bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of a Diverse Array of Ca2+‐Activated K+ Channels are Functionally Coupled to the Mechanosensitive TRPV4 Channel in the Late Distal Tubule
Authors: Li Yue , Hu Hongxiang , O'Neil Roger G 
Published: November 2023

The voltage‐ and Ca2+‐activated, big conductance K+ channel (BK, maxi‐K channel) is known to be expressed in the distal tubule of kidney where it underlies flow‐ and Ca2+‐dependent K+ secretion in the connecting tubule (CNT) and cortical collecting duct (CCD). While the mechanosensitive TRPV4 channel is central for controlling Ca2+ influx, the potential role of other Ca2+‐activated K+ channels, KCa, in regulating K+ secretion in these cells is largely unknown. Using qPCR mRNA analysis and immunoblots of mouse kidney and the CCD cell line, mCCDcl1 (a K+ secreting cell line; Fodstad H et al., Am J Physiol 296:F966–75, 2009) an array of KCa channels was identified. Subsequent immunohistochemical staining for expressed ion channel proteins in CCD, CNT, and the mCCDcl1 cells, revealed expression of TRPV4 and three subfamilies of KCa channels: the small‐conductance SK channels (SK1 and SK3), the intermediate conductance IK channel (IK1), and the BK‐alpha channel. Intracellular Ca2+ imaging of mCCDcl1 cells demonstrated that the KCa channels were functionally regulated by TRPV4 and that each of the KCa channels displayed a potent functional cross‐talk with TRPV4 upon TRPV4 activation (GSK1016790A, 3–10 nM). Indeed, upon KCa channel activation induced by TRPV4, intracellular Ca2+ levels were suppressed upon inhibition of each given KCa channel subtype, SK, IK, BK, by addition of selective K+ channel inhibitors‐‐apamin (300 nM), TRAM‐34 (300 nM), or Iberiotoxin (100 nM), respectively. Further, for mCCDcl1 cells grown on permeable supports, activation of TRPV4 (GSK1016790A) induced a rapid decrease in transepithelial electrical resistance (TEER) that was partially restored by selective inhibition of each KCa subtype, SK, IK, and BK, as above. Immunohistochemcial staining of mouse CCD demonstrated expression of these channels in most cells, although significant differential staining of principle cells (PC) versus intercalated cells (IC) was apparent with the following staining patterns: TRPV4 PC>IC, BKa PC=IC, IK1 PC=IC, SK1 IC>>PC, and SK3 PC>>IC. It is concluded that an array of KCa channels are expressed in CNT and CCD and, like BK‐alpha, are functionally regulated by TRPV4‐mediated Ca2+ influx where strong cross‐talk between the KCa and TRPV4 exists. Further, it is postulated that with the high Ca2+ binding affinity of SK1/3 and IK1, these KCa channels will be activated early on during the initial induction of TRPV4. The observed functional cross‐talk will lead to early elevation in intracellular Ca2+ levels which will enhance activation of the low Ca2+ binding affinity channel, BK, and, in turn, give rise to enhanced BK‐mediated K+ secretion. Whether SK1/3 and IK1 directly participate in K+ secretion is likely, but remains to be verified in future studies.Support or Funding InformationNIH R01 DK098401 AHA 14GRNT20380916
Rapid Internalization and Trafficking of GC‐A/NPRA via Endo‐lysosomal Compartments with Concurrent Generation of cGMP in Mouse Mesangial Cells: Role of FQQI Motif
Authors: Mani Indra , Garg Renu , Tripathi Satyabha , Pandey Kailash N 
Published: November 2023

The objective of this study was to delineate the critical role of endocytic signal in intracellular sorting and signaling of receptor into these compartments. We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AAAA) using QuickChange II site‐directed mutagenesis kit in enhanced green fluorescence protein (eGFP)‐tagged NPRA (eGFP‐NPRA) cDNA sequence. Mouse mesangial cells (MMCs) were transiently transfected with both mutant and wild‐type constructs and cultured at 370C in an atmosphere of 5% CO2 and 95% O2. We utilized immunofluorescence staining and co‐immunoprecipitation (co‐IP) of plasma membrane, adaptor protein‐1, endosomal, lysosomal, and Rab 11 markers to follow subcellular trafficking and signaling by confocal immunofluorescence microscopy and immunoblotting. The treatment of cells with ANP at different times accelerated the internalization of receptor from cell surface to cell interior. The internalization studies revealed the immunofluorescence colocalization of mutated motif (FQQI/AAAA) receptor with pan‐Cadherin, a plasma membrane marker, and showed a decreased internalization of eGFP‐NPRA by 49%±3.4 (P< 0.001) compared with the wild‐type receptor. Interestingly, we show that μ1B subunit of adaptor protein‐1 (AP‐1, μ1B), binds directly to a phenylalanine based FQQI motif in the cytoplasmic tail of receptor. However, subcellular trafficking studies indicated that colocalization of mutated receptor with μ1B of AP‐1, early endosome antigen‐1 (EEA‐1), lysosome‐associated membrane protein‐1 (LAMP‐1), and Rab 11 marker, was decreased by 50%±3.2 (P< 0.001), into early endosome by 57%±4.7 (P< 0.001), lysosome by 32%±1.4 (P< 0.01), and recycling endosome by 40%±1.8 (P< 0.01) respectively, compared with wild‐type receptor. The receptor containing mutated motif (FQQI/AAAA) also produced a significantly decreased level 64%±3.6 (P< 0.001) intracellular cGMP during subcellular trafficking compared to WT receptor. The co‐immunoprecipitation assay confirmed a decreased level of immunofluorescence colocalization of mutant receptor with subcellular compartments during endocytic processes. The results suggest that Phe790 and Ile793 constitute important element in FQQI motif, which are essential for the internalization and trafficking of NPRA. The findings may provide the basis for new molecular association of single nucleotide polymorphisms in Npr1gene with susceptibility to diseases and /or resistance to drugs in translational studies.Support or Funding InformationThis work was supported by NIH grants R01HL057531 and R01HL062147
Localization and Regulation of the Disease‐Causing H+‐ATPase B1 Subunit in the Early Distal Nephron of Human and Murine Kidney
Authors: Dimke Henrik , Chambrey Régine , Zamani Reza , Marcussen Niels , Svenningsen Per , Frische Sebastian 
Published: November 2023

The H+‐ATPase B1 subunit is abundantly expressed in intercalated cells (IC) of the collecting system. Loss of function mutations in the gene results in distal renal tubular acidosis, due to insufficient acid secretion into urine. Curiously, mutations in the disease‐causing H+‐ATPase subunits has been reported to cause more severe forms of distal renal tubular acidosis, as compared to dominant mutations in AE1. Using 2 antibodies directed against the N‐terminal domain of the B1 subunit, we found abundant immunoreactivity in human kidney. As expected IC showed strongest signal, but significant signal was also observed in apical membrane aspects of the early distal nephron. Colocalization using known cellular markers confirmed expression of the B1 subunit in NCC positive distal convoluted tubules and NKCC2 positive cortical thick ascending limbs, but much less abundant in ENaC positive connecting tubule cells. Further immunohistochemical evaluation using an antibody directed against the other disease causing H+‐ATPase subunit A4, also showed localization to segments in the early distal nephron in human kidney. Expression was also detectable in macula densa for both subunits. In mouse and rat, B1 localization was investigated using antibodies directed against the N‐ or C‐terminal domains of the B1 subunit. Here subunit expression could be detected in apical membrane domains, beginning from the medullary thick ascending limb and stretching to the distal convoluted segment 2, in addition to the previously described IC. Using mice lacking the B1 subunit to validate the antibodies, complete loss of antigenicity was observed for two antibodies, while one showed minor staining in connecting tubules and IC, likely representing overlap with the B2 subunit epitope. To address whether the B1 subunit was subjected to regulation in the distal tubules, tissue from rats subjected to NH4Cl‐ and hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis were studied by immunohistochemistry. A mildly increased expression of the B1 subunit was noted in early distal nephron segments in kidneys of NH4Cl‐induced acidotic rats. In contrast, no change in expression could be observed in hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis. In conclusion, the B1 subunit is expressed in the early distal nephron in humans and rodents. The physiological consequences of H+‐ATPase expression in these segments remain to be delineated in detail.Support or Funding InformationThis work was funded by grants from the Novo Nordisk Foundation, the Carlsberg Foundation, the A.P. Møller Foundation, and the Lundbeck foundation.
