scholarly journals AS160 Modulates Aldosterone-stimulated Epithelial Sodium Channel Forward Trafficking

2010 ◽  
Vol 21 (12) ◽  
pp. 2024-2033 ◽  
Author(s):  
Xiubin Liang ◽  
Michael B. Butterworth ◽  
Kathryn W. Peters ◽  
Raymond A. Frizzell
Keyword(s):  
Protein Expression ◽  
Sodium Channel ◽  
Time Course ◽  
Plasma Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Rab Protein ◽  
Adipocyte Plasma Membranes

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (Isc). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and Isc 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and Isc under basal conditions to ∼80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, β and ε. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3β and ε and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.

scholarly journals Peroxisome proliferator-activated receptor-γ agonists repress epithelial sodium channel expression in the kidney

2012 ◽  
Vol 302 (5) ◽  
pp. F540-F551 ◽  
Author(s):  
Emily Borsting ◽  
Vicki Pei-Chun Cheng ◽  
Chris K. Glass ◽  
Volker Vallon ◽  
Robyn Cunard
Keyword(s):  
Protein Expression ◽  
Sodium Channel ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Duct Cell ◽  
Kidney Cortex ◽  
Peroxisome Proliferator ◽  
Cortical Collecting Duct ◽  
Peroxisome Proliferator Activated Receptor

Thiazolidinediones (TZDs), known as peroxisome proliferator-activated receptor (PPAR) agonists, are used to treat type 2 diabetes. However, ∼5% of patients experience the treatment-limiting side effect of edema. Studies have implicated activation of the epithelial sodium channel (ENaC) as a cause of TZD-induced fluid retention, although there have been conflicting reports. The goal of this study was to resolve the role of PPARγ in control of ENaC isoforms in the kidney. Herein, we demonstrate in mice that rosiglitazone (RGZ), a PPARγ ligand, increases body weight and abdominal fat pad fluid content and reduces hematocrit. Seven days of RGZ decreases ENaCα and ENaCβ mRNA and ENaCγ protein expression in the kidney cortex, and acute treatment for 5 h with pioglitazone, another potent TZD, does not increase renal ENaC isoform mRNA or protein expression. Pioglitazone also decreases ENaCα and ENaCγ mRNA expression in a cortical collecting duct cell line. As no direct transcriptional studies had been conducted, we examined the PPARγ-dependent regulation of ENaC. Pioglitazone represses ENaCγ promoter activity, and this repression is partially relieved by inhibition of protein synthesis. Chromatin immunoprecipitation assays revealed that repression is associated with a decrease in histone H4K5 acetylation at the proximal ENaCγ promoter. In summary, TZDs do not increase ENaC mRNA expression in the kidney, and in fact repress the ENaCγ promoter via an indirect transcriptional mechanism.

scholarly journals The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

2007 ◽  
Vol 282 (52) ◽  
pp. 37402-37411 ◽  
Author(s):  
Warren G. Hill ◽  
Michael B. Butterworth ◽  
Huamin Wang ◽  
Robert S. Edinger ◽  
Jonathan Lebowitz ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Lipid Rafts ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Sodium Channel Enac

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron

2012 ◽  
Vol 303 (9) ◽  
pp. F1289-F1299 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Anke Dahlmann ◽  
Bettina Krueger ◽  
Marko Bertog ◽  
Johannes Loffing ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Salt Diet ◽  
Low Salt ◽  
Immunohistochemical Evidence ◽  
Epithelial Sodium Channel Enac

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na+ channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (Δ Iami) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased Δ Iami in CNT/CCD but had little effect on Δ Iami in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS−/−) mice that lack aldosterone. In CNT/CCD of AS−/− mice, Δ Iami was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS−/− mice, Δ Iami was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

Cloning and functional expression of the mouse epithelial sodium channel

1999 ◽  
Vol 277 (1) ◽  
pp. F121-F129 ◽  
Author(s):  
Yoon J. Ahn ◽  
David R. Brooker ◽  
Farhad Kosari ◽  
Brian J. Harte ◽  
Jinqing Li ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Distal Colon ◽  
Dominant Negative ◽  
Full Length ◽  
Cortical Collecting Duct ◽  
Race Pcr

