scholarly journals Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

1994 ◽  
Vol 127 (6) ◽  
pp. 1907-1921 ◽  
Author(s):  
C Duc ◽  
N Farman ◽  
C M Canessa ◽  
J P Bonvalet ◽  
B C Rossier
Keyword(s):  
In Situ Hybridization ◽  
Sodium Channel ◽  
Sodium Transport ◽  
Epithelial Sodium Channel ◽  
Rat Colon ◽  
Sodium Reabsorption ◽  
Gamma Subunit ◽  
Collecting Ducts ◽  
Alpha Beta

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

scholarly journals Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

1997 ◽  
Vol 8 (12) ◽  
pp. 1813-1822 ◽  
Author(s):  
A May ◽  
A Puoti ◽  
H P Gaeggeler ◽  
J D Horisberger ◽  
B C Rossier
Keyword(s):  
Sodium Channel ◽  
Sodium Transport ◽  
Epithelial Sodium Channel ◽  
Mrna Abundance ◽  
Alpha Subunit ◽  
Gamma Subunit ◽  
Early Effect ◽  
Alpha Beta ◽  
Epithelial Sodium Channel Enac ◽  
In Cells

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

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

2000 ◽  
Vol 279 (1) ◽  
pp. F46-F53 ◽  
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.

scholarly journals Regulation of the epithelial sodium channel by accessory proteins

2003 ◽  
Vol 371 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Kelly GORMLEY ◽  
Yanbin DONG ◽  
Giuseppe A. SAGNELLA
Keyword(s):  
Blood Pressure ◽  
Cystic Fibrosis ◽  
Sodium Channel ◽  
Sodium Transport ◽  
Genetic Disorders ◽  
Epithelial Sodium Channel ◽  
Sodium Balance ◽  
Sodium Reabsorption ◽  
Channel Activity ◽  
Cellular Mechanisms

The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium fluxes in epithelial cells. Modulation of sodium reabsorption through the distal nephron ENaC is an important component in the overall control of sodium balance, blood volume and thereby of blood pressure. This is clearly demonstrated by rare genetic disorders of sodium-channel activity (Liddle's syndrome and pseudohypoaldosteronism type 1), associated with contrasting effects on blood pressure. The mineralocorticoid aldosterone is a well-established modulator of sodium-channel activity. Considerable insight has now been gained into the intracellular signalling pathways linking aldosterone-mediated changes in gene transcription with changes in ion transport. Activating pathways include aldosterone-induced proteins and especially the serum- and glucocorticoid-inducible kinase (SGK) and the small G-protein, K-Ras 2A. Targeting of the ENaC for endocytosis and degradation is now emerging as a major mechanism for the down-regulation of channel activity. Several proteins acting in concert are an intrinsic part of this process but Nedd4 (neural precursor cell expressed developmentally down-regulated 4) is of central importance. Other mechanisms known to interact with ENaC and affect sodium transport include channel-activating protease 1 (CAP-1), a membrane-anchored protein, and the cystic fibrosis transmembrane regulator. The implications of research on accessory factors controlling ENaC activity are wide-ranging. Understanding cellular mechanisms controlling ENaC activity may provide a more detailed insight not only of ion-channel abnormalities in cystic fibrosis but also of the link between abnormal renal sodium transport and essential hypertension.

scholarly journals Epithelial Sodium Channel Is a Key Mediator of Growth Hormone-Induced Sodium Retention in Acromegaly

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3294-3305 ◽  
Author(s):  
Peter Kamenicky ◽  
Say Viengchareun ◽  
Anne Blanchard ◽  
Geri Meduri ◽  
Philippe Zizzari ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Transport ◽  
Epithelial Sodium Channel ◽  
Janus Kinase ◽  
Sodium Reabsorption ◽  
Sodium Retention ◽  
Signal Transducer ◽  
Distal Nephron ◽  
Igf I ◽  
Gc Rats

Acromegalic patients present with volume expansion and arterial hypertension, but the renal sites and molecular mechanisms of direct antinatriuretic action of GH remain unclear. Here, we show that acromegalic GC rats, which are chronically exposed to very high levels of GH, exhibited a decrease of furosemide-induced natriuresis and an increase of amiloride-stimulated natriuresis compared with controls. Enhanced Na+,K+-ATPase activity and altered proteolytic maturation of epithelial sodium channel (ENaC) subunits in the cortical collecting ducts (CCDs) of GC rats provided additional evidence for an increased sodium reabsorption in the late distal nephron under chronic GH excess. In vitro experiments on KC3AC1 cells, a murine CCD cell model, revealed the expression of functional GH receptors and IGF-I receptors coupled to activation of Janus kinase 2/signal transducer and activator of transcription 5, ERK, and AKT signaling pathways. That GH directly controls sodium reabsorption in CCD cells is supported by: 1) stimulation of transepithelial sodium transport inhibited by GH receptor antagonist pegvisomant; 2) induction of α-ENaC mRNA expression; and 3) identification of signal transducer and activator of transcription 5 binding to a response element located in the α-ENaC promoter, indicative of the transcriptional regulation of α-ENaC by GH. Our findings provide the first evidence that GH, in concert with IGF-I, stimulates ENaC-mediated sodium transport in the late distal nephron, accounting for the pathogenesis of sodium retention in acromegaly.

