Generic placeholder image

Current Medicinal Chemistry


ISSN (Print): 0929-8673
ISSN (Online): 1875-533X

Research Article

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine

Author(s): Matthias Wörn, Hubert Kalbacher and Ferruh Artunc*

Volume 29, Issue 42, 2022

Published on: 28 July, 2022

Page: [6433 - 6445] Pages: 13

DOI: 10.2174/0929867329666220608162256

Price: $65


Background: Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK).

Objective: We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC.

Methods: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract.

Results: No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC.

Conclusion: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Keywords: Epithelial sodium channel, ENaC, proteolytic activation, nephrotic syndrome, proteasuria, polybasic tract.

Artunc, F. Proteolytic activation of the epithelial sodium channel in nephrotic syndrome by proteasuria: Concept and therapeutic potential. Turk J Nephrol, 2020, 29(1), 59-65.
Artunc, F.; Wörn, M.; Schork, A.; Bohnert, B.N. Proteasuria-The impact of active urinary proteases on sodium retention in nephrotic syndrome. Acta Physiol. (Oxf.), 2019, 225(4), e13249.
[] [PMID: 30597733]
Kleyman, T.R.; Eaton, D.C. Regulating ENaC’s gate. Am. J. Physiol. Cell Physiol., 2020, 318(1), C150-C162.
[] [PMID: 31721612]
Althaus, M.; Lawong, R.Y. Proteolytic ENaC activation in health and disease-a complicated puzzle. Pflugers Arch., 2022, 4(474), 177-179.
[PMID: 34799769]
Hughey, R.P.; Bruns, J.B.; Kinlough, C.L.; Harkleroad, K.L.; Tong, Q.; Carattino, M.D.; Johnson, J.P.; Stockand, J.D.; Kleyman, T.R. Epithelial sodium channels are activated by furin-dependent proteolysis. J. Biol. Chem., 2004, 279(18), 18111-18114.
[] [PMID: 15007080]
Bruns, J.B.; Carattino, M.D.; Sheng, S.; Maarouf, A.B.; Weisz, O.A.; Pilewski, J.M.; Hughey, R.P.; Kleyman, T.R. Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit. J. Biol. Chem., 2007, 282(9), 6153-6160.
[] [PMID: 17199078]
Adachi, M.; Kitamura, K.; Miyoshi, T.; Narikiyo, T.; Iwashita, K.; Shiraishi, N.; Nonoguchi, H.; Tomita, K. Activation of epithelial sodium channels by prostasin in Xenopus oocytes. J. Am. Soc. Nephrol., 2001, 12(6), 1114-1121.
[] [PMID: 11373334]
Carattino, M.D.; Mueller, G.M.; Palmer, L.G.; Frindt, G.; Rued, A.C.; Hughey, R.P.; Kleyman, T.R. Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease. Am. J. Physiol. Renal Physiol., 2014, 307(9), F1080-F1087.
[] [PMID: 25209858]
Anand, D.; Hummler, E.; Rickman, O.J. ENaC activation by proteases. Acta physiologica (Oxford, England), 2022, e13811.
Wang, X.P.; Balchak, D.M.; Gentilcore, C.; Clark, N.L.; Kashlan, O.B. Activation by cleavage of the epithelial Na+ channel α and γ subunits independently coevolved with the vertebrate terrestrial migration. eLife, 2022, 11, 11.
[] [PMID: 34984981]
Bohnert, B.N.; Menacher, M.; Janessa, A.; Wörn, M.; Schork, A.; Daiminger, S.; Kalbacher, H.; Häring, H.U.; Daniel, C.; Amann, K.; Sure, F.; Bertog, M.; Haerteis, S.; Korbmacher, C.; Artunc, F. Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome. Kidney Int., 2018, 93(1), 159-172.
[] [PMID: 29042083]
Xiao, M.; Bohnert, B.N.; Aypek, H.; Kretz, O.; Grahammer, F.; Aukschun, U.; Wörn, M.; Janessa, A.; Essigke, D.; Daniel, C.; Amann, K.; Huber, T.B.; Plow, E.F.; Birkenfeld, A.L.; Artunc, F. Plasminogen deficiency does not prevent sodium retention in a genetic mouse model of experimental nephrotic syndrome. Acta Physiol. (Oxf.), 2021, 231(1), e13512.
[] [PMID: 32455507]
Bohnert, B.N.; Essigke, D.; Janessa, A.; Schneider, J.C.; Wörn, M.; Kalo, M.Z.; Xiao, M.; Kong, L.; Omage, K.; Hennenlotter, J.; Amend, B.; Birkenfeld, A.L.; Artunc, F. Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney. Am. J. Physiol. Renal Physiol., 2021, 321(4), F480-F493.
[] [PMID: 34423678]
Wörn, M.; Bohnert, B.N.; Alenazi, F.; Boldt, K.; Klose, F.; Junger, K.; Ueffing, M.; Birkenfeld, A.L.; Kalbacher, H.; Artunc, F. Proteasuria in nephrotic syndrome-quantification and proteomic profiling. J. Proteomics, 2021, 230, 103981.
[] [PMID: 32927112]
Baechle, D.; Cansier, A.; Fischer, R.; Brandenburg, J.; Burster, T.; Driessen, C.; Kalbacher, H. Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity. J. Pept. Sci., 2005, 11(3), 166-174.
[] [PMID: 15635643]
Jores, T.; Klinger, A.; Groß, L.E.; Kawano, S.; Flinner, N.; Duchardt-Ferner, E.; Wöhnert, J.; Kalbacher, H.; Endo, T.; Schleiff, E.; Rapaport, D. Characterization of the targeting signal in mitochondrial β-barrel proteins. Nat. Commun., 2016, 7(1), 12036.
[] [PMID: 27345737]
Zaidi, N.; Herrmann, T.; Baechle, D.; Schleicher, S.; Gogel, J.; Driessen, C.; Voelter, W.; Kalbacher, H. A new approach for distinguishing cathepsin E and D activity in antigen-processing organelles. FEBS J., 2007, 274(12), 3138-3149.
[] [PMID: 17521331]
MEROPS Peptidase S1 family. Available from:
Levey, A.S.; Coresh, J.; Greene, T.; Marsh, J.; Stevens, L.A.; Kusek, J.W.; Van Lente, F. Expressing the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin. Chem., 2007, 53(4), 766-772.
[] [PMID: 17332152]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy