Hydrogen peroxide (H(2)O(2)) mediated activation of mTORC2 increases intracellular Na(+) concentration in the renal medullary thick ascending limb of Henle

Hydrogen peroxide (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2) (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 H(2)O(2) induced increase of [Na(+)](i). mTAL cell volume remained unchanged (± 1%) by H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na(+)](i) despite associated increases of basolateral pump activity.