Human Cell

, Volume 32, Issue 3, pp 297–305 | Cite as

ClC-5 alleviates renal fibrosis in unilateral ureteral obstruction mice

  • Shi-Xia YangEmail author
  • Zheng-Chang Zhang
  • Hui-Ling Bai
Research Article


Renal fibrosis is the major feature of end-stage renal disease with high mortality. Chloride (Cl) moving along Cl channels has been suggested to play to an important role in renal function. This study aims to investigate the role of ClC-5 in renal fibrosis in unilateral ureteral occlusion (UUO) mice. C57BL/6 mice received UUO surgery followed by delivery of adeno-associated virus encoding ClC-5 cDNA (AAVClC-5). Western blotting, real-time PCR and histological analysis were used to investigate the effects of ClC-5 on renal fibrosis and underlying mechanisms. The expression of ClC-5 was significantly decreased in renal cortex of UUO mice and transforming growth factor-β1 (TGF-β1)-stimulated HK2 cells. Overexpression of ClC-5 in vivo markedly ameliorated UUO-induced renal injury and fibrosis. The increased expressions of plasminogen activator inhibitor type 1, connective tissue growth factor, collagen III and collagen IV were also inhibited by ClC-5 upregulation. Moreover, UUO-induced immune cell infiltration and inflammatory cytokines release were attenuated in mice infected with AAVClC-5. In addition, the in vivo and in vitro results showed that ClC-5 overexpression prevented epithelial-to-mesenchymal transition (EMT), concomitantly with a restoration of E-cadherin expression and a decrease of vimentin, α-SMA and S100A4 expressions. Furthermore, ClC-5 overexpression inhibited UUO- or TGF-β1-induced increase in nuclear factor kappa B (NF-κB) acetylation and matrix metalloproteinases-9 (MMP-9) expression. However, downregulation of ClC-5 in HK2 cells further potentiated TGF-β1-induced EMT and increase in NF-κB acetylation and MMP-9 expression. ClC-5 upregulation ameliorates renal fibrosis via inhibiting NF-κB/MMP-9 pathway signaling activation, suggesting that ClC-5 may be a novel therapeutic target for treating renal fibrosis and chronic kidney disease.


Renal fibrosis Inflammation Epithelial-to-mesenchymal transition NF-κB/MMP-9 signaling ClC-5 



Unilateral ureteral occlusion


Adeno-associated virus


Transforming growth factor-β1


Nuclear factor kappa B


Matrix metalloproteinases-9


Extracellular matrix


Epithelial-to-mesenchymal transition


Plasminogen activator inhibitor type 1


Connective tissue growth factor






Monocyte chemoattractant protein-1


Intercellular adhesion molecule-1


Tumor necrosis factor-α


Compliance with ethical standards

Ethics approval

All animal experiments were carried out according to the institutional guidelines from the Principles of Laboratory Animal Care of Gansu Provincial Hospital of Traditional Chinese Medicine.

Conflict of interest

The authors declared that they have no competing interests.

Supplementary material

13577_2019_253_MOESM1_ESM.docx (1.6 mb)
Supplementary material 1 (DOCX 1601 kb)


