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states is challenging. In addition, it is likely that we have not Cutting It Out: ENaC identified all of the proteases that can cleave and activate Processing in the Human ENaC. Multiple lines of evidence suggest that ENaC processing Nephron and activation is a physiologically relevant phenomenon. In the context of extracellular volume depletion or aldosterone † Evan C. Ray* and Thomas R. Kleyman* administration in the absence volume depletion, increases † *Department of Medicine and Department of Cell Biology, in channel expression at the cell surface and proteolytic University of Pittsburgh, Pittsburgh, Pennsylvania processing of the a and g subunits have been described.7,8 J Am Soc Nephrol 26: ccc–ccc, 2014. Prostasin and/or kallikrein could have a role in cleaving the g doi: 10.1681/ASN.2014060618 subunit in these settings. In the setting of proteinuria, filtra- tion of plasminogen by damaged glomeruli and its sub- sequent conversion to plasmin by tubular urokinase may Studies over the past decade have shed light on how proteases activate ENaC, contributing to the renal sodium retention activate the epithelial sodium channel (ENaC). Two ENaC seen in this setting.9,10 subunits (a and g) have short stretches of embedded amino Antibodies directed against the amino- or carboxyl-termini acid residues that inhibit the channel. Following protease- of ENaC subunits can readily differentiate cleaved from non- dependent cleavage of these subunits at sites flanking these cleaved ENaC subunits.11 The challenge is demonstrating that a inhibitory tracks, fragments are released and channels are subunit has been cleaved twice, shedding an inhibitory activated. track.10,12 In this issue of JASN, Zachar et al. address important The serine protease furin is expressed in the trans-Golgi questions regarding the proteolytic processing of the g subunit network and processes proteins transiting through the bio- in human kidney.13 Specifically, they examined whether g synthetic pathway. Furin cleaves the a subunit of ENaC twice subunit processing in human kidney is affected by the ad- at sites flanking an inhibitory track, transitioning channels ministration of an angiotensin-converting enzyme inhibitor, from a low- to a moderate-activity state. Furin cleaves the g angiotensin-receptor blocker, or a diuretic, and whether subunit once, preceding its inhibitory track. Subsequent proteolytic processing is altered in the setting of nephrotic cleavage by a second protease at a site distal to the g subunit syndrome. inhibitory track releases another inhibitory track and tran- Two antibodies were used to answer these questions. One sitions ENaC to a high activity state.1,2 Apopulationof antibody, directed against the g subunit inhibitory track, was channels that have bypassed proteolytic processing has previously described by Svenningsen et al.14 Zachar et al. been observed at the cell surface, adding a layer of complexity generated a new antibody that is directed against residues regarding channel regulation by proteases (for reviews, see immediately following the prostasin/kallikrein cleavage site. Kashlan and Kleyman3 and Kleyman et al.4). Surprisingly, this antibody appears to require cleavage at the A growing number of proteases have been shown to cleave prostasin/kallikrein site in order to recognize its epitope. the g subunit at sites distal to the inhibitory track, including The authors asked whether individuals on diuretics ex- prostasin, transmembrane protease serine 4 (TMPRSS4), hibit altered g subunit expression or proteolytic cleavage. matriptase, kallikrein, plasmin, and elastase. While there is Increases in full-length and presumably furin-cleaved g sub- some evidence that kallikrein and prostasin may have a role units were noted in individuals on diuretics compared with in activating ENaC in the distal nephron or in other epithe- controls. The ratio of furin-cleaved to full-length g subunit lia,5,6 the key proteases responsible for cleaving the g subunit was unchanged, suggesting that diuretics did do not alter the at a site distal to the inhibitory track under specificpatho- extent of furin-dependent channel processing. Individuals physiologic conditions have not been clearly defined. To