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Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel
The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8764147/ https://www.ncbi.nlm.nih.gov/pubmed/35058797 http://dx.doi.org/10.3389/fphys.2021.787521 |
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author | Chen, Bruce Fluitt, Maurice B. Brown, Aaron L. Scott, Samantha Gadicherla, Anirudh Ecelbarger, Carolyn M. |
author_facet | Chen, Bruce Fluitt, Maurice B. Brown, Aaron L. Scott, Samantha Gadicherla, Anirudh Ecelbarger, Carolyn M. |
author_sort | Chen, Bruce |
collection | PubMed |
description | The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis. |
format | Online Article Text |
id | pubmed-8764147 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-87641472022-01-19 Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel Chen, Bruce Fluitt, Maurice B. Brown, Aaron L. Scott, Samantha Gadicherla, Anirudh Ecelbarger, Carolyn M. Front Physiol Physiology The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis. Frontiers Media S.A. 2022-01-04 /pmc/articles/PMC8764147/ /pubmed/35058797 http://dx.doi.org/10.3389/fphys.2021.787521 Text en Copyright © 2022 Chen, Fluitt, Brown, Scott, Gadicherla and Ecelbarger. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Physiology Chen, Bruce Fluitt, Maurice B. Brown, Aaron L. Scott, Samantha Gadicherla, Anirudh Ecelbarger, Carolyn M. Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title | Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title_full | Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title_fullStr | Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title_full_unstemmed | Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title_short | Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel |
title_sort | selective deletion of the mechanistic target of rapamycin from the renal collecting duct principal cell in mice down-regulates the epithelial sodium channel |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8764147/ https://www.ncbi.nlm.nih.gov/pubmed/35058797 http://dx.doi.org/10.3389/fphys.2021.787521 |
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