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Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy
INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaem...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9828766/ https://www.ncbi.nlm.nih.gov/pubmed/36256487 http://dx.doi.org/10.1111/dme.14963 |
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author | Williams, Bethany M. Cliff, Chelsy L. Demirel, Isak Squires, Paul E. Hills, Claire E. |
author_facet | Williams, Bethany M. Cliff, Chelsy L. Demirel, Isak Squires, Paul E. Hills, Claire E. |
author_sort | Williams, Bethany M. |
collection | PubMed |
description | INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell–cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. METHODS: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta‐1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel‐mediated ATP release, while immunoblotting determined protein expression. Co‐incubation with the ATP‐diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP‐P2X7R signalling. Indirect co‐culture with conditioned media from the alternate cell type evaluated paracrine‐mediated heterotypic interactions. RESULTS: Tonabersat partially negated glucose/TGFβ1‐induced increases in Cx43 hemichannel‐mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP‐P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co‐culture studies suggest that epithelial cell secretome increases Tonabersat‐sensitive hemichannel‐mediated dye uptake in fibroblasts and downstream protein expression. CONCLUSION: Tonabersat‐sensitive hemichannel‐mediated ATP release enhances TGFβ1‐driven heterotypic cell–cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late‐stage diabetic nephropathy. |
format | Online Article Text |
id | pubmed-9828766 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-98287662023-01-10 Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy Williams, Bethany M. Cliff, Chelsy L. Demirel, Isak Squires, Paul E. Hills, Claire E. Diabet Med RESEARCH: BASIC SCIENCE SPECIAL ISSUE INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell–cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. METHODS: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta‐1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel‐mediated ATP release, while immunoblotting determined protein expression. Co‐incubation with the ATP‐diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP‐P2X7R signalling. Indirect co‐culture with conditioned media from the alternate cell type evaluated paracrine‐mediated heterotypic interactions. RESULTS: Tonabersat partially negated glucose/TGFβ1‐induced increases in Cx43 hemichannel‐mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP‐P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co‐culture studies suggest that epithelial cell secretome increases Tonabersat‐sensitive hemichannel‐mediated dye uptake in fibroblasts and downstream protein expression. CONCLUSION: Tonabersat‐sensitive hemichannel‐mediated ATP release enhances TGFβ1‐driven heterotypic cell–cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late‐stage diabetic nephropathy. John Wiley and Sons Inc. 2022-10-25 2022-12 /pmc/articles/PMC9828766/ /pubmed/36256487 http://dx.doi.org/10.1111/dme.14963 Text en © 2022 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | RESEARCH: BASIC SCIENCE SPECIAL ISSUE Williams, Bethany M. Cliff, Chelsy L. Demirel, Isak Squires, Paul E. Hills, Claire E. Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title | Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title_full | Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title_fullStr | Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title_full_unstemmed | Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title_short | Blocking connexin 43 hemichannel‐mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
title_sort | blocking connexin 43 hemichannel‐mediated atp release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy |
topic | RESEARCH: BASIC SCIENCE SPECIAL ISSUE |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9828766/ https://www.ncbi.nlm.nih.gov/pubmed/36256487 http://dx.doi.org/10.1111/dme.14963 |
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