Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron

Nesterov, Viatcheslav; Dahlmann, Anke; Bertog, Marko; Korbmacher, Christoph

doi:10.1152/ajprenal.00031.2008

Cited by 45 publications

(47 citation statements)

References 61 publications

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“…35 It was recently shown that rats fed a 1% sodium diet expressed both full-length and cleaved ␥ENaC at the plasma membrane, whereas Na-depleted or aldosteronetreated rats mainly expressed the cleaved subunit. 36 In agreement, we 37 and others 36 recently demonstrated that trypsin can activate ENaC in microdissected mouse and rat distal nephron and that the stimulatory effect of trypsin was more pronounced in tubules from animals maintained on a standardsodium diet than animals maintained on a low-sodium diet. These findings demonstrate that ENaC can be activated by extracellular proteases in the native renal tubule under normal physiologic conditions and that the degree of proteolytic preactivation of ENaC by endogenous proteases will determine /ml), in a combination of plasminogen (1 mg/ml) and uPA (150 U/ml), in uPA (150 U/ml) alone, or in plasminogen (1 mg/ml) alone.…”

Section: Discussionsupporting

confidence: 84%

“…28 As recently reported, pretreatment of M-1 cells with a furin inhibitor largely augmented the stimulatory effect of trypsin on ENaC currents, whereas pretreatment with aprotinin had little effect. 37 Thus, to minimize ENaC activation by endogenous proteases, the confluent M-1 cells grown on filters were preincubated for 6 to 8 h before the experiment with the furin inhibitor dec-RVKR-cmk (decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone from Calbiochem/Merck Biosciences GmbH, Schwalbach, Germany) which was added to the apical bath solution. A 10 mM stock solution of dec-RVKR-cmk was prepared in DMSO and was diluted to a final concentration of 40 M.…”

Section: Ussing Chambermentioning

confidence: 99%

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Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Svenningsen

Bistrup²,

Friis³

et al. 2009

Journal of the American Society of Nephrology

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366

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Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC ␥ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

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Section: Discussionsupporting

confidence: 84%

Section: Ussing Chambermentioning

confidence: 99%

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Svenningsen

Bistrup²,

Friis³

et al. 2009

Journal of the American Society of Nephrology

Self Cite

228

366

View full text Add to dashboard Cite

show abstract

“…Thus, trypsin IV may proteolytically activate ENaC expressed at the apical membrane of the renal collecting duct. We previously have shown that trypsin I can activate ENaC in microdissected mouse distal nephron tissue (14). Here, we have demonstrated the expression of trypsinogen IV mRNA in human proximal tubular epithelial cells.…”

Section: Discussionmentioning

confidence: 50%

“…In contrast, the sites at which trypsins cleave and activate ENaC have not yet been identified. The prototypical serine proteases trypsin I and chymotrypsin are commonly used as experimental tools to achieve maximal proteolytic ENaC activation (13,14). There is evidence that trypsin I is expressed in epithelial cells (15) and can be detected in human urine (16) (Human Urine, PeptideAtlas, June 2010).…”

mentioning

confidence: 99%

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Haerteis

Krappitz

et al. 2014

Journal of Biological Chemistry

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Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC) but is not yet fully understood. The serine protease trypsin is known to activate ENaC in vitro but may not be relevant in vivo. In this study, we wanted to investigate whether the trypsin‐like serine protease trypsin IV, known to be expressed in several epithelial tissues, can activate human ENaC. Moreover, using site‐directed mutagenesis we wanted to identify functionally relevant cleavage sites in the γ‐subunit of the channel. In the Xenopus laevis oocyte expression system, we monitored the proteolytic activation of ENaC currents and the appearance of γ‐ENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC and that this activation involves a critical cleavage site (K189) in the extracellular domain of the γ‐subunit. Moreover, we showed that channel activation by trypsin was prevented by mutating three putative trypsin cleavage sites (K168; K170; R172) in addition to mutating previously described prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) sites. In conclusion, we demonstrated for the first time that trypsin IV can activate ENaC and that trypsin and trypsin IV use specific cleavage sites to achieve channel activation. Preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue‐specific proteases. Grant Funding Source: Supported by grants of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (S.H., M.K.)

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“…Thus, in both cases the loss of the segment between the two furin cleavage sites is not likely to be an obligatory mechanism of ENaC activation by ␣ cleavage. Such built-in redundancy into channel cleavage would certainly be consistent with the ability of multiple diverse proteases such as trypsin, subtilisin, elastase, prostasin, furin, and plasmin to activate ENaC in different systems with a similar final outcome (9,24,(31)(32)(33)(34)(35).…”

Section: Discussionmentioning

confidence: 68%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...

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Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron

Abstract: Nesterov V, Dahlmann A, Bertog M, Korbmacher C. Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron.

Cited by 45 publications

References 61 publications

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

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