Hydrogen Peroxide Increases Intracellular Sodium Concentration in the Medullary Thick Ascending Limb Isolated from Sprague‐Dawley Rats

Abstract

Elevations of renal medullary hydrogen peroxide (H2O2) produce a salt‐sensitive form of hypertension as shown by chronic infusion of H2O2 into the renal medulla of a single remaining kidney of normotensive Sprague‐Dawley (SD) rats (Makino et al., Hypertension, 2003) and salt‐resistant consomic SS.13BN rats. Conversely, chronic medullary infusion of catalase (H2O2 scavenger) into the renal medulla of Dahl salt‐sensitive rats significantly reduced salt induced hypertension (Taylor and Cowley, Am J Physiol Regul Integr Comp Physiol, 2005). The goal of this study was to assess the effects of H2O2 upon intracellular sodium concentration [Na+]i and the role of mTORC1 and mTORC2 signaling in the medullary thick ascending limb (mTAL) isolated from the outer medulla (OM) of SD rats.Methodsrats were anesthetized, the kidney flushed with saline, removed, and mTALs dissected from the inner stripe of the OM at 4°C under a stereomicroscope. The mTALs were attached to poly‐Lysine coated cover slips and incubated with HBSS containing 10 nM of the Na+‐sensitive fluorescence dye NaGreen and 0.05% Pluronic acid F‐127 for 30 min at room temperature. The mTALs were washed and then placed in a heated chamber (37°C) mounted on the stage of an inverted Nikon TE‐2000U microscope for recording.ResultsIt was found that 100 uM H2O2 resulted in a significant increase in [Na+]i (p<0.05). The responses to pretreatment of mTAL with 10 uM PP242 (mTORC1/2 inhibitor), 200 nM rapamycin (mTORC1 specific inhibitor), or 1 mM furosemide (Na+/K+/2Cl− inhibitor) were then studied. We found that each of these inhibitors nearly abolished H2O2 induced increases in intracellular Na+. Western blot analysis of isolated mTAL from SD rats for each of the treatments found that 100 uM H2O2 increased the activation of AKT (pAKTSer473; a surrogate for mTORC1/2 activation) and S6 (pS6Ser235/236; a surrogate for mTORC1 activation) and were inhibited by their respective inhibitors.ConclusionTogether these observations indicate that H2O2 acts through the mTOR signaling pathways to inhibit NKCC2 activity, which would in turn be expected to lead to sodium retention and hypertension.Support or Funding InformationHL 122662This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.