Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Download Full-text

Hydrogen Sulfide Prevents Advanced Glycation End-Products Induced Activation of the Epithelial Sodium Channel

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/976848 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Qiushi Wang ◽

Binlin Song ◽

Shuai Jiang ◽

Chen Liang ◽

Xiao Chen ◽

...

Keyword(s):

Sodium Channel ◽

Advanced Glycation End Products ◽

Epithelial Sodium Channel ◽

Sodium Reabsorption ◽

Metabolic Memory ◽

Advanced Glycation ◽

Open Probability ◽

A6 Cells ◽

Glycation End Products ◽

End Products

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds implicated in diabetes. Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal nephron plays an important role in diabetic hypertension. Here, we report that H2S antagonizes AGEs-induced ENaC activation in A6 cells. ENaC open probability(PO)in A6 cells was significantly increased by exogenous AGEs and that this AGEs-induced ENaC activity was abolished by NaHS (a donor of H2S) and TEMPOL. Incubating A6 cells with the catalase inhibitor 3-aminotriazole (3-AT) mimicked the effects of AGEs on ENaC activity, but did not induce any additive effect. We found that the expression levels of catalase were significantly reduced by AGEs and both AGEs and 3-AT facilitated ROS uptake in A6 cells, which were significantly inhibited by NaHS. The specific PTEN and PI3K inhibitors, BPV(pic) and LY294002, influence ENaC activity in AGEs-pretreated A6 cells. Moreover, after removal of AGEs from AGEs-pretreated A6 cells for 72 hours, ENaCPOremained at a high level, suggesting that an AGEs-related “metabolic memory” may be involved in sodium homeostasis. Our data, for the first time, show that H2S prevents AGEs-induced ENaC activation by targeting the ROS/PI3K/PTEN pathway.

Download Full-text

Interaction between Epithelial Sodium Channel γ-Subunit and Claudin-8 Modulates Paracellular Sodium Permeability in Renal Collecting Duct

Journal of the American Society of Nephrology ◽

10.1681/asn.2019080790 ◽

2020 ◽

Vol 31 (5) ◽

pp. 1009-1023 ◽

Cited By ~ 1

Author(s):

Ali Sassi ◽

Yubao Wang ◽

Alexandra Chassot ◽

Olga Komarynets ◽

Isabelle Roth ◽

...

Keyword(s):

Sodium Channel ◽

Knockout Mice ◽

Collecting Duct ◽

Epithelial Sodium Channel ◽

Paracellular Permeability ◽

Sodium Reabsorption ◽

Kidney Tubule ◽

Sodium Permeability ◽

Tubular Lumen ◽

Renal Collecting Duct

BackgroundWater and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient.MethodsTo investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule–specific knockout mice lacking ENaC subunits to assess the ENaC’s effect on claudin-8 expression.ResultsOverexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule–specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC β-subunit or α-subunit silencing or kidney tubule–specific β-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance.ConclusionsOur data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.

Download Full-text

Aldosterone-Induced Sodium Reabsorption in Pregnant Rat Kidney Distal Nephron: Expression and Activity of Epithelial Sodium Channel.

10.1210/endo-meetings.2010.part3.p13.p3-617 ◽

2010 ◽

pp. P3-617-P3-617

Author(s):

V Houde ◽

G Frindt ◽

M Provencher ◽

J St-Louis ◽

LG Palmer ◽

...

Keyword(s):

Sodium Channel ◽

Rat Kidney ◽

Epithelial Sodium Channel ◽

Sodium Reabsorption ◽

Distal Nephron ◽

Pregnant Rat ◽

Expression And Activity

Download Full-text

Abstract 076: Urinary Exosomal Epithelial Sodium Channel And Sodium-chloride Cotransporter Measurement In Humans

Hypertension ◽

10.1161/hyp.64.suppl_1.076 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

TAOPHEEQ A MUSTAPHA ◽

VICTOR NWAZUE ◽

KEVIN SCHEY ◽

RAJ SATISH ◽

JAMES M LUTHER

Keyword(s):

