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Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells

BACKGROUND: Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined. METHODS: To investigate the contributory role of TJ proteins in the leaky proximal...

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Autores principales: Kim, Sua, Kim, Gheun-Ho
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734727/
https://www.ncbi.nlm.nih.gov/pubmed/29252987
http://dx.doi.org/10.1371/journal.pone.0189221
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author Kim, Sua
Kim, Gheun-Ho
author_facet Kim, Sua
Kim, Gheun-Ho
author_sort Kim, Sua
collection PubMed
description BACKGROUND: Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined. METHODS: To investigate the contributory role of TJ proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and ZO-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2. For this, small-interfering RNAs (siRNAs) against claudin-2, occludin and ZO-1 were transfected into HK-2 cells. TER and transepithelial flux rates of dextrans (4 and 70 kDa) were determined after 24 h. RESULTS: Transfection of siRNAs (25 nM) knocked down TJ protein expression. Control HK-2 monolayers achieved a steady-state TER of 6–8 Ω·cm(2) when grown in 12-well Transwell filters, which are compatible with leaky epithelia. Knockdown of claudin-2 decreased in TER and increased occludin expression. Transfection with siRNA against either occludin or ZO-1 increased TER and decreased claudin-2 expression. TER was decreased by co-inhibition of claudin-2 and ZO-1 but increased by co-inhibition of claudin-2 and occludin. TER was suppressed when claudin-2, occludin, and ZO-1 were all inhibited. Dextran flux rate was increased by claudin-2, occludin, or ZO-1 siRNA transfection. Increased dextran flux was enhanced by co-transfection of claudin-2, ZO-1, and occludin siRNA. CONCLUSIONS: The depletion of claudin-2, occludin and ZO-1 in HK-2 cells had differential effects on TER and macromolecule flux. We demonstrated that integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium.
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spelling pubmed-57347272017-12-22 Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells Kim, Sua Kim, Gheun-Ho PLoS One Research Article BACKGROUND: Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined. METHODS: To investigate the contributory role of TJ proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and ZO-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2. For this, small-interfering RNAs (siRNAs) against claudin-2, occludin and ZO-1 were transfected into HK-2 cells. TER and transepithelial flux rates of dextrans (4 and 70 kDa) were determined after 24 h. RESULTS: Transfection of siRNAs (25 nM) knocked down TJ protein expression. Control HK-2 monolayers achieved a steady-state TER of 6–8 Ω·cm(2) when grown in 12-well Transwell filters, which are compatible with leaky epithelia. Knockdown of claudin-2 decreased in TER and increased occludin expression. Transfection with siRNA against either occludin or ZO-1 increased TER and decreased claudin-2 expression. TER was decreased by co-inhibition of claudin-2 and ZO-1 but increased by co-inhibition of claudin-2 and occludin. TER was suppressed when claudin-2, occludin, and ZO-1 were all inhibited. Dextran flux rate was increased by claudin-2, occludin, or ZO-1 siRNA transfection. Increased dextran flux was enhanced by co-transfection of claudin-2, ZO-1, and occludin siRNA. CONCLUSIONS: The depletion of claudin-2, occludin and ZO-1 in HK-2 cells had differential effects on TER and macromolecule flux. We demonstrated that integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium. Public Library of Science 2017-12-18 /pmc/articles/PMC5734727/ /pubmed/29252987 http://dx.doi.org/10.1371/journal.pone.0189221 Text en © 2017 Kim, Kim http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kim, Sua
Kim, Gheun-Ho
Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title_full Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title_fullStr Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title_full_unstemmed Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title_short Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells
title_sort roles of claudin-2, zo-1 and occludin in leaky hk-2 cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734727/
https://www.ncbi.nlm.nih.gov/pubmed/29252987
http://dx.doi.org/10.1371/journal.pone.0189221
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