Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle

Abstract Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we h...

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Autores principales:	Nadezhda N. Zheleznova, Vikash Kumar, Theresa Kurth, Allen W. Cowley
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f966b2aa348d4e829710cefd4542fe03
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spelling	oai:doaj.org-article:f966b2aa348d4e829710cefd4542fe032021-12-02T18:18:06ZHydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle10.1038/s41598-021-86678-12045-2322https://doaj.org/article/f966b2aa348d4e829710cefd4542fe032021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-86678-1https://doaj.org/toc/2045-2322Abstract Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.Nadezhda N. ZheleznovaVikash KumarTheresa KurthAllen W. CowleyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nadezhda N. Zheleznova Vikash Kumar Theresa Kurth Allen W. Cowley Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
description	Abstract Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.
format	article
author	Nadezhda N. Zheleznova Vikash Kumar Theresa Kurth Allen W. Cowley
author_facet	Nadezhda N. Zheleznova Vikash Kumar Theresa Kurth Allen W. Cowley
author_sort	Nadezhda N. Zheleznova
title	Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
title_short	Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
title_full	Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
title_fullStr	Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
title_full_unstemmed	Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle
title_sort	hydrogen peroxide (h2o2) mediated activation of mtorc2 increases intracellular na+ concentration in the renal medullary thick ascending limb of henle
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f966b2aa348d4e829710cefd4542fe03
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Hydrogen peroxide (H2O2) mediated activation of mTORC2 increases intracellular Na+ concentration in the renal medullary thick ascending limb of Henle

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