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 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 (pAKT^Ser473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6^Ser235/236/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 (pAKT^Ser473/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⁺-ATPase^Ser16 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.