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Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl(-) permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigate...

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Autores principales: Gerbino, Andrea, De Zio, Roberta, Russo, Daniela, Milella, Luigi, Milano, Serena, Procino, Giuseppe, Pusch, Michael, Svelto, Maria, Carmosino, Monica
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314819/
https://www.ncbi.nlm.nih.gov/pubmed/32581267
http://dx.doi.org/10.1038/s41598-020-67219-8
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author Gerbino, Andrea
De Zio, Roberta
Russo, Daniela
Milella, Luigi
Milano, Serena
Procino, Giuseppe
Pusch, Michael
Svelto, Maria
Carmosino, Monica
author_facet Gerbino, Andrea
De Zio, Roberta
Russo, Daniela
Milella, Luigi
Milano, Serena
Procino, Giuseppe
Pusch, Michael
Svelto, Maria
Carmosino, Monica
author_sort Gerbino, Andrea
collection PubMed
description The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl(-) permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl(-) currents time-dependently within several minutes. Of note, the same effect on Cl(-) currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca(2+) chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl(-) current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.
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spelling pubmed-73148192020-06-26 Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka Gerbino, Andrea De Zio, Roberta Russo, Daniela Milella, Luigi Milano, Serena Procino, Giuseppe Pusch, Michael Svelto, Maria Carmosino, Monica Sci Rep Article The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl(-) permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl(-) currents time-dependently within several minutes. Of note, the same effect on Cl(-) currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca(2+) chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl(-) current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic. Nature Publishing Group UK 2020-06-24 /pmc/articles/PMC7314819/ /pubmed/32581267 http://dx.doi.org/10.1038/s41598-020-67219-8 Text en © The Author(s) 2020 Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Gerbino, Andrea
De Zio, Roberta
Russo, Daniela
Milella, Luigi
Milano, Serena
Procino, Giuseppe
Pusch, Michael
Svelto, Maria
Carmosino, Monica
Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title_full Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title_fullStr Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title_full_unstemmed Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title_short Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka
title_sort role of pkc in the regulation of the human kidney chloride channel clc-ka
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314819/
https://www.ncbi.nlm.nih.gov/pubmed/32581267
http://dx.doi.org/10.1038/s41598-020-67219-8
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