ‐β and/or ‐ε significantly inhibited high NaCl‐induced ORE‐X and TAD activities, however the effects on TAD activity were minimal. Knockdown of β and/or ε decreased mRNA abundances of AR and BGT1. The effects of knocking down β and ε were not additive. Knock down of β and ε did not affect the nuclear localization of NFAT5 but significantly decreased protein abundance of NFAT5.We conclude 14‐3‐3 ‐β and ‐ε contribute to NFAT5 transcriptional activity by regulating NFAT5 protein expression.Grant Funding Source: Supported by The Intramural Research Program of NHLBI, NIH.
Interaction of TM4SF4 with the human thiamine (vitamin B1) transporter‐2 (hTHTR‐2) in human intestinal epithelial cells (896.1)
Authors: Subramanian Veedamali , Nabokina Svetlana , Said Hamid 
Published: June 2021

The human thiamine transporter‐2 (hTHTR‐2) is involved in intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients have shown that these transporters have associated proteins that affect different aspects of their physiology and cell biology. Nothing is currently known about protein(s) that may interact with hTHTR‐2 in intestinal cells and influence its physiological function and cell biology.We addressed this issue using the yeast split‐ubiquitin two‐hybrid approach to screen a human intestinal cDNA library with the full‐length hTHTR‐2 as bait. We also performed GST‐pull‐down assay and co‐localization to confirm the existence of protein‐protein interaction. Our screening results have identified the human tetraspanin (transmembrane 4 super family member 4, TM4SF4) protein (which is abundantly expressed in the intestine) as a potential interactor with hTHTR‐2. The interaction between hTHTR‐2 and TM4SF4 were further confirmed by in vitro GST‐pull‐down assay and by live‐cell confocal imaging of HuTu‐80 cells co‐expressing hTHTR‐2‐GFP and Cherry‐TM4SF4. Co‐expression of hTHTR‐2 with TM4SF4 in HuTu‐80 cells led to a significant induction in hTHTR‐2 ‐ mediated thiamine uptake. These results show for the first time the existence of an accessory protein that interacts with hTHTR‐2 and influence its physiological function. (Supported by NIH grant DK 56061‐15).Grant Funding Source: NIH‐NIDDK
Bacterial immunomodulators in the treatment of patients with nonspecific ulcerative colitis (898.1)
Authors: Omarov T. , Tell L. , Dalenov E. 
Published: June 2021

Inflammatory bowel disease to date remains one of the most important and unsolved problems in gastroenterology.The purpose is to study the immune status of patients with nonspecific ulcerative colitis taking bacterial immunomodulators.Materials. 20 patients with left‐sided nonspecific ulcerative colitis in the acute stage as an immunomodulator on the background of basic therapy NUC received synbiotics BION 3 per 1 tablet a day for a month.Results. The study of the immune status before treatment revealed decreasing To‐lymphocytes 42,7±1,2%, Ts 8,3±0,7%, respectively, and indicators of spontaneous NST‐ stimulated 4,5±0,8%, and 6,6±1 6% and increasing in Th‐39, 4±1,5, T act 49,3±3,1% and the ratio of Th/Ts 4,8±0,4 indicating a violation of cellular immunity and phagocyte system depression. Increasing the level of PNA cells 21,0±1,2% and the CIC more than two times 30±2,5 conv unit indicates the severity of the chronic inflammatory process. After treatment, the cell‐mediated immunity returns to normal To 47,4±1,8%, Ts 12,9±0,6%, Tact 37,7±0,8%, Th 35,9±1,0% and the ratio of Th/Ts 2,8±014. Indicators NST test of spontaneous and stimulated significantly normalized and are 5,9±0,5% and 14,9±0,6% and showed activation of phagocytic activity of neutrophils. CIC after treatment is 19,9±2,4 conv units.Conclusions: 1. Inclusion to the treatment the bacterial immunomodulators normalizes the immune status.