receiving an angiotensin-converting enzyme inhibitor or complicate matters, proteases function in cascades (e.g., clot- angiotensin-receptor blocker had levels of expression of full- ting factors). Differentiating upstream proteases from those length and presumably furin-cleaved g subunits that were that directly cleave and activate the channel under different similar to controls. Evidence of cleavage at the prostasin/ kallikrein site, corresponding to a channel-activating event, Published online ahead of print. Publication date available at www.jasn.org. was noted in some individuals receiving these medications, Correspondence: Dr. Thomas R. Kleyman, Renal-Electrolyte Division, Uni- while absent in controls. The increased g subunit expression versity of Pittsburgh, A919 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA in individuals receiving diuretics could reflect relative volume 15261. Email: [email protected] depletion. However, caveats in interpreting this work are that Copyright © 2014 by the American Society of Nephrology the reason these drugs were administered, and details

J Am Soc Nephrol 26: ccc–ccc,2014 ISSN : 1046-6673/2601-ccc 1 EDITORIAL www.jasn.org regarding the extracellular volume status of these patients were the g subunit in proteinuric states is cleaved by plasmin at the not provided. lysine residue mentioned above. The antibodies used by the Individuals with proteinuria also had more full-length and authors would likely not detect a plasmin-cleaved fragment. presumably furin-cleaved g subunit. A decreased ratio of Fourth, on the basis of previous work it is difficult to explain furin-cleaved to full-length g subunit was also noted, suggest- how the 37-kDa fragment recognized by the antibody against ing that a second processing event had occurred releasing the g the inhibitory track was generated. This is not consistent subunit inhibitory track. Evidence of cleavage at the prostasin/ with a single cleavage at or in close proximity to the prostasin/ kallikrein site was noted in five of six individuals with pro- kallikrein site.11 In summary, Zachar et al. have generated a teinuria, providing additional support of a second processing novel antibody and presented new findings regarding human event. It is interesting, but not surprising, that the two antibodies kidney g subunit processing in in the setting of proteinuria. localized g subunits to different sites within principal cells in the Their work also raises new questions. distal nephron of kidneys from individuals with proteinuria. While the antibody directed against the inhibitory track showed somewhat diffuse intracellular staining, the antibody recognizing a putatively prostasin/kallikrein-cleaved g subunit ACKNOWLEDGMENTS showed punctate staining. These results provide intriguing clues regarding g subunit Supported by a grant from the National Institutes of Health (R01- processing in humans under different states. First, there is DK065161) furin-dependent processing of the g subunit in the human kidney. Second, under certain conditions there appears to be cleavage at the prostasin/kallikrein site. This is particularly DISCLOSURES evident in individuals with proteinuria and provides an im- None. portant clue regarding proteases that cleave the g subunit and activate the channel in proteinuric states. If plasmin, gener- REFERENCES ated by filtered plasminogen, is primarily responsible for cleaving the g subunit in this setting, cleavage would occur 1. Bruns JB, Carattino MD, Sheng S, Maarouf AB, Weisz OA, Pilewski JM, 1 following a lysine residue that is eight residues distal to the Hughey RP, Kleyman TR: Epithelial Na channels are fully activated 9 by furin- and prostasin-dependent release of an inhibitory peptide prostasin/kallikrein site. The resulting fragment would be from the gamma-subunit. J Biol Chem 282: 6153–6160, 2007 unlikely to be recognized by the new antibody used by the 2. Carattino MD, Hughey RP, Kleyman TR: Proteolytic processing of authors because it would contain only two of the residues in the epithelial sodium channel gamma subunit has a dominant role in the antigen used to raise the antibody. A simple test could channel activation. JBiolChem283: 25290–25295, 2008 confirm that the antibody would not recognize a plasmin- 3. Kashlan OB, Kleyman TR: ENaC structure and function in the wake of a resolved structure of a family member. Am J Physiol Renal Physiol 301: cleaved fragment. Given the previous work of Svenningsen F684–F696, 2011 14 et al. showing that plasmin cleaves and activates prostasin, 4. Kleyman TR, Carattino MD, Hughey RP: ENaC at the cutting edge: the findings of Zachar et al. are consistent with prostasin- Regulation of epithelial sodium channels by proteases. JBiolChem dependent cleavage of the g subunit in proteinuric states. 