Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome

Proteolytic activation of the renal epithelial sodium channel ENaC involves cleavage events in its α- and γ-subunits and is thought to mediate sodium retention in nephrotic syndrome (NS). However, detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α- and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the N-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed sodium retention and increased expression of fragments of α- and γ-ENaC cleaved at both the proximal and more prominently at the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented sodium retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α- and γ-ENaC was similarly found in healthy mice treated with a low salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and more prominently distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

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5′ Heterogeneity in epithelial sodium channel α-subunit mRNA leads to distinct NH2-terminal variant proteins

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.5.c1312 ◽

1998 ◽

Vol 274 (5) ◽

pp. C1312-C1323 ◽

Cited By ~ 43

Author(s):

Christie P. Thomas ◽

Scott Auerbach ◽

John B. Stokes ◽

Kenneth A. Volk

Keyword(s):

Sodium Channel ◽

Xenopus Oocytes ◽

Epithelial Sodium Channel ◽

Open Reading Frame ◽

Splice Sites ◽

Α Subunit ◽

Exon 2 ◽

Reading Frame ◽

Subunit Mrna ◽

Epithelial Sodium Channel Enac

The amiloride-sensitive epithelial sodium channel (ENaC) is composed of three subunits: α, β, and γ. The human α-ENaC subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. Proc. Natl. Acad. Sci. USA91: 247–251, 1994). To determine the origin of these transcripts, we characterized the 5′ end of the α-ENaC gene. Four transcripts that differ at their first exon were identified. Exon 1A splices to exon 2 to form the 5′ end of α-ENaC1, whereas exon 1B arises separately and continues into exon 2 to form α-ENaC2. Other variant mRNAs, α-ENaC3 and α-ENaC4, are formed by activating 5′ splice sites within exon 1B. Although α-ENaC3 and -4 did not change the open reading frame for α-ENaC, α-ENaC2 contains upstream ATGs that add 59 amino acids to the previous (α-ENaC1) protein. To address the significance of these isoforms, both proteins were expressed in Xenopus oocytes. The cRNA for each α-ENaC transcript when combined with β- and γ-ENaC cRNA reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant α-ENaC mRNAs that lead to functional ENaC peptides.

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Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome

Kidney International ◽

10.1016/j.kint.2017.07.023 ◽

2018 ◽

Vol 93 (1) ◽

pp. 159-172 ◽

Cited By ~ 34

Author(s):

Bernhard N. Bohnert ◽

Martina Menacher ◽

Andrea Janessa ◽

Matthias Wörn ◽

Anja Schork ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Epithelial Sodium Channel ◽

Volume Retention ◽

Epithelial Sodium Channel Enac

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Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome

Clinical Science ◽

10.1042/cs1040389 ◽

2003 ◽

Vol 104 (4) ◽

pp. 389-395 ◽

Cited By ~ 13

Author(s):

A. AUDIGÉ ◽

Z.R. YU ◽

B.M. FREY ◽

D.E. UEHLINGER ◽

F.J. FREY ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Mrna Expression ◽

Sodium Excretion ◽

Early Phase ◽

Urinary Sodium Excretion ◽

Urinary Sodium ◽

Sodium Retention ◽

Puromycin Aminonucleoside ◽

Epithelial Sodium Channel Enac

In experimental nephrotic syndrome, urinary sodium excretion is decreased during the early phase of the disease. The molecular mechanism(s) leading to salt retention has not been completely elucidated. The rate-limiting constituent of collecting duct sodium transport is the epithelial sodium channel (ENaC). We examined the abundance of ENaC subunit mRNAs and proteins in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. The time courses of urinary sodium excretion, plasma aldosterone concentration and proteinuria were studied in male Sprague–Dawley rats treated with a single dose of either PAN or vehicle. The relative amounts of αENaC, βENaC and γENaC mRNAs were determined in kidneys from these rats by real-time quantitative TaqMan PCR, and the amounts of proteins by Western blot. The kinetics of urinary sodium excretion and the appearance of proteinuria were comparable with those reported previously. Sodium retention occurred on days 2, 3 and 6 after PAN injection. A significant up-regulation of αENaC and βENaC mRNA abundance on days 1 and 2 preceded sodium retention on days 2 and 3. Conversely, down-regulation of αENaC, βENaC and γENaC mRNA expression on day 3 occurred in the presence of high aldosterone concentrations, and was followed by a return of sodium excretion to control values. The amounts of αENaC, βENaC and γENaC proteins were not increased during PAN-induced sodium retention. In conclusion, ENaC mRNA expression, especially αENaC, is increased in the very early phase of the experimental model of PAN-induced nephrotic syndrome in rats, but appears to escape from the regulation by aldosterone after day 3.

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Perspectives on edema in childhood nephrotic syndrome

AJP Renal Physiology ◽

10.1152/ajprenal.00229.2015 ◽

2015 ◽

Vol 309 (7) ◽

pp. F575-F582 ◽

Cited By ~ 10

Author(s):

Chia Wei Teoh ◽

Lisa A. Robinson ◽

Damien Noone

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Epithelial Sodium Channel ◽

Childhood Nephrotic Syndrome ◽

Glomerular Filtrate ◽

Epithelial Sodium Channel Enac

There have been two major theories surrounding the development of edema in nephrotic syndrome (NS), namely, the under- and overfill hypotheses. Edema is one of the cardinal features of NS and remains one of the principal reasons for admission of children to the hospital. Recently, the discovery that proteases in the glomerular filtrate of patients with NS are activating the epithelial sodium channel (ENaC), resulting in intrarenal salt retention and thereby contributing to edema, might suggest that targeting ENaC with amiloride might be a suitable strategy to manage the edema of NS. Other potential agents, particularly urearetics and aquaretics, might also prove useful in NS. Recent evidence also suggests that there may be other areas involved in salt storage, especially the skin, and it will be intriguing to study the implications of this in NS.

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