  1. 1.
    Becker GJ, Hewitson TD. The role of tubulointerstitial injury in chronic renal failure. Curr Opin Nephrol Hypertens. 2000;9(2):133–8.CrossRefGoogle Scholar
  2. 2.
    Waasdorp M, de Rooij DM, Florquin S, Duitman J, Spek CA. Protease-activated receptor-1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy. J Cell Mol Med. 2018. Scholar
  3. 3.
    Qi R, Yang C. Renal tubular epithelial cells: the neglected mediator of tubulointerstitial fibrosis after injury. Cell Death Dis. 2018;9(11):1126. Scholar
  4. 4.
    Buchtler S, Grill A, Hofmarksrichter S, Stockert P, Schiechl-Brachner G, Rodriguez Gomez M, Neumayer S, Schmidbauer K, Talke Y, Klinkhammer BM, Boor P, Medvinsky A, Renner K, Castrop H, Mack M. Cellular origin and functional relevance of Collagen I production in the kidney. JASN. 2018;29(7):1859–73. Scholar
  5. 5.
    Li Z, Hong Z, Peng Z, Zhao Y, Shao R. Acetylshikonin from Zicao ameliorates renal dysfunction and fibrosis in diabetic mice by inhibiting TGF-beta1/Smad pathway. Hum Cell. 2018;31(3):199–209. Scholar
  6. 6.
    Yin J, Wang Y, Chang J, Li B, Zhang J, Liu Y, Lai S, Jiang Y, Li H, Zeng X. Apelin inhibited epithelial-mesenchymal transition of podocytes in diabetic mice through downregulating immunoproteasome subunits beta5i. Cell Death Dis. 2018;9(10):1031. Scholar
  7. 7.
    Meng XM, Tang PM, Li J, Lan HY. TGF-beta/Smad signaling in renal fibrosis. Front Physiol. 2015;6:82. Scholar
  8. 8.
    Gao R, Chen J, Hu Y, Li Z, Wang S, Shetty S, Fu J. Sirt1 deletion leads to enhanced inflammation and aggravates endotoxin-induced acute kidney injury. PLoS One. 2014;9(6):e98909. Scholar
  9. 9.
    Yang H, Liao D, Tong L, Zhong L, Wu K. MiR-373 exacerbates renal injury and fibrosis via NF-kappaB/MatrixMetalloproteinase-9 signaling by targeting Sirtuin1. Genomics. 2018. Scholar
  10. 10.
    Huang XZ, Wen D, Zhang M, Xie Q, Ma L, Guan Y, Ren Y, Chen J, Hao CM. Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-beta/Smad3 pathway. J Cell Biochem. 2014;115(5):996–1005. Scholar
  11. 11.
    Rajagopal M, Wallace DP. Chloride secretion by renal collecting ducts. Curr Opin Nephrol Hypertens. 2015;24(5):444–9. Scholar
  12. 12.
    Souza-Menezes J, Morales MM, Tukaye DN, Guggino SE, Guggino WB. Absence of ClC5 in knockout mice leads to glycosuria, impaired renal glucose handling and low proximal tubule GLUT2 protein expression. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol. 2007;20(5):455–64. Scholar
  13. 13.
    Lin Z, Jin S, Duan X, Wang T, Martini S, Hulamm P, Cha B, Hubbard A, Donowitz M, Guggino SE. Chloride channel (Clc)-5 is necessary for exocytic trafficking of Na+/H+ exchanger 3 (NHE3). J Biol Chem. 2011;286(26):22833–45. Scholar
  14. 14.
    Carraro-Lacroix LR, Lessa LM, Bezerra CN, Pessoa TD, Souza-Menezes J, Morales MM, Girardi AC, Malnic G. Role of CFTR and ClC-5 in modulating vacuolar H+-ATPase activity in kidney proximal tubule. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol. 2010;26(4–5):563–76. Scholar
  15. 15.
    Tan XH, Zheng XM, Yu LX, He J, Zhu HM, Ge XP, Ren XL, Ye FQ, Bellusci S, Xiao J, Li XK, Zhang JS. Fibroblast growth factor 2 protects against renal ischaemia/reperfusion injury by attenuating mitochondrial damage and proinflammatory signalling. J Cell Mol Med. 2017;21(11):2909–25. Scholar
  16. 16.
    Meng XM, Wang S, Huang XR, Yang C, Xiao J, Zhang Y, To KF, Nikolic-Paterson DJ, Lan HY. Inflammatory macrophages can transdifferentiate into myofibroblasts during renal fibrosis. Cell Death Dis. 2016;7(12):e2495. Scholar
  17. 17.
    Laverty G, Anttila A, Carty J, Reddy V, Yum J, Arnason SS. CFTR mediated chloride secretion in the avian renal proximal tubule. Comp Biochem Physiol A Mol Integr Physiol. 2012;161(1):53–60. Scholar
