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

Nadezhda N Zheleznova,Theresa Kurth,Allen W Cowley,Vikash Kumar

doi:10.1038/s41598-021-86678-1

Nadezhda N Zheleznova, Theresa Kurth + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-86678-1

Copy DOI

Journal: Scientific Reports	Publication Date: Mar 31, 2021
Citations: 2	License type: open-access

Affiliation: Medical College of Wisconsin

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.

Highlights

Reactive oxygen species (ROS) are implicated in the renal dysfunction and hypertension and hydrogen peroxide (H2O2) is a major component of ROS1,2
To determine the effects of chronic elevation of renal medullary H2O2 for another min (H2O2) on mTOR complex 2 (mTORC2) activity, 3 days of control mean arterial pressure (MAP) measurements were obtained in Sprague Dawley (SD) rats followed by continuous intrarenal infusion of either H2O2 (347 nmol/kg/min) or isotonic saline for another 3 days
The present study found that chronic 3-day infusion of H 2O2 within the physiological range in the renal medulla resulted in stimulation of the mTORC2 pathway in SD rats

Summary

Introduction

Reactive oxygen species (ROS) are implicated in the renal dysfunction and hypertension and hydrogen peroxide (H2O2) is a major component of ROS1,2. Chronic infusion of H2O2 in the renal medulla of normotensive Sprague Dawley (SD) rats resulted in sustained hypertension in rats fed a normal salt diet[3]. With chronic renal interstitial infusion of catalase (H2O2 scavenger)[4] Together, these data indicate that renal levels of H2O2 play an important role in determining BP salt-sensitivity. Administration of mTORC1/2 pathway inhibitor PP242 completely prevented and reversed salt-induced hypertension and kidney injury in SS r ats[18]. This study focused upon the medullary thick ascending limb (mTAL) since this segment of the nephron reabsorbs ~ 25% of the filtered Na+ and has been found to transport and reabsorb excess amounts of NaCl in the SS rats compared to salt-resistant Dahl R r ats[24,25]. Studies in our laboratory have found that increased mTAL luminal flow results in increases in intracellular and mitochondrial H 2O2, which is dependent on the presence of NOX4 in contrast to NOX2 which could account solely for increases in O 2− production[26]

Objectives

Methods

Results

Conclusion