Sodium Chloride ◽

Sodium Channel ◽

Multiple Reaction Monitoring ◽

Epithelial Sodium Channel ◽

Sodium Balance ◽

Sodium Reabsorption ◽

Dependent Manner ◽

Creatinine Concentration ◽

Spot Urine ◽

Sodium Chloride Cotransporter

Sodium reabsorption in the distal nephron is tightly regulated in part by epithelial sodium channel (ENaC) and sodium chloride cotransporter (NCC), although non-invasive measure of these proteins in humans has not previously been feasible. We recently analyzed the urinary exosomal proteome and identified candidate targets for quantification of ENaC and NCC using targeted mass spectrometry. To test the hypothesis that urinary exosomal ENaC and NCC are altered during renin-angiotensin-aldosterone system activation, we activated the endogenous RAAS using a low sodium diet (LS) in two separate studies. We provided 8 subjects LS diet (10mmol/day for 7days) to assess urinary protein excretion at 7 days (study 1) and longitudinally over the course of 1 week (study 2). Daily 24-hour urine was collected to monitor sodium balance, and spot urine samples were obtained each morning on days 0, 2, 4, and 6 of LS diet. Urinary exosomal ENaC-α, ENaC-γ, and NCC peptides were analyzed using targeted multiple-reaction-monitoring analysis quantified with stable-isotope peptide standards, and results were normalized to urine creatinine concentration. In study 1, urinary ENaCγ increased after 8 days of LS diet (Figure A). In study 2, urinary exosomal ENaCγ (Figure B) and NCC peptides (Figure C) increased in a time-dependent manner during LS diet. These measures of urinary sodium channel expression may provide further insight into distal sodium reabsorption in human hypertension.

Download Full-text

Cyclosporine stimulates the renal epithelial sodium channel by elevating cholesterol

AJP Renal Physiology ◽

10.1152/ajprenal.90647.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. F284-F290 ◽

Cited By ~ 21

Author(s):

Jing Wang ◽

Zhi-Ren Zhang ◽

Chu-Fang Chou ◽

You-You Liang ◽

Yuchun Gu ◽

...

Keyword(s):

Sodium Channel ◽

Epithelial Sodium Channel ◽

Disulfonic Acid ◽

Severe Side Effect ◽

Open Probability ◽

A6 Cells ◽

Induced Hypertension ◽

Microscopy Data ◽

Abca1 Protein ◽

Epithelial Sodium Channel Enac

Cyclosporine A (CsA) is an efficient immunosuppressant used for reducing allograft rejection but with a severe side effect of causing hypertension. We hypothesize that the renal epithelial sodium channel (ENaC) may participate in CsA-induced hypertension. In the present study, we used the patch-clamp cell-attached configuration to examine whether and how CsA stimulates ENaC in A6 distal nephron cells. The data showed that CsA significantly increased ENaC open probability. Since CsA is an inhibitor of the ATP-binding cassette A1 (ABCA1) transporter, we employed 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), another ABCA1 inhibitor, and found that DIDS mimicked the effects of CsA on ENaC basal and cholesterol-induced activity but without any additive effect if combined with CsA. CsA and DIDS also had an identical effect on reduced ENaC activity caused by cholesterol extraction. ABCA1 protein was detected in A6 cells by Western blot analysis. Confocal microscopy data showed that both CsA and DIDS facilitated A6 cells to uptake cholesterol. Since enhanced ENaC activity is known to cause hypertension, these data together suggest that CsA may cause hypertension by stimulating ENaC through a pathway associated with inhibition of ABCA1 and consequent elevation of cholesterol in the cells.

Download Full-text

Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.1.f46 ◽

2000 ◽

Vol 279 (1) ◽

pp. F46-F53 ◽

Cited By ~ 169

Author(s):

Carolyn A. Ecelbarger ◽

Gheun-Ho Kim ◽

James Terris ◽

Shyama Masilamani ◽

Carter Mitchell ◽

...

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Collecting Duct ◽

Rat Kidney ◽

Epithelial Sodium Channel ◽

Sodium Reabsorption ◽

Acute Administration ◽

Brattleboro Rats ◽

Water Restriction ◽

Short Term

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (α, β, and γ) in rat kidney. Seven-day infusion of 1-deamino-[8-d-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of β- and γ-ENaC (to 238% and 288% of vehicle, respectively), whereas α-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of β- and γ- but no significant change in α-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

Download Full-text