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Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease
BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disasse...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249671/ https://www.ncbi.nlm.nih.gov/pubmed/32450899 http://dx.doi.org/10.1186/s12964-020-00558-1 |
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author | Price, Gareth W. Chadjichristos, Christos E. Kavvadas, Panagiotis Tang, Sydney C. W. Yiu, Wai Han Green, Colin R. Potter, Joe A. Siamantouras, Eleftherios Squires, Paul E. Hills, Claire E. |
author_facet | Price, Gareth W. Chadjichristos, Christos E. Kavvadas, Panagiotis Tang, Sydney C. W. Yiu, Wai Han Green, Colin R. Potter, Joe A. Siamantouras, Eleftherios Squires, Paul E. Hills, Claire E. |
author_sort | Price, Gareth W. |
collection | PubMed |
description | BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43(+/−) mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43(+/−) mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. GRAPHICAL ABSTRACT: In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-β1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP](e)), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability. [Image: see text] |
format | Online Article Text |
id | pubmed-7249671 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-72496712020-06-04 Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease Price, Gareth W. Chadjichristos, Christos E. Kavvadas, Panagiotis Tang, Sydney C. W. Yiu, Wai Han Green, Colin R. Potter, Joe A. Siamantouras, Eleftherios Squires, Paul E. Hills, Claire E. Cell Commun Signal Research BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43(+/−) mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43(+/−) mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. GRAPHICAL ABSTRACT: In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-β1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP](e)), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability. [Image: see text] BioMed Central 2020-05-25 /pmc/articles/PMC7249671/ /pubmed/32450899 http://dx.doi.org/10.1186/s12964-020-00558-1 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Price, Gareth W. Chadjichristos, Christos E. Kavvadas, Panagiotis Tang, Sydney C. W. Yiu, Wai Han Green, Colin R. Potter, Joe A. Siamantouras, Eleftherios Squires, Paul E. Hills, Claire E. Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title | Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title_full | Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title_fullStr | Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title_full_unstemmed | Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title_short | Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
title_sort | blocking connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249671/ https://www.ncbi.nlm.nih.gov/pubmed/32450899 http://dx.doi.org/10.1186/s12964-020-00558-1 |
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