Sexual Dimorphic Expression of Renal Claudins, Water Channels and Transporters accounts for the Downstream Shift in Salt and Volume Reabsorption along the Nephron in Female vs. Male Rats
Authors: Veiras Luciana Cecilia , Pei Lei , Yu Alan S.L. , McDonough Alicia Ann 
Published: November 2023

Recently, we reported that female rats, as well as mice, have lower abundance of apical proximal tubule (PT) sodium transporters (NHE3, NaPi2) as well as lower abundance of myosin VI, the motor that drives regulated redistribution of the apical transporters. This reduction in PT transporters in females is complemented by higher abundance and phosphorylation of distal tubule Na‐Cl cotransporter (NCC) and higher abundance of cleaved (activated) forms of epithelial sodium channels’ (ENaC) alpha and gamma subunits. At the physiologic level, the impact of the differences is evident as more rapid excretion of a saline load, higher clearance rate of endogenous lithium (a marker of flow from the proximal tubule), and no difference in thiazide sensitive natriuresis in females vs. males. The aim of the current study was to test the hypothesis that a sexual dimorphism in claudins (Cld‐) and water channels (AQP‐) accompanies the observed differences in transporters. Sprague Dawley rats were fasted overnight with water provided before termination. Abundance of claudins and water channels were determine by quantitative immunoblot of cortical or medullary homogenates. Claudin‐2, expressed from the PT through the thin descending limb, forms high‐conductance cation‐selective paracellular pores that play an important role in the paracellular reabsorption of salt in the PT (>32% of the filtered load). Claudin‐2 expression in cortex was lower in females vs. males: 0.32 ± 0.03 vs. 1.0 ± 0.1 (p< 0.002). Aquaporin 1, which confers “leakiness” to the PT, expressed in both apical and basolateral membranes of the PT, was also lower in females vs. males: 37 kD band: 0.44 ± 0.014 vs. 1.0 ± 0.023; 25 kD band: 0.84 ± 0.22 vs. 1.0 ± 0.26 (both p<0.001). Claudin‐7, expressed in the DCT and CCD, functions as either a Cl− barrier or pore; in vivo knockout provokes lethal salt wasting. Claudin‐7 expression in cortex was higher in females vs. males: 1.4 ± 0.01 vs. 1.0 ± 0.03 (p< 0.001). Claudin‐10, detected all along the nephron, and medullary apical Aquaporin‐2 were expressed at similar levels in females and males.ConclusionsLower abundance of both Cld‐2 and AQP‐1 in females corresponds to lower Na+ transporter abundance in the proximal nephron and may contribute to their improved ability to excrete a saline load. These factors may contribute to the lower blood pressures and resistance to experimental hypertension in females vs. males. In the distal nephron, higher abundance of Cld‐7 corresponds to the higher NCC and ENaC activities where it may facilitate salt reabsorption in response to the increased volume flow from the proximal nephron.Support or Funding InformationAHA 15GRNT23160003 and NIH DK083785
Nitric Oxide (NO) Regulates Paracellular Resistance in Isolated, Perfused Rat Thick Ascending Limbs through cGMP
Authors: Monzon Casandra M , Garvin Jeffrey L 
Published: November 2023

Thick ascending limbs reabsorb30% of the filtered Na. About 50% traverses the paracellular pathway. We showed that exogenously‐added nitric oxide reduces the Na/Cl permeability ratio(PNa/PCl) of the paracellular pathway via cGMP/PKG, but the effects on absolute per meabilities are unknown. We hypothesized that endogenously‐produced NO decreases paracellular PNa in thick ascending limbs. To test this we measured the effect of L‐arginine (0.5 mM; NO synthase substrate) and cGMP (0.5 mM; NO's second messenger) on PNa and PCl calculated from PNa/PCl and transepithelial resistance. PNa/PCl was 2.0 ± 0.2 in the control period and 1.6± 0.1 after L‐arginine treatment (p<0.05). Transepithelial resistance (Rt) was 7722 ± 1554 ohm‐cm in the control period and 6318 ± 1757 ohm‐cm after L‐arginine (p<0.05). Calculated PNa and PCl were 0.361 ± 0.049 μm/sec and 0.181 ± 0.025μm/sec, respectively, in the control period. After L‐arginine they were 0.621 ± 0.158μm/sec and 0.388± 0.099 μm/sec, respectively. In the presence of the NOS inhibitor L‐NAME (5mM), L‐arginine had no significant effect on