Urokinase‐type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)‐mediated sodium retention in experimental 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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Aberrant glomerular filtration of urokinase-type plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine

AJP Renal Physiology ◽

10.1152/ajprenal.00138.2015 ◽

2015 ◽

Vol 309 (3) ◽

pp. F235-F241 ◽

Cited By ~ 21

Author(s):

Mette Stæhr ◽

Kristian B. Buhl ◽

René F. Andersen ◽

Per Svenningsen ◽

Flemming Nielsen ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Glomerular Filtration ◽

Plasminogen Activator ◽

Protease Activity ◽

Urine Samples ◽

Plasminogen Activation ◽

Sodium Retention ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in preurine are thought to activate proteolytically epithelial sodium channel (ENaC) and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator (uPA) in vitro. It was hypothesized that uPA is abnormally filtered to preurine and is inhibited in urine by amiloride in nephrotic syndrome. This was tested by determination of Na+ balance, uPA protein and activity, and amiloride concentration in urine from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Urine samples from 6 adult and 18 pediatric patients with nephrotic syndrome were analyzed for uPA activity and protein. PAN treatment induced significant proteinuria in rats which coincided with increased urine uPA protein and activity, increased urine protease activity, and total plasminogen/plasmin concentration and Na+ retention. Amiloride (2 mg·kg−1·24 h−1) concentration in urine was in the range 10–20 μmol/l and reduced significantly urine uPA activity, plasminogen activation, protease activity, and sodium retention in PAN rats, while proteinuria was not altered. In paired urine samples, uPA protein was significantly elevated in urine from children with active nephrotic syndrome compared with remission phase. In six adult nephrotic patients, urine uPA protein and activity correlated positively with 24 h urine protein excretion. In conclusion, nephrotic syndrome is associated with aberrant filtration of uPA across the injured glomerular barrier. Amiloride inhibits urine uPA activity which attenuates plasminogen activation and urine protease activity in vivo. Urine uPA is a relevant target for amiloride in vivo.

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

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-021-02639-7 ◽

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.

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Association of Urinary Plasminogen-Plasmin with Edema and Epithelial Sodium Channel Activation in Patients with Nephrotic Syndrome

American Journal of Nephrology ◽

10.1159/000501059 ◽

2019 ◽

Vol 50 (2) ◽

pp. 92-104 ◽

Cited By ~ 3

Author(s):

Jun-Liang Chen ◽

Li Wang ◽

Xing-Mei Yao ◽

Ying-Jun Zang ◽

Yi Wang ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Influencing Factor ◽

Fractional Excretion ◽

Epithelial Sodium Channel ◽

Pathophysiological Mechanism ◽

Sodium Retention ◽

Edema Formation ◽

Fractional Excretion Of Sodium ◽

Serial Samples

Background: Previous animal experiments and small human studies suggest that urinary plasmin can activate the epithelial sodium channel (ENaC) and contribute to sodium retention in nephrotic syndrome (NS), but this however is not well studied in clinical settings, and its relevance to edema formation is not well characterized in humans. We have investigated the association between urinary plasmin and clinical phenotypes in a large group of patients with NS from multiple etiologies, aiming to assess the role of urinary plasmin in sodium handling and edema formation. Methods: Two hundred and three NS patients with urine and blood samples were divided into mild and severe symptom groups based on their edema severity. Twenty six of them had serial samples collected during the course of immunosuppressive therapy. The plasminogen-plasmin level and other key parameters were assayed, and their association with clinical manifestations were analyzed. Results: One hundred and one of the 203 patients had renal biopsies performed, the results of which had included all the common types of primary NS and various types of secondary NS. Quantitative comparison and multivariate logistic regression analysis identified urinary plasminogen-plasmin to creatinine ratio (uPLG-PL/C), serum albumin, D-Dimer, and cardiac dysfunction history, but not albuminuria or 24-h urine protein, as independent risk factors for edema (p < 0.01). In patients who were treated and had serial samples, a decrease in uPLG-PL/C was identified as an independent influencing factor of edema remission (p < 0.01). Finally, the urinary fractional excretion of sodium (FENa) in patients was inversely correlated with the fractional excretion of potassium (FEK; p< 0.001), and FEK/FENa ratio was positively correlated with uPLG-PL/C (p < 0.001), suggesting a close association between uPLG-PL and ENaC activation. Conclusions: Our study identifies uPLG-PL abundance as an independent influencing factor of edema in adult NS patients, and supports the conclusion that plasmin-dependent ENaC activation is an important pathophysiological mechanism of sodium retention and edema formation in humans with NS.

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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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Secretion of the epithelial sodium channel chaperone PCSK9 from the cortical collecting duct links sodium retention with hypercholesterolemia in nephrotic syndrome

Kidney International ◽

10.1016/j.kint.2020.06.045 ◽

2020 ◽

Vol 98 (6) ◽

pp. 1449-1460 ◽

Cited By ~ 1

Author(s):

Eduardo Molina-Jijon ◽

Stéphanie Gambut ◽

Camille Macé ◽

Carmen Avila-Casado ◽

Lionel C. Clement

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Collecting Duct ◽

Epithelial Sodium Channel ◽

Sodium Retention ◽

Cortical Collecting Duct

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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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