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

2003 ◽  
Vol 104 (4) ◽  
pp. 389-395 ◽  
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.

Collecting Duct Na+/K+-ATPase Activity Is Correlated with Urinary Sodium Excretion in Rat Nephrotic Syndromes

2000 ◽  
Vol 11 (4) ◽  
pp. 604-615 ◽  
Author(s):  
GEORGES DESCHÊNES ◽  
ALAIN DOUCET
Keyword(s):  
Nephrotic Syndrome ◽  
Atpase Activity ◽  
Sodium Excretion ◽  
Collecting Duct ◽  
Urinary Sodium ◽  
Sodium Balance ◽  
Brattleboro Rats ◽  
Sodium Retention ◽  
Puromycin Aminonucleoside ◽  
Stimulation Of

Abstract. In puromycin aminonucleoside (PAN)-treated nephrotic rats, sodium retention is associated with increased Na+/K+-ATPase activity in the cortical collecting ducts (CCD). This study was undertaken to determine whether stimulation of Na+/K+-ATPase in the CCD is a feature of other experimental nephrotic syndromes, whether it might be responsible for renal sodium retention, and whether it is mediated by increased plasma vasopressin levels or activation of calcineurin. For this purpose, the time courses of urinary excretion of sodium and protein, sodium balance, ascites, and Na+/K+-ATPase activities in microdissected CCD were studied in rats with PAN or adriamycin nephrosis or HgCl2nephropathy. The role of vasopressin and calcineurin in PAN nephrosis were evaluated by measuring these parameters in Brattleboro rats and in rats treated with cyclosporin or tacrolimus. Despite different patterns of changes in urinary sodium and protein excretion in the three nephrotic syndrome models, there was a linear relationship between CCD Na+/K+-ATPase activities and sodium excretion in all three cases. The results also indicated that there was no correlation between proteinuria and sodium retention, but ascites was present only when proteinuria was associated with marked reduction of sodium excretion. Finally, the lack of vasopressin in Brattleboro rats or the inhibition of calcineurin by administration of either cyclosporin or tacrolimus did not prevent development of the nephrotic syndrome in PAN-treated rats or stimulation of CCD Na+/K+-ATPase. It is concluded that stimulation of Na+/K+-ATPase in the CCD of nephrotic rats might be responsible for sodium retention and that this phenomenon is independent of proteinuria and vasopressin and calcineurin activities.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial sodium channel (ENaC) in the mouse kidney

2021 ◽  
Author(s):  
Bernhard N. Bohnert ◽  
Daniel Essigke ◽  
Andrea Janessa ◽  
Jonas C Schneider ◽  
Matthias Wörn ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Sodium Channel ◽  
Serine Protease ◽  
Cleavage Site ◽  
Mineralocorticoid Receptor ◽  
Epithelial Sodium Channel ◽  
Sodium Retention ◽  
Cleavage Sites ◽  
Proteolytic Activation ◽  
Epithelial Sodium Channel Enac

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.

Increased apical targeting of renal ENaC subunits and decreased expression of 11βHSD2 in HgCl2-induced nephrotic syndrome in rats

2006 ◽  
Vol 290 (3) ◽  
pp. F674-F687 ◽  
Author(s):  
Soo Wan Kim ◽  
Sophie de Seigneux ◽  
Martin C. Sassen ◽  
JongUn Lee ◽  
Jin Kim ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Sodium Excretion ◽  
Collecting Duct ◽  
Urinary Sodium Excretion ◽  
Brown Norway ◽  
Apical Plasma Membrane ◽  
Control Group ◽  
Sodium Retention ◽  
Outer Medulla ◽  
Outer Stripe

Nephrotic syndrome is often accompanied by sodium retention and generalized edema. We hypothesize that dysregulation of the epithelial sodium channel (ENaC) and/or of sodium (co)transporters may be responsible for the increased sodium retention associated with HgCl2-induced nephropathy. In addition, we examined the hypothesis that the expression of type 2 11β-hydroxysteroid dehydrogenase (11βHSD2) is reduced, contributing to the enhanced mineralocorticoid activity. Membranous nephropathy was induced in Brown Norway rats by repeated injections of HgCl2 (1 mg/kg sc), whereas the control group received only vehicle. After 13 days of treatment, the abundance of ENaC subunits, sodium (co)transporters, and 11βHSD2 in the kidney was examined by immunoblotting and immunohistochemistry. HgCl2 treatment induced marked proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and ascites. The protein abundance of α-ENaC was increased in the cortex/outer stripe of outer medulla (OSOM) and inner stripe of the outer medulla (ISOM). The protein abundances of β-ENaC and γ-ENaC were decreased in the cortex/OSOM while increased in the ISOM. Immunoperoxidase microscopy demonstrated increased targeting of ENaC subunits to the apical plasma membrane in the distal convoluted tubule, connecting tubule, and cortical and medullary collecting duct segments. Moreover, 11βHSD2 abundance was decreased in cortex/OSOM and ISOM. The protein abundances of type 3 Na/H exchanger (NHE3), Na-K-2Cl cotransporter (NKCC2), and thiazide-sensitive Na-Cl cotransporter (NCC) were decreased. Moreover, the abundance of the α-1 subunit of the Na-K-ATPase was decreased in the cortex/OSOM and ISOM but remained unchanged in the inner medulla. These results suggest that increased apical targeting of ENaC subunits combined with diminished abundance of 11βHSD2 may contribute to sodium retention associated with HgCl2-induced nephrotic syndrome. The decreased abundance of NHE3, NKCC2, NCC, and Na-K-ATPase may play a compensatory role in promoting sodium excretion.

scholarly journals Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice

2021 ◽  
Author(s):  
Ferruh Artunc ◽  
Bernhard N. Bohnert ◽  
Jonas C. Schneider ◽  
Tobias Staudner ◽  
Florian Sure ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Sodium Channel ◽  
Serine Protease ◽  
Cleavage Site ◽  
Factor Vii ◽  
Sodium Retention ◽  
Protease Domain ◽  
Proteolytic Activation ◽  
Serine Protease Domain ◽  
Epithelial Sodium Channel Enac

AbstractProteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2−/−) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2−/− mice. However, Habp2−/− mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2−/− mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

scholarly journals MO061PLASMINOGEN DEFIENCY DOES NOT PROTECT MICE FROM SODIUM RETENTION IN EXPERIMENTAL NEPHROTIC SYNDROME

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Mengyun Xiao ◽  
Bernhard Nikolaus Bohnert ◽  
Matthias Woern ◽  
Edward Plow ◽  
Tobias B Huber ◽  
...  
Keyword(s):  
Body Weight ◽  
Nephrotic Syndrome ◽  
Sodium Excretion ◽  
Serine Proteases ◽  
Essential Role ◽  
Urinary Sodium Excretion ◽  
Sodium Retention ◽  
Plasmin Activity ◽  
Edema Formation

Abstract Background and Aims Sodium retention and edema formation are the hallmarks of nephrotic syndrome and thought to be mediated by proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in nephrotic syndrome. However, there is not enough evidence to demonstrate the essential role of plasmin in mediating sodium retention in an experimental nephrotic model. Method We investigated sodium retention and edema formation in a mouse model of nephrotic syndrome based on an inducible podocyte-specific podocin knockout (Bl6-Nphs2tm3.1Antc or Δipod * Tg(Nphs1-rtTA*3G8Jhm)* Tg(tetO-cre) 1Jaw). To generate an inducible podocin knockout with plasminogen deficient model (Nphs2Δipod * Plg-/-, hereafter referred to as Plg-/-), plasminogen deficient mice (Bl6-Plgtm1Jld or -/-) were intercrossed with Nphs2Δipod mice. Nephrotic syndrome was induced after oral doxycycline treatment for 14 days. Body weight, urinary protein, urinary sodium excretion, as well as urinary plasmin activity were daily determined 14 days after end of induction. To determine if sodium retention can be prevented by serine protease inhibitor aprotinin in Plg+/+ and Plg-/- mice after induction of nephrotic syndrome, sustained-release pellets containing aprotinin (2 mg per day) or placebo pellets were implanted to either Plg+/+ or Plg-/- mice (n=4 per group). Results Uninduced Plg+/+ (n=6-13) and Plg-/- mice (n=6-14) had normal kidney function and sodium handling. After end of doxycycline induction, there was no significant differencein proteinuria increase between Plg+/+ (from 2 ± 0 to 161 ± 16 mg/mg creatinine, p<0.05) and Plg-/- mice (from 9 ± 8 to 146 ± 44 mg/mg creatinine, p<0.05) leading to similar hypoalbuminemia. In urine samples from Plg+/+ mice, Western blot revealed urinary excretion of plasminogen/plasmin which was completely absent in Plg-/- mice. Accordingly, urinary plasmin activity was only detectable in Plg+/+ mice using a chromogenic substrate. After onset of proteinuria, amiloride-sensitive natriuresis was increased compared to uninduced mice indicating ENaC activation. While urinary sodium excretion dropped in both genotypes indicating sodium retention (from 193 ± 16 to 16 ± 6 µmol/24h in Plg+/+ mice, p<0.05; from 229 ± 11 to 26 ± 7 µmol/24h in Plg-/-mice, p<0.05). As a consequence, body weight maximum increased in both genotypes 21 ± 1% in Plg+/+ and 17 ± 2% in Plg-/- mice (p=0.616) and was paralleled by development of ascites. Urinary amidolytic activity were completely prevented by the presence of aprotinin, as well as sodium retention and ascites in both Plg+/+and Plg-/- mice. Conclusion This study shows for the first time that mice lacking urinary plasmin are not protected from ENaC-mediated sodium retention in experimental nephrotic syndrome, however it can be prevented by aprotinin. These findings point to an essential role of other hitherto unknown serine proteases excreted in nephrotic urine.

