scholarly journals Acute effects of aldosterone on the epithelial Na channel in rat kidney

2015 ◽  
Vol 308 (6) ◽  
pp. F572-F578 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Surface Protein ◽  
Early Response ◽  
Surface Expression ◽  
Na Channel ◽  
Channel Activity ◽  
Acute Effects ◽  
Chronic Stimulation ◽  
Epithelial Na Channels ◽  
Epithelial Na Channel

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 μg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current ( INa). INa averaged 100 pA/cell, 20–30% of that reported for the same preparation under conditions of chronic stimulation. INa was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of β-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

scholarly journals Surface Expression of Epithelial Na Channel Protein in Rat Kidney

2008 ◽  
Vol 131 (6) ◽  
pp. 617-627 ◽  
Author(s):  
Gustavo Frindt ◽  
Zuhal Ergonul ◽  
Lawrence G. Palmer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Surface Expression ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Renal Cells ◽  
Patch Clamp Recording ◽  
Membrane Fractionation ◽  
Whole Cell Patch Clamp ◽  
Epithelial Na Channel

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

scholarly journals Regulation of ENaC trafficking in rat kidney

2016 ◽  
Vol 147 (3) ◽  
pp. 217-227 ◽  
Author(s):  
Gustavo Frindt ◽  
Diego Gravotta ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Surface Expression ◽  
Na Channel ◽  
Salt Diet ◽  
Golgi Protein ◽  
Endocytic Compartments ◽  
Gradient Separation ◽  
Na Depletion ◽  
Epithelial Na Channel ◽  
Salt Homeostasis

The epithelial Na channel (ENaC) forms a pathway for Na+ reabsorption in the distal nephron, and regulation of these channels is essential for salt homeostasis. In the rat kidney, ENaC subunits reached the plasma membrane in both immature and fully processed forms, the latter defined by either endoglycosidase H–insensitive glycosylation or proteolytic cleavage. Animals adapted to a low-salt diet have increased ENaC surface expression that is specific for the mature forms of the subunit proteins and is similar (three- to fourfold) for α, β, and γENaC. Kidney membranes were fractionated using differential centrifugation, sucrose-gradient separation, and immunoabsorption. Endoplasmic reticulum membranes, isolated using an antibody against calnexin, expressed immature γENaC, and the content decreased with Na depletion. Golgi membranes, isolated with an antibody against the cis-Golgi protein GM130, expressed both immature and processed γENaC; Na depletion increased the content of processed γENaC in this fraction by 3.8-fold. An endosomal compartment isolated using an antibody against Rab11 contained both immature and processed γENaC; the content of processed subunit increased 2.4-fold with Na depletion. Finally, we assessed the content of γENaC in the late endocytic compartments indirectly using urinary exosomes. All of the γENaC in these exosomes was in the fully cleaved form, and its content increased by 4.5-fold with Na depletion. These results imply that stimulation of ENaC surface expression results at least in part from increased rates of formation of fully processed subunits in the Golgi and subsequent trafficking to the apical membrane.

scholarly journals Effects of dietary K on cell-surface expression of renal ion channels and transporters

2010 ◽  
Vol 299 (4) ◽  
pp. F890-F897 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Ion Channels ◽  
Surface Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Secretory Cells ◽  
Na Channel ◽  
Apical Surface ◽  
High K ◽  
Low K ◽  
Epithelial Na Channel

Changes in apical surface expression of ion channels and transporters in the superficial rat renal cortex were assessed using biotinylation and immunoblotting during alterations in dietary K intake. A high-K diet increased, and a low-K diet decreased, both the overall and surface abundance of the β- and γ-subunits of the epithelial Na channel (ENaC). In the case of γ-ENaC, the effect was specific for the 65-kDa cleaved form of the protein. The overall amount of α-ENAC was also increased with increasing K intake. The total expression of the secretory K+ channels (ROMK) increased with a high-K diet and decreased with a low-K diet. The surface expression of ROMK increased with high K intake but was not significantly altered by a low-K diet. In contrast, the amounts of total and surface protein representing the thiazide-sensitive NaCl cotransporter (NCC) decreased with increasing K intake. We conclude that modulation of K+ secretion in response to changes in dietary K intake involves changes in apical K+ permeability through regulation of K+ channels and in driving force subsequent to alterations in both Na delivery to the distal nephron and Na+ uptake across the apical membrane of the K+ secretory cells.

scholarly journals Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium

2009 ◽  
Vol 297 (5) ◽  
pp. F1249-F1255 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Biochemical Parameter ◽  
Surface Expression ◽  
Dietary Sodium ◽  
Na Channel ◽  
Apical Surface ◽  
Deficient Diet ◽  
Luminal Membrane ◽  
Epithelial Na Channel

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) β-subunit (β-ENaC) and γ-subunit (γ-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of β-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of γ-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of γ-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Regulation of maturation and processing of ENaC subunits in the rat kidney

2006 ◽  
Vol 291 (3) ◽  
pp. F683-F693 ◽  
Author(s):  
Zuhal Ergonul ◽  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
Salt Restriction ◽  
High K ◽  
Electrophysiological Measurements ◽  
Immature Form ◽  
Epithelial Na Channel

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7–9 days, the abundance of two forms of the α-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the β-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the γ-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in α- and γ-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in α- and γ- but not β-subunits when animals were treated with exogenous aldosterone, and in β- and γ- but not α-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.

scholarly journals Epithelial Na+ channel activation and processing in mice lacking SGK1

2008 ◽  
Vol 294 (6) ◽  
pp. F1298-F1305 ◽  
Author(s):  
Geza Fejes-Tóth ◽  
Gustavo Frindt ◽  
Aniko Náray-Fejes-Tóth ◽  
Lawrence G. Palmer
Keyword(s):  
Full Length ◽  
Na Channel ◽  
Channel Activity ◽  
Salt Wasting ◽  
Optimal Processing ◽  
Collecting Ducts ◽  
Cell Current ◽  
Dependent Protein ◽  
Knockout Animals ◽  
Epithelial Na Channel

Amiloride-sensitive Na+ channel activity was examined in the cortical collecting ducts of a mouse line (SGK1−/−) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na+ channel (ENaC) protein. Neither SGK1−/− mice nor paired SGK1+/+ animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current ( INa), under control conditions with standard chow. Administration of aldosterone (0.5 μg/h via osmotic minipump for 7 days) increased INa to a similar extent in SGK1+/+ (378 ± 61 pA/cell at −100 mV) and in SGK1−/− (350 ± 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of γ-ENaC, was more pronounced in SGK+/+ mice. The SGK1−/− animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, INa was enhanced more in SGK1−/− (94 ± 14 pA/cell) than in SGK+/+ (23 ± 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length γ-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na+-Cl− cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.

scholarly journals Expression of epithelial Na channels in Xenopus oocytes.

1990 ◽  
Vol 96 (1) ◽  
pp. 23-46 ◽  
Author(s):  
L G Palmer ◽  
I Corthesy-Theulaz ◽  
H P Gaeggeler ◽  
J P Kraehenbuhl ◽  
B Rossier
Keyword(s):  
Sedimentation Coefficient ◽  
High Sensitivity ◽  
Sucrose Density Gradient ◽  
Na Channel ◽  
Channel Activity ◽  
Membrane Expression ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Epithelial Na Channels ◽  
Confluent Cell

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.

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