284: 20447–20451, 2009 There are several caveats regarding these findings. First, the 5. Picard N, Eladari D, El Moghrabi S, Planès C, Bourgeois S, Houillier P, Wang Q, Burnier M, Deschenes G, Knepper MA, Meneton P, Chambrey prostasin/kallikrein cleavage site consists of a track of four basic R: Defective ENaC processing and function in tissue kallikrein-deficient residues, and it is likely that other proteases are present in the mice. J Biol Chem 283: 4602–4611, 2008 lumen of the distal nephron and are capable of cleaving the 6. Frateschi S, Keppner A, Malsure S, Iwaszkiewicz J, Sergi C, Merillat AM, channel at this site. Second, the size of the fragment identified Fowler-Jaeger N, Randrianarison N, Planès C, Hummler E: Mutations of g the serine protease CAP1/Prss8 lead to reduced embryonic viability, with the new antibody (45 kD) is not consistent with a sub- – 1 skin defects, and decreased ENaC activity. Am J Pathol 181: 605 615, unit that is simply cleaved at the prostasin/kallikrein site. The 2012 size of the fragment should be about 5 kD less than the furin 7. Ergonul Z, Frindt G, Palmer LG: Regulation of maturation and pro- cleaved fragment, or about 70 kD. Possible explanations for cessing of ENaC subunits in the rat kidney. Am J Physiol Renal Physiol this discrepancy include additional cleavage at a site preceding 291: F683–F693, 2006 the second transmembrane domain, as has been observed by 8. Frindt G, Palmer LG: Surface expression of sodium channels and 15 transporters in rat kidney: effects of dietary sodium. Am J Physiol Renal others, or that this 45-kD fragment is a cross-reacting poly- Physiol 297: F1249–F1255, 2009 peptide and not related to the g subunit. Immunolocalization 9. Passero CJ, Mueller GM, Rondon-Berrios H, Tofovic SP, Hughey RP, with this antibody is consistent with the antibody recognizing Kleyman TR: Plasmin activates epithelial Na1 channels by cleaving the ENaC. Coimmunoprecipitation studies with this antibody gamma subunit. J Biol Chem 283: 36586–36591, 2008 and an antibody directed against the carboxyl terminus of 10. Svenningsen P, Bistrup C, Friis UG, Bertog M, Haerteis S, Krueger B, fi Stubbe J, Jensen ON, Thiesson HC, Uhrenholt TR, Jespersen B, the g subunit could address the rst possibility (the 45-kD Jensen BL, Korbmacher C, Skøtt O: Plasmin in nephrotic urine acti- fragment should not coimmunoprecipitate). Third, these vates the epithelial sodium channel. JAmSocNephrol20: 299–310, findings do not exclude the possibility that a component of 2009

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11. Hughey RP, Mueller GM, Bruns JB, Kinlough CL, Poland PA, Harkleroad 13. Zachar RM, Skjødt K, Marcussen N, Walter S, Toft A, Nielsen MR, Jensen KL, Carattino MD, Kleyman TR: Maturation of the epithelial Na1 BL, Svenningsen P: The epithelial sodium channel g-subunit is processed channel involves proteolytic processing of the alpha- and gamma- proteolytically in human kidney. J Am Soc Nephrol xx: xx–xx, 2014 subunits. JBiolChem278: 37073–37082, 2003 14. Svenningsen P, Uhrenholt TR, Palarasah Y, Skjødt K, Jensen BL, Skøtt 12. Uchimura K, Kakizoe Y, Onoue T, Hayata M, Morinaga J, Yamazoe R, O: Prostasin-dependent activation of epithelial Na1 channels by low Ueda M, Mizumoto T, Adachi M, Miyoshi T, Shiraishi N, Sakai Y, Tomita plasmin concentrations. Am J Physiol Regul Integr Comp Physiol 297: K, Kitamura K: In vivo contribution of serine proteases to the proteolytic R1733–R1741, 2009 activation of gENaC in aldosterone-infused rats. Am J Physiol Renal 15. García-Caballero A, Dang Y, He H, Stutts MJ: ENaC proteolytic regulation Physiol 303: F939–F943, 2012 by channel-activating protease 2. J Gen Physiol 132: 521–535, 2008

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