The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed α-, β-, and γENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse α-, β-, and γENaC (α-, β-, and γmENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length βmENaC and partial-length α- and γmENaC clones were isolated. Full-length α- and γmENaC cDNAs were subsequently obtained by 5′-rapid amplification of cDNA ends (5′-RACE) PCR. Injection of mouse α-, β-, and γENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive ( K i = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that α-, β-, and γmENaC were expressed in lung and kidney. Interestingly, αmENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5′-RACE PCR. Its sequence was found to be nearly identical to αmENaC. To begin to identify regions within αmENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of αmENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type α-, β-, and γmENaC with the intracellular NH2 terminus of αmENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of αmENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.

Regulation of blood pressure, the epithelial sodium channel (ENaC), and other key renal sodium transporters by chronic insulin infusion in rats

2006 ◽  
Vol 290 (5) ◽  
pp. F1055-F1064 ◽  
Author(s):  
Jian Song ◽  
Xinqun Hu ◽  
Shahla Riazi ◽  
Swasti Tiwari ◽  
James B. Wade ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Sodium Channel ◽  
Insulin Infusion ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Kidney Cortex ◽  
Sodium Reabsorption ◽  
Cortical Collecting Duct ◽  
Sprague Dawley ◽  
Epithelial Sodium Channel Enac

Hyperinsulinemia is associated with hypertension. Dysregulation of renal distal tubule sodium reabsorption may play a role. We evaluated the regulation of the epithelial sodium channel (ENaC) and the thiazide-sensitive Na-Cl cotransporter (NCC) during chronic hyperinsulinemia in rats and correlated these changes to blood pressure as determined by radiotelemetry. Male Sprague-Dawley rats (∼270 g) underwent one of the following three treatments for 4 wk ( n = 6/group): 1) control; 2) insulin-infused plus 20% dextrose in drinking water; or 3) glucose water-drinking (20% dextrose in water). Mean arterial pressures were increased by insulin and glucose (mmHg at 3 wk): 98 ± 1 (control), 107 ± 2 (insulin), and 109 ± 3 (glucose), P < 0.01. Insulin (but not glucose) increased natriuretic response to benzamil (ENaC inhibitor) and hydrochlorothiazide (NCC inhibitor) on average by 125 and 60%, respectively, relative to control rats, suggesting increased activity of these reabsorptive pathways. Neither insulin nor glucose affected the renal protein abundances of NCC or the ENaC subunits (α, β, and γ) in kidney cortex, outer medulla, or inner medulla in a major way, as determined by immunoblotting. However, insulin and to some extent glucose increased apical localization of these subunits in cortical collecting duct principal cells, as determined by immunoperoxidase labeling. In addition, insulin decreased cortical “with no lysine” kinase (WNK4) abundance (by 16% relative to control), which may have increased NCC activity. Overall, insulin infusion increased blood pressure, and NCC and ENaC activity in rats. Increased apical targeting of ENaC and decreased WNK4 expression may be involved.

Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex

2000 ◽  
Vol 278 (4) ◽  
pp. F530-F539 ◽  
Author(s):  
Johannes Loffing ◽  
Dominique Loffing-Cueni ◽  
Andreas Macher ◽  
Steven C. Hebert ◽  
Beatriz Olson ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Water Channel ◽  
Distribution Patterns ◽  
Epithelial Sodium Channel ◽  
Transport Systems ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Cortical Collecting Duct ◽  
Aquaporin 2

The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species.

scholarly journals Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

2012 ◽  
Vol 302 (5) ◽  
pp. F581-F590 ◽  
Author(s):  
Michael B. Butterworth ◽  
Robert S. Edinger ◽  
Mark R. Silvis ◽  
Luciana I. Gallo ◽  
Xiubin Liang ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Fluorescent Microscopy ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cortical Collecting Duct ◽  
Interfering Rna ◽  
Epithelial Sodium Channel Enac

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na+) transport. The greatest reduction in Na+ transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na+ transport in mpkCCD cells.

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