scholarly journals Activation of the Hypoxia-Inducible Factor Pathway Inhibits Epithelial Sodium Channel-Mediated Sodium Transport in Collecting Duct Principal Cells

2021 ◽  
pp. ASN.2021010046
Author(s):  
Eva Dizin ◽  
Valerie Olivier ◽  
Isabelle Roth ◽  
Ali Sassi ◽  
Grégoire Arnoux ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Knockout Mice ◽  
Sodium Transport ◽  
Sodium Intake ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Sodium Reabsorption ◽  
Active Sodium ◽  
Principal Cells ◽  
Hif Pathway

Background Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxiainducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)-dependent sodium transport. Methods We investigated HIF's effect on ENaC expression in mpkCCDcl4 cells (a model of collecting duct principal cells) using real-time PCR and Western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule-specific Hif1α knockout mice. Results In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of βENaC and γENaC, as well as of Na,K-ATPase. HIF1α silencing increased βENaC and γENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γENaC abundance under high sodium intake. Conclusions This study reveals that γENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.

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

2020 ◽  
Vol 31 (5) ◽  
pp. 1009-1023 ◽  
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.

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

Hypertension ◽  
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.

Localization of rat CLC-K2 chloride channel mRNA in the kidney

1999 ◽  
Vol 276 (4) ◽  
pp. F552-F558 ◽  
Author(s):  
Momono Yoshikawa ◽  
Shinichi Uchida ◽  
Atsushi Yamauchi ◽  
Akiko Miyai ◽  
Yujiro Tanaka ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chloride Channel ◽  
Rat Kidney ◽  
Water Channel ◽  
Physiological Role ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Collecting Ducts

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+exchanger, Na+-Cl−cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle’s loop, but the signal in the cortical thick ascending limb of Henle’s loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.

Evolution of the epithelial sodium channel and the sodium pump as limiting factors of aldosterone action on sodium transport

2011 ◽  
Vol 43 (13) ◽  
pp. 844-854 ◽  
Author(s):  
Romain A. Studer ◽  
Emilie Person ◽  
Marc Robinson-Rechavi ◽  
Bernard C. Rossier
Keyword(s):  
Sodium Channel ◽  
Sodium Transport ◽  
Salt Intake ◽  
Epithelial Sodium Channel ◽  
Cell Junctions ◽  
Limiting Factors ◽  
Last Common Ancestor ◽  
Β Subunit ◽  
Α Subunit ◽  
Additional Function

Despite large changes in salt intake, the mammalian kidney is able to maintain the extracellular sodium concentration and osmolarity within very narrow margins, thereby controlling blood volume and blood pressure. In the aldosterone-sensitive distal nephron (ASDN), aldosterone tightly controls the activities of epithelial sodium channel (ENaC) and Na,K-ATPase, the two limiting factors in establishing transepithelial sodium transport. It has been proposed that the ENaC/degenerin gene family is restricted to Metazoans, whereas the α- and β-subunits of Na,K-ATPase have homologous genes in prokaryotes. This raises the question of the emergence of osmolarity control. By exploring recent genomic data of diverse organisms, we found that: 1) ENaC/degenerin exists in all of the Metazoans screened, including nonbilaterians and, by extension, was already present in ancestors of Metazoa; 2) ENaC/degenerin is also present in Naegleria gruberi , an eukaryotic microbe, consistent with either a vertical inheritance from the last common ancestor of Eukaryotes or a lateral transfer between Naegleria and Metazoan ancestors; and 3) The Na,K-ATPase β-subunit is restricted to Holozoa, the taxon that includes animals and their closest single-cell relatives. Since the β-subunit of Na,K-ATPase plays a key role in targeting the α-subunit to the plasma membrane and has an additional function in the formation of cell junctions, we propose that the emergence of Na,K-ATPase, together with ENaC/degenerin, is linked to the development of multicellularity in the Metazoan kingdom. The establishment of multicellularity and the associated extracellular compartment (“internal milieu”) precedes the emergence of other key elements of the aldosterone signaling pathway.

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