  18. 18.
    Jouret F, Bernard A, Hermans C, Dom G, Terryn S, Leal T, Lebecque P, Cassiman JJ, Scholte BJ, de Jonge HR, Courtoy PJ, Devuyst O. Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney. JASN. 2007;18(3):707–18. Scholar
  19. 19.
    Mansour-Hendili L, Blanchard A, Le Pottier N, Roncelin I, Lourdel S, Treard C, Gonzalez W, Vergara-Jaque A, Morin G, Colin E, Holder-Espinasse M, Bacchetta J, Baudouin V, Benoit S, Berard E, Bourdat-Michel G, Bouchireb K, Burtey S, Cailliez M, Cardon G, Cartery C, Champion G, Chauveau D, Cochat P, Dahan K, De la Faille R, Debray FG, Dehoux L, Deschenes G, Desport E, Devuyst O, Dieguez S, Emma F, Fischbach M, Fouque D, Fourcade J, Francois H, Gilbert-Dussardier B, Hannedouche T, Houillier P, Izzedine H, Janner M, Karras A, Knebelmann B, Lavocat MP, Lemoine S, Leroy V, Loirat C, Macher MA, Martin-Coignard D, Morin D, Niaudet P, Nivet H, Nobili F, Novo R, Faivre L, Rigothier C, Roussey-Kesler G, Salomon R, Schleich A, Sellier-Leclerc AL, Soulami K, Tiple A, Ulinski T, Vanhille P, Van Regemorter N, Jeunemaitre X, Vargas-Poussou R. Mutation update of the CLCN5 Gene responsible for dent disease 1. Hum Mut. 2015;36(8):743–52. Scholar
  20. 20.
    Zhang H, Pang Y, Ma C, Li J, Wang H, Shao Z. ClC5 decreases the sensitivity of multiple myeloma cells to bortezomib via promoting prosurvival autophagy. Oncol Res. 2018;26(3):421–9. Scholar
  21. 21.
    Figueira MF, Castiglione RC, de Lemos Barbosa CM, Ornellas FM, da Silva Feltran G, Morales MM, da Fonseca RN, de Souza-Menezes J. Diabetic rats present higher urinary loss of proteins and lower renal expression of megalin, cubilin, ClC-5, and CFTR. Physiol Rep. 2017. Scholar
  22. 22.
    Ruiz-Lafuente N, Alcaraz-Garcia MJ, Sebastian-Ruiz S, Garcia-Serna AM, Gomez-Espuch J, Moraleda JM, Minguela A, Garcia-Alonso AM, Parrado A. IL-4 up-regulates MiR-21 and the MiRNAs hosted in the CLCN5 gene in chronic lymphocytic leukemia. PLoS One. 2015;10(4):e0124936. Scholar
  23. 23.
    Vaughan DE, Rai R, Khan SS, Eren M, Ghosh AK. Plasminogen activator inhibitor-1 is a marker and a mediator of senescence. Arterioscler Thromb Vasc Biol. 2017;37(8):1446–52. Scholar
  24. 24.
    Weston BS, Wahab NA, Mason RM. CTGF mediates TGF-beta-induced fibronectin matrix deposition by upregulating active alpha5beta1 integrin in human mesangial cells. JASN. 2003;14(3):601–10.CrossRefGoogle Scholar
  25. 25.
    Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig. 2009;119(6):1420–8. Scholar
  26. 26.
    Lovisa S, Zeisberg M, Kalluri R. Partial epithelial-to-mesenchymal transition and other new mechanisms of kidney fibrosis. TEM. 2016;27(10):681–95. Scholar
  27. 27.
    Lovisa S, LeBleu VS, Tampe B, Sugimoto H, Vadnagara K, Carstens JL, Wu CC, Hagos Y, Burckhardt BC, Pentcheva-Hoang T, Nischal H, Allison JP, Zeisberg M, Kalluri R. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med. 2015;21(9):998–1009. Scholar
  28. 28.
    Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. JASN. 2004;15(1):1–12.CrossRefGoogle Scholar
  29. 29.
    Oba S, Kumano S, Suzuki E, Nishimatsu H, Takahashi M, Takamori H, Kasuya M, Ogawa Y, Sato K, Kimura K, Homma Y, Hirata Y, Fujita T. miR-200b precursor can ameliorate renal tubulointerstitial fibrosis. PLoS One. 2010;5(10):e13614. Scholar
  30. 30.
    Salminen A, Kaarniranta K. NF-kappaB signaling in the aging process. J Clin Immunol. 2009;29(4):397–405. Scholar

Copyright information

© Japan Human Cell Society and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of NephrologyGansu Provincial Hospital of Traditional Chinese MedicineLanzhouChina
  2. 2.Department of NeurologyLanzhou University Second HospitalLanzhouChina

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