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 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 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 Cleavage state of γENaC in mouse and rat kidney

2021 ◽  
Author(s):  
Gustavo Frindt ◽  
Shujie Shi ◽  
Thomas R Kleyman ◽  
Lawrence G Palmer
Keyword(s):  
Molecular Mass ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Mouse Kidney ◽  
Apparent Molecular Mass ◽  
Na Channel ◽  
Extracellular Proteases ◽  
Salt Diet ◽  
Heterologous System ◽  
Membrane Bound

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the g subunit. Here we investigate the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in FRT cells by co-expressing the apical-membrane bound protease CAP1 (prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest C-terminal fragment of gENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in FRT cells. Similar cleaved peptides were observed in total and cell surface fractions of rat kidney. This suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using non-reducing gels, in which the N- and C-terminal fragments of gENaC are linked by a disulfide bond. Under these conditions the major cleaved form in rat kidney had an apparent molecular mass of 56 kDa, ~4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant gENaC species in the apical membrane of rat and mouse kidney on a low-Na diet is the twice-cleaved, presumably activated form.

Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na+ channel (ENaC)

1997 ◽  
Vol 272 (6) ◽  
pp. C1871-C1880 ◽  
Author(s):  
O. Staub ◽  
H. Yeger ◽  
P. J. Plant ◽  
H. Kim ◽  
S. A. Ernst ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Renal Hypertension ◽  
Distal Colon ◽  
Surface Epithelium ◽  
Na Channel ◽  
Channel Stability ◽  
Interacting Protein ◽  
Ubiquitin Protein Ligase ◽  
Collecting Tubules ◽  
Epithelial Na Channel

The epithelial Na+ channel (ENaC) was previously shown to be expressed in several Na(+)- and fluid-absorbing epithelia, particularly those of the kidney, colon, and lung. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein with ENaC and demonstrated that Nedd4 binds by its WW domains to the proline-rich PY motifs of ENaC. These PY motifs were recently shown to be deleted/mutated in patients afflicted with Liddle's syndrome, a hereditary form of systemic renal hypertension. Such mutations cause elevated channel activity by an increase in channel number/stability at the plasma membrane and by increased channel opening. We then proposed that Nedd4, by regulating channel stability/ degradation, may be a suppressor of ENaC. To test whether Nedd4 is localized to those tissues/regions that express ENaC, we performed immunocytochemical analysis of rat Nedd4 (rNedd4) distribution in rat kidney, colon, and lung tissues. Our results show that, in the kidney, rNedd4 is primarily localized to the cortical collecting tubules and outer and inner medullary collecting ducts. These tubular segments were previously shown to express ENaC. The epithelium lining medullary calyxes was also intensely stained, and microvillar borders of proximal convoluted tubules expressed variable amounts of rNedd4. In the lung, rNedd4 was mainly expressed in the epithelia lining the airways, in the submucosal glands and ducts, and in the distal respiratory epithelium. These sites resemble the pattern of ENaC expression. In contrast, in the distal colon, rNedd4 was strongly expressed in the epithelia lining the crypts but not in the ENaC-expressing surface epithelium. Low-salt diet (to elevate serum aldosterone levels) had no effect on rNedd4 distribution in the kidney or colon. Thus Nedd4 is coexpressed and likely colocalizes with ENaC in specific regions within the kidney and lung but not in the colon. We speculate this difference in colocalization may reflect differences in the regulation of channel stability in those tissues.

Paraoxonase 2 is an ER chaperone that regulates the epithelial Na+ channel

2021 ◽  
Author(s):  
Shujie Shi ◽  
Teresa M. Buck ◽  
Andrew J. Nickerson ◽  
Jeffrey L. Brodsky ◽  
Thomas R. Kleyman
Keyword(s):  
Surface Expression ◽  
Inhibitory Effect ◽  
Na Channel ◽  
Dependent Manner ◽  
Er Chaperone ◽  
Channel Surface ◽  
Biochemical Analyses ◽  
Rat Thyroid ◽  
Epithelial Na Channel ◽  
Epithelial Sodium Channel Enac

The mammalian paraoxonases have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2 and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in Fisher Rat Thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts prior to ENaC subunits ubiquitination. As a result of the enhanced ENaC subunits ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirect the channel for ER associated degradation.

scholarly journals Ras Pathway Activates Epithelial Na+ Channel and Decreases Its Surface Expression in Xenopus Oocytes

1998 ◽  
Vol 9 (12) ◽  
pp. 3417-3427 ◽  
Author(s):  
Luca Mastroberardino ◽  
Benjamin Spindler ◽  
Ian Forster ◽  
Jan Loffing ◽  
Roberta Assandri ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Expression System ◽  
Regulatory Protein ◽  
Surface Expression ◽  
Na Channel ◽  
Target Cells ◽  
Ras Pathway ◽  
A6 Epithelia ◽  
The Mean ◽  
Epithelial Na Channel

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together withXK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control,XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12Vled to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC′s was also decreased; however, in comparison with progesterone-treated oocytes,XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressedXENaC was increased byXK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting thatXK-Ras2AG12V increased the mean activity ofXENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

Sign in / Sign up

Export Citation Format

Share Document