Pathogenesis of Salt Retention in Dogs with Chronic Bile-Duct Ligation

1982 ◽  
Vol 62 (1) ◽  
pp. 65-70 ◽  
Author(s):  
C. Chaimovitz ◽  
U. Alon ◽  
O. S. Better
Keyword(s):  
Bile Duct ◽  
Sodium Excretion ◽  
Control Period ◽  
Extracellular Volume ◽  
Urinary Sodium Excretion ◽  
Urinary Sodium ◽  
Sodium Balance ◽  
Sodium Retention ◽  
Plasma Aldosterone Concentration ◽  
Duct Ligation

1. The present study investigates the role of mineralocorticoids in the pathogenesis of salt retention and ascites in dogs with chronic ligation of the common bile duct (CBDL). 2. After CBDL the natriuretic response to an intravenous sodium load [0.9% sodium chloride solution (150 mmol/l): saline; 10% of body weight] was markedly depressed. Urinary sodium excretion was 285 ± 62 vs 960 ± 58 μmol/min in the control period before CBDL (P < 0.001). This antinatriuresis was associated with a significant rise in plasma aldosterone concentration, from 52.5 ± 5.5 pg/ml before CBDL to 177 ± 50 pg/ml after CBDL (P < 0.02). Ascites was present in all salt-retaining CBDL dogs. 3. Bilateral adrenalectomy resulted in disappearance of ascites and in a rise in the natriuretic response to extracellular volume expansion. Urinary sodium excretion was 770 ± 124 μmol/min, a value significantly higher than in the CBDL dogs with intact adrenals (P < 0.001). Sodium balance studies in the adrenalectomized CBDL dogs during chronic deoxycorticosterone acetate (DOCA) treatment (25 mg/day) showed that in these animals there was failure to escape from the mineralocorticoid-induced sodium retention. Glomerular filtration rate and renal plasma flow did not change during the studies. 4. The present evidence supports the thesis that sodium retention in the CBDL dog results from a dual mechanism: (a) excess of circulating aldosterone and (b) an extra-adrenal factor which prevents escape from the salt-retaining effect of mineralocorticoids, in the CBDL dogs, thereby perpetuating the antinatriuresis in these animals.

Increased expression and apical targeting of renal ENaC subunits in puromycin aminonucleoside-induced nephrotic syndrome in rats

2004 ◽  
Vol 286 (5) ◽  
pp. F922-F935 ◽  
Author(s):  
Soo Wan Kim ◽  
Weidong Wang ◽  
Jakob Nielsen ◽  
Jeppe Praetorius ◽  
Tae-Hwan Kwon ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Plasma Membrane ◽  
Sodium Excretion ◽  
Collecting Duct ◽  
Apical Plasma Membrane ◽  
Control Group ◽  
Na Channel ◽  
Sodium Retention ◽  
Puromycin Aminonucleoside ◽  
Connecting Tubule

Nephrotic syndrome is often accompanied by sodium retention and generalized edema. However, the molecular basis for the decreased renal sodium excretion remains undefined. We hypothesized that epithelial Na channel (ENaC) subunit dysregulation may be responsible for the increased sodium retention. An experimental group of rats was treated with puromycin aminonucleoside (PAN; 180 mg/kg iv), whereas the control group received only vehicle. After 7 days, PAN treatment induced significant proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and extensive ascites. The protein abundance of α-ENaC and β-ENaC was increased in the inner stripe of the outer medulla (ISOM) and in the inner medulla (IM) but was not altered in the cortex. γ-ENaC abundance was increased in the cortex, ISOM, and IM. Immunoperoxidase brightfield- and laser-scanning confocal fluorescence microscopy demonstrated increased targeting of α-ENaC, β-ENaC, and γ-ENaC subunits to the apical plasma membrane in the distal convoluted tubule (DCT2), connecting tubule, and cortical and medullary collecting duct segments. Immunoelectron microscopy further revealed an increased labeling of α-ENaC in the apical plasma membrane of cortical collecting duct principal cells of PAN-treated rats, indicating enhanced apical targeting of α-ENaC subunits. In contrast, the protein abundances of Na+/H+ exchanger type 3 (NHE3), Na+-K+-2Cl- cotransporter (BSC-1), and thiazide-sensitive Na+-Cl- cotransporter (TSC) were decreased. Moreover, the abundance of the α1-subunit of the Na-K-ATPase was decreased in the cortex and ISOM, but it remained unchanged in the IM. In conclusion, the increased or sustained expression of ENaC subunits combined with increased apical targeting in the DCT2, connecting tubule, and collecting duct are likely to play a role in the sodium retention associated with PAN-induced nephrotic syndrome. The decreased abundance of NHE3, BSC-1, TSC, and Na-K-ATPase may play a compensatory role to promote sodium excretion.

Sign in / Sign up

Export Citation Format

Share Document