Acute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells

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.

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Intrarenal Ghrelin Receptor Antagonism Prevents High-Fat Diet-Induced Hypertension in Male Rats

Endocrinology ◽

10.1210/en.2013-2177 ◽

2014 ◽

Vol 155 (7) ◽

pp. 2658-2666 ◽

Cited By ~ 6

Author(s):

Brandon A. Kemp ◽

Nancy L. Howell ◽

John J. Gildea ◽

Shetal H. Padia

Keyword(s):

Growth Hormone ◽

High Fat Diet ◽

Collecting Duct ◽

Male Rats ◽

Na Channel ◽

Standard Diet ◽

High Fat ◽

Ghrelin Receptor ◽

Induced Hypertension ◽

Epithelial Na Channel

Excess weight gain contributes up to 65% of the risk of primary hypertension, and the increase in blood pressure in response to high-fat diet (HFD) is preceded by significant increases in renal tubular sodium (Na+) reabsorption. In normal rats, intrarenal ghrelin infusion increases distal nephron-dependent Na+ reabsorption via activation of the intrarenal ghrelin receptor (GHSR). This study focusses on the role of intrarenal GHSR-mediated Na+ reabsorption in HFD-induced hypertension. Dahl salt-sensitive rats received standard diet or HFD for 6 weeks. Rats underwent uninephrectomy and osmotic minipump implantation for chronic intrarenal delivery of vehicle (0.25 μL/h × 28 d), selective GHSR antagonist [D-Lys-3]-growth hormone releasing peptide-6 (0.2μM/d), or GHSR inverse agonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SUB-P) (3.6μM/d). HFD rats with vehicle pumps had significantly increased renal GHSR expression compared with standard diet (0.092 ± 0.005 vs 0.065 ± 0.004 arbitrary units; P < .05), whereas acyl ghrelin levels were similar (16.3±6.2 vs 15.7±8.7 pg/g tissue). HFD rats with vehicle pumps became hypertensive after 2 weeks (P < .05) and showed a significant reduction in 24-hour urine Na+ before hypertension. At this time, these rats showed an increase in collecting duct α-epithelial Na+ channel, thereby providing a potential mechanism for the excess Na+ reabsorption. In contrast, HFD rats with [D-Lys-3]-growth hormone releasing peptide-6 or SUB-P pumps never became hypertensive and did not show the reduction in urine Na+. Because SUB-P blocks the constitutive, but not ghrelin-dependent, activity of the GHSR, and HFD-induced α-epithelial Na+ channel up-regulation was abolished during GHSR antagonism, these data suggest that HFD increases the constitutive activity of renal GHSR to increase Na+ reabsorption and induce hypertension in rats.

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Surface Expression of Epithelial Na Channel Protein in Rat Kidney

The Journal of General Physiology ◽

10.1085/jgp.200809989 ◽

2008 ◽

Vol 131 (6) ◽

pp. 617-627 ◽

Cited By ~ 76

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.

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Immunocytochemical localization of Na+ channels in rat kidney medulla

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.2.f366 ◽

1989 ◽

Vol 256 (2) ◽

pp. F366-F369 ◽

Cited By ~ 3

Author(s):

D. Brown ◽

E. J. Sorscher ◽

D. A. Ausiello ◽

D. J. Benos

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Collecting Duct ◽

Rat Kidney ◽

Apical Plasma Membrane ◽

Na Channel ◽

Thick Ascending Limb ◽

Na Channels ◽

Kidney Medulla ◽

Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

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Overexpression of the Epithelial Na+Channel γ Subunit in Collecting Duct Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m413689200 ◽

2005 ◽

Vol 280 (18) ◽

pp. 18348-18354 ◽

Cited By ~ 12

Author(s):

Kenneth A. Volk ◽

Russell F. Husted ◽

Rita D. Sigmund ◽

John B. Stokes

Keyword(s):

Collecting Duct ◽

Na Channel ◽

Γ Subunit ◽

Duct Cells ◽

Collecting Duct Cells ◽

Epithelial Na Channel

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Faculty Opinions recommendation of Oxygen regulation of the epithelial Na channel in the collecting duct.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.7848956.8190054 ◽

2011 ◽

Author(s):

Bellamkonda Kishore

Keyword(s):

Collecting Duct ◽

Na Channel ◽

Oxygen Regulation ◽

Epithelial Na Channel

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Oxygen regulation of the epithelial Na channel in the collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00245.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. F412-F424 ◽

Cited By ~ 6

Author(s):

Russell F. Husted ◽

Hongyan Lu ◽

Rita D. Sigmund ◽

John B. Stokes

Keyword(s):

Kinase Activity ◽

Collecting Duct ◽

Hypoxia Inducible Factor ◽

Na Channel ◽

Mrna Levels ◽

Apical Surface ◽

Amp Kinase ◽

Na Transport ◽

Protein Levels ◽

Epithelial Na Channel

The Po2 within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing Po2 on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O2 concentration from 20 to 8% decreased ENaC activity by 40%; increasing O2 content to 40% increased ENaC activity by 50%. The O2 effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O2 medium. Corticosteroids increased ENaC activity at each O2 concentration; there was no interaction. The pathways for O2 and steroid regulation of ENaC are different since O2 did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2–45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O2 appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O2 concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O2 induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O2; both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

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Cloning of a bovine renal epithelial Na+ channel subunit

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.3.c641 ◽

1995 ◽

Vol 269 (3) ◽

pp. C641-C654 ◽

Cited By ~ 34

Author(s):

C. M. Fuller ◽

M. S. Awayda ◽

M. P. Arrate ◽

A. L. Bradford ◽

R. G. Morris ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Collecting Duct ◽

In Vitro Translation ◽

Equilibrium Potential ◽

Na Channel ◽

Xenopus Laevis Oocytes ◽

Cdna Expression Library ◽

Kinase C ◽

Epithelial Na Channel

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

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