paracellular resistance (L‐NAME alone: 7924 ± 1964 ohm‐cm; vs L‐NAME + L‐arginine: 8463 ± 1725ohm‐cm). Next we tested the effect of cGMP. PNa/PCl was 2.6 ± 0.6 in the control period and 1.6 ± 0.2 after cGMP treatment (p<0.06). Rt in the control period was 7592 ± 1470 ohm‐cm and 4796 ± 847 ohm‐cm after cGMP (p<0.035). PNa and PCl were 0.390 ± 0.071 μm/sec and 0.150± 0.028 μm/sec, respectively, for the control period, and 0.550± 0.170 μm/sec and 0.340 ± 0.110 μm/sec, respectively, after db‐cGMP. We conclude that contrary to our hypothesis NO increased both PNa and PCl, and thus decreased transepithelial resistance. NO's effects are likely mediated by cGMP. The increases in PNa and PCl most likely contribute to elevated NaCl flux from bath to lumen along the thick ascending limb due to their electrochemical gradients in vivo and thus reduce net NaCl reabsorption.Support or Funding InformationHL‐28982
Extreme blood pressure polymorphisms influence the bicarbonate‐independent conductance of NBCn1 but not its Na/HCO3 cotransport activity
Authors: Moss Fraser J , Zeise Brian , Parker Mark D , Yu Bing , Morrison Alanna C , Boron Walter F 
Published: November 2023

Genome‐wide association studies identified SLC4A7 as a locus associated with extreme blood pressure levels (Ehret G.B. et al. Nature. 2011 vol. 478 p.103). The product of this gene, the electroneutral Na+‐coupled HCO3− cotransporter NBCn1 is an important mediator of acid–base movement wherever it is expressed, which includes epithelial tissues, neurons and vascular smooth muscle. In addition to Na/HCO3 cotransport, NBCn1 mediates a steady‐state conductance (Gn1) that is independent of HCO3− transport. Exome sequencing of SLC4A7 was conducted among African and European American individuals from the Atherosclerosis Risk in Communities (ARIC) study. Individuals with whole exome sequence data were selected from the extremes (above upper quartile and below lower quartile) of the systolic and diastolic blood pressure distributions. In total, 42 single‐nucleotide polymorphisms (SNPs) associated with extremes of blood pressure were found within SLC4A7. Six SNPs located in the NBCn1 coding sequence (these result in the E252D, L261M, E326K, T563A, N756S, and P896S missense mutations) were selected for insertion into the human NBCn1‐B variant to test their function when expressed in Xenopus oocytes. All six NBCn1‐B mutants exhibited a near‐normal Na/HCO3 cotransport activity as determined by the rate of intracellular pH (pHi) recovery from a CO2‐induced acidification. However, when the Gn1 is normalized to cotransport activity to control for surface presentation, five of the mutants displayed a 12–35% reduction in the measured Gn1. The sixth mutation, P896S, which resides in the fourth extracellular loop (EL4) of NBCn1, caused a reduction in Gn1 of 84% on the wild‐type E326 background and 76% on the extreme blood‐pressure–associated K326 background. We conclude from this work that the polymorphisms we tested have little, if any effect on the Na/HCO3 cotransport rate, but can have substantial effects on the Gn1. Thus, these polymorphisms would not be expected to have a major, direct effect on pHi, but would be expected to affect cell membrane voltage (Vm). In particular, reduced Gn1 would be expected to hyperpolarize cells (i.e., shift Vm toward more negative voltages). Such shifts in Vm would be expected to secondarily affect a wide range of ion channels and electrogenic solute carriers in the SLC superfamily.Support or Funding InformationR01‐HL090969, R01‐DK30344, R01‐DK81567
TRPM4 Is Expressed in and Carries a Nonselective Cation Current Through the Apical Membrane of Mouse Cortical Collecting Duct Principal Cells
Authors: Wu Ming Ming , Thai Tiffany L , Zhai Yujia , Eaton Douglas C , Zhang Zhiren , Ma Heping 
Published: November 2023

