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.