Zymogen‐locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice

Essigke, Daniel; Ilyaskin, Alexandr V.; Wörn, Matthias; Bohnert, Bernhard N.; Xiao, Mengyun; Daniel, Christoph; Amann, Kerstin; Birkenfeld, Andreas L.; Szabo, Roman; Bugge, Thomas H.; Korbmacher, Christoph; Artunc, Ferruh

doi:10.1111/apha.13640

Cited by 21 publications

(24 citation statements)

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“…This substrate detects the activity of a wide range of trypsin-like proteases. The experimental protocol was similar to that described in previous reports [14,34]. The fluorescence signal resulting from substrate hydrolysis at the cell surface was continuously recorded over a time period up to 90 min using a TECAN plate reader (360 nm excitation/465 nm emission wavelength).…”

Section: Determination Of the Proteolytic Activity Of Fsapmentioning

confidence: 99%

“…Subsequently, 20 µg of sample was loaded on an 8% (γ-ENaC) or 4 −1 5% (α-and β-ENaC) polyacrylamide gel for electrophoresis under reducing conditions. After transfer of the proteins onto a nitrocellulose membrane (Amersham, UK), the blocked blots were incubated with primary rabbit antibodies against murine α-, β-(custom-made, Pineda, Berlin), and γ-ENaC (SPC-403, Stressmarq, Viktoria, Canada) overnight at 4 °C after 1:1000 dilution in blocking buffer (Licor, Lincoln, USA) [4,14,28]. Antibodies were based on the peptide sequences described by Masilamani et al [27] and validated using lysates from oocytes expressing murine αβγENaC against lysates from sham-injected oocytes as described previously [7].…”

Section: Western Blot From Kidney Tissue Of Micementioning

confidence: 99%

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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

Artunc

Bohnert

Schneider

et al. 2021

Pflugers Arch - Eur J Physiol

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Proteolytic 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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Section: Determination Of the Proteolytic Activity Of Fsapmentioning

confidence: 99%

Section: Western Blot From Kidney Tissue Of Micementioning

confidence: 99%

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

Artunc

Bohnert

Schneider

et al. 2021

Pflugers Arch - Eur J Physiol

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“…Interestingly, in both models, proteolysis of αand γ-ENaC subunits as well as their subcellular localization were conserved in kidney (Table 1). 50 The adult epithelial phenotype caused by tissue-specific gene deletion clearly confirmed an implication of CAP1 in ENaC-mediated sodium transport in lung and colon 43,45,47 but not in skin. 42,47,53,54 Alveolar-specific CAP1 knockout mice displayed a 40% reduction in the ENaC-mediated Na + current and impairment in alveolar fluid clearance.…”

Section: Cap1/prss8 (Prostasin)mentioning

confidence: 84%

“…Interestingly, in both models, proteolysis of α‐ and γ‐ENaC subunits as well as their subcellular localization were conserved in kidney (Table 1). 50 …”

Section: Enac Regulation By Proteasesmentioning

confidence: 99%

ENaC activation by proteases

2022

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Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.

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“…Among the redundant regulatory mechanism, proteolytic processing is a unique feature of ENaC, leading to the removal of inhibitory peptide tracts from the αand γsubunits by serine proteases. [98][99][100][101][102][103][104] After intracellular cleavage at two sites in α-ENaC and one in γ-ENaC during channel maturation in the trans-Golgi network by the serine protease furin, final proteolytic ENaC activation takes place at the plasma membrane where γ-ENaC is cleaved by membrane-bound proteases such as prostasin 105 and/ or other extracellular proteases in a region C-terminal to the furin site. [98][99][100][101][102][103][104] During proteolytic processing, the open probability of ENaC increases and reaches almost 100% in the fully cleaved state.…”

Section: F I G U R E 2 Comparison Of Nephrotic Features and Enac Acti...mentioning

confidence: 99%

Rodent models to study sodium retention in experimental nephrotic syndrome

et al. 2022

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Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2 Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2 Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown.With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

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Zymogen‐locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 21 publications

References 50 publications

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

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

ENaC activation by proteases

Rodent models to study sodium retention in experimental nephrotic syndrome

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