Maintaining daily water, K+‐ and Na+‐balance is an important function of principal cells of the mouse cortical collecting duct (CCD). Under physiological conditions, these cells express a Ca2+ ‐activated nonselective cation channel (NSCCa), the molecular identity of which has been unknown. This study sought to identify whether the nonselective cation channel, TRPM4, functions as a NSCCa in principal cells of mouse cortical collecting duct.Using immunohistochemistry and western blotting, we show the presence of TRPM4 channel protein in the mouse cortical collecting duct cell line mpkCCDc14. Single channel recordings showed a current that is carried by monovalent cations and is Ca2+ sensitive and exhibits equal permeability for Na+ and K+. The corresponding current–voltage relationship of this channel was linear with a slope conductance 23.4 pS, which is the same as that observed from heterologously expressed TRPM4 channels. TRPM4 is inhibited by ATP and stimulated by PIP2. Application of 1 mM ATP to the “cytoplasmic” bath solution of an inside‐out patch almost completely inhibited channel activity but washing out ATP fully restored the channel activity. The channel activity significantly increased after adding 1 μM PI(4,5)P2 to the “cytoplasmic” bath solution. The TRPM4 inhibitor 9‐phenanthrol (100 μM) strongly inhibited the endogenous current. These biophysical properties of NSCCa are identical to those described for TRPM4. Acute application of H2O2 (100 μM and 500 μM) was unable to stimulate TRPM4 activity inside‐out patches; however, using a cell surface biotinylation assay, we showed that treating the mpkCCDc14 with 100 μM H2O2 for 24hrs significantly decreased cell surface levels of TRPM4 protein.Overall, we conclude that TRPM4 is the molecular identity of NSCCa in mpkCCDc14. Further study will be needed to determine the physiological relevance of this channel.Support or Funding InformationNIH R01‐DK100582 to HM
Elevated Level of Hypoxia Inducible Factor 1α Can Alter Renal Epithelial Function
Authors: Nag Subhra S , Resnick Andrew 
Published: November 2023

In the United States, about 600,000 people have Polycystic Kidney Disease (PKD) characterized by fluid filled structures (cysts) formed inside the kidney. Cysts interfere with normal renal function and lead to kidney failure. In the cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were found to be elevated. It is hypothesized that kidney cystic epithelia transform from normal absorption to secretion leading to accumulation of fluid within the cyst lumen. The fluid accumulation and insufficient vascular supply result in localized chronic hypoxia i.e., elevated HIF1α levels which may further promote cyst growth. We hypothesize that increased level of HIF1α can promote switching of renal epithelial function from absorptive to a secretory phenotype, possibly due to loss of epithelial tight junction integrity and altered active ion transport.HIF1α level was pharmacologically elevated by cobalt chloride (CoCl2) in monolayers of mouse cortical collecting duct cell line (mCCD 1296 (d)) grown on suspended permeable membranes in the presence or absence of fluid flow. In our analysis, transepithelial electrical resistance (TEER) and voltage values were recorded using an Endohm chamber. Higher TEER values reflect reduced paracellullar permeability indicating tight junction integrity which was verified by paracellular permeability assay using FITC‐conjugated dextran molecules. Short circuit current (Isc) was calculated using Ohm's law (I= V/R) to compare active ion transport. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. We found that elevated levels of HIF1α by CoCl2 caused loss of tight junction integrity and increased paracellular permeability. Increased HIF1α also resulted in decreased sodium transport which was determined from ameloride‐sensitive Isc. Currently, we are investigating the effect of increased HIFα by CoCl2 or deferoxamine on possible shift of active ion transport across the epithelial monolayer using specific sodium and chloride ion channel and transporter blockers. We are trying to understand the effect of increased level of HIF 1α on the localization and expression of tight junction proteins (e.g., zonula occludin 1 (ZO1) and claudins) and cotransporter proteins (e.g., Na+K+ATPase and NKCC1), leading to altered paracellular permeability and active ion transport. Thus, this study may provide a basis to understand how HIF1α or hypoxia may cause loss of normal epithelial function leading to cyst progression. This work could provide fresh insight into new treatment strategies for PKD.Support or Funding InformationThe project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN