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

Figures

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  Figure 1. H2O2 activates mTORC2 pathways in the absence of changes in blood pressure (BP). (A) Average 24 h mean arterial pressure (MAP) of single kidney SD rats (n = 9; fed 0.4% NaCl diet) with implanted renal interstitial infusion catheters receiving continuous infusion of isotonic saline (days 1–3) followed by infusion of H2O2 (347 nmol/kg/min; open squares; n = 5) or continued infusion of saline (closed squares; n = 4). *—small but significant post-surgical differences between control groups on day 1 and 2 (determined by two-way analysis of variance (ANOVA) for repeated measures) were observed which converged to a similar stable BP after several days, at which time the H2O2 infusion was begun. (B, C) Western Blot analysis of pAKTSer473 and AKT proteins in renal cortical (Cortex, B), outer medullar (OM, C) tissue and pS6Ser235/236 and S6 proteins in OM tissue (D) homogenates of SD rats fed a 0.4% NaCl diet receiving a renal interstitial infusion of saline (gray bar; n = 4), or after 3 days of H2O2 (347 nmol/kg/min; black bar; n = 5). * P < 0.05 value is shown; determined by Student’s T-test.

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  Figure 2. Measurement of intracellular Na+ concentrations ([Na+]i) in mTAL isolated from SD rats. (A) Representative tracings of Sodium Green (Na Green) fluorescence normalized to the first point in mTAL permeabilized with Gramicidin (20 µM) followed by addition to the bath of 5, 10, 15 and 20 mM Na+ concentrations to create a standard curve. Ouabain (4 mM) was added with Gramicidin to inhibit Na+-K+-ATPase. (B) Representative standard curve of Na Green fluorescence as a function of bath [Na+]i. (C) Representative images of isolated mTAL from SD rat. Left image (BF): bright field image of mTAL. Center image (control): mTAL loaded with Na Green dye for 30 min. Right image (+ 100 µM H2O2): response of Na Green loaded mTAL to application of 100 µM H2O2 for 5 min. (D) H2O2 dose–response graph (n = 3) measured by Na-Green dye (Na+ changes) in mTAL freshly isolated from SD rat. Here is represented the cumulative concentrations of H2O2. (E) Measurements of mTAL area using 3D reconstruction images on confocal microscope before (control) and 2 min after 100 µM H2O2 application (n = 17 mTAL, 3 SD rats).

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  Figure 3. Intracellular [Na+]i responses in isolated mTAL determined using Na+ sensitive dye, Na Green. (A) Responses to 100 µM of H2O2 (open circles; shown as A on the right; n = 16); (B) Responses after pretreatment of mTAL with 10 µM PP242 in HBSS bath for 10 min followed by addition of final 100 µM of H2O2 concentration (black circles; shown as B on the right; n = 8); (C) Time control recording in HBSS medium (gray squares; shown as C on the right; n = 10); (D) Pretreatment of mTAL with 10 µM PP242 in HBSS bath for 10 min and throughout all measurements (black triangles; shown as D on the right; n = 6). *P < 0.05 between mTAL treated with H2O2 compared to those pretreated with PP242; determined by Student’s t-Test.

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  Figure 4. (A) Representative Western Blot of total AKT and phosphorylated AKT (pAKTSer473) in mTAL isolated from SD rats. mTAL incubated in HBSS for 20 min (control); incubated 10 min in HBSS and then with 100 µM of H2O2 for another 10 min (H2O2); incubated with 10 µM of PP242 for 10 min and then with 100 µM of H2O2 for another 10 min (H2O2 + PP242). (B) Activity of mTORC2 determined by densitometric quantification expressed as the ratio of pAKTSer473 relative to total AKT. Data were normalized to control and represented as mean ± S.E.M (n = 8). *P < 0.05 between control and H2O2 treated mTAL; #P < 0.05 between H2O2 treated and H2O2 + PP242 treated mTAL; determined by Student’s t-Test.

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  Figure 5. (A, C) Representative Western Blot of total NKCC2 and phosphorylated NKCC2 cotransporters (pNKCC2Ser126 (A) and pNKCC2Thr96/101 (C)) in mTAL isolated from SD rats. mTAL incubated in HBSS for 20 min (control); incubated 10 min in HBSS and then with 100 µM of H2O2 for another 10 min (H2O2); incubated with 10 µM of PP242 for 10 min and with 100 µM of H2O2 for another 10 min (H2O2 + PP242). (B, D) Activity of NKCC2 cotransporters determined by densitometric quantification expressed as the ratio of pNKCC2Ser126 (B) and pNKCC2Thr96/101 (D) relative to total NKCC2 (n = 8 and n = 4, respectively). Data were normalized to control and represented as mean ± S.E.M. *P < 0.05 between control and H2O2 treated mTAL; #P < 0.05 between H2O2 treated and H2O2 + PP242 treated mTAL; determined by Student’s t-Test.

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  Figure 6. (A) Representative Western Blot of total Na+-K+-ATPase and phosphorylated Na+-K+-ATPase (pNa+-K+-ATPaseSer16) in mTAL isolated from SD rats. mTAL incubated in HBSS for 20 min (control); incubated 10 min in HBSS and then with 100 µM of H2O2 for another 10 min (H2O2); incubated with 10 µM of PP242 for 10 min and with 100 µM of H2O2 for another 10 min (H2O2 + PP242). (B) Activity of Na+-K+-ATPase pump determined by densitometric quantification expressed as the ratio of pNa+-K+-ATPaseSer16 relative to total Na+-K+-ATPase. Data were normalized to control and represented as mean ± S.E.M (n = 12). *P< 0.05 between control and H2O2 treated mTAL; #P < 0.05 between H2O2 treated and H2O2 + PP242 treated mTAL; determined by Student’s t-Test.

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  Figure 7. (A) Representative Western Blot of phosphorylated NHE-3 (pNHE-3Ser522) and total NHE-3 in mTAL isolated from SD rats. mTAL incubated in HBSS for 20 min (control); incubated 10 min in HBSS and then with 100 µM of H2O2 for another 10 min (H2O2); incubated with 10 µM of PP242 for 10 min and with 100 µM of H2O2 for another 10 min (H2O2 + PP242). (B) Activity of NHE-3 determined by densitometric quantification expressed as the ratio of pNHE-3Ser522 relative to total NHE-3. Data were normalized to control and represented as mean ± S.E.M (n = 6). *P < 0.05 between control and H2O2 treated mTAL, #P < 0.05 between H2O2 treated and H2O2 + PP242 treated mTAL; determined by Student’s t-Test.