Hydrogen sulfide prevents hydrogen peroxide-induced activation of epithelial sodium channel through a PTEN/PI(3,4,5)P3 dependent pathway

Abstract

Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2) stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO ) was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS). Pre-treatment of A6 cells with H2S slightly decreased ENaC P(O); however, in these cells H2O2 failed to elevate ENaC PO . Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS) level and induced accumulation of PI(3,4,5)P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5)P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN) which is a negative regulator of PI(3,4,5)P3. Moreover, BPV(pic), a specific inhibitor of PTEN, elevated PI(3,4,5)P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5)P3 dependent pathway.

Figure 1. H₂S inhibits H₂O₂-induced enhancement of ENaC activity in A6 cells.

(A) and (B) Representative single-channel ENaC traces respectively recorded from two A6 cells before and after addition of 3 mM H₂O₂ and 3 mM H₂O₂+0.1 mM NaHS to the basolateral bath; the breaks between the traces indicate omitted 20 min of the recording periods; downward events represent channel openings (the dashed gray line); the currents were monitored for at least 30 min before and after chemical application for both in (A) and (B). (C) and (D) Summarized Po of ENaC before and after application of different reagents as indicated (n = 6 paired experiments). **P<0.01 compared with control group, ^## P<0.01 compared with H₂O₂ treatment group.

Figure 2. H₂S ameliorates H₂O₂-elicited oxidative stress in A6 cells.

All experiments were assessed in the presence of 50 µM 2,2′-dipyridyl. The images represent the level of intracellular ROS detected by the membrane-permeable fluorescent probe, carboxy-H₂DCFDA, under indicated conditions in A6 cells. (A) The left image shows that there was a certain residual level of intracellular ROS under control condition; middle image shows a significant elevation of intracellular ROS level upon application of 3 mM H₂O₂; right image shows that this H₂O₂-induced increase in intracellular ROS was abolished by additional 0.1 mM NaHS. (B) The images represent that the intracellular ROS levels under control (left), treatment of A6 cells with 0.1 mM NaHS for 30 min (middle) followed by additional 0.1 mM H₂O₂ for 30 min (right). (C) and (D) Summarized fluorescent intensity of (A) and (B), respectively. Data were from three independent paired experiments. **P<0.01 compared with control group, ^## P<0.01 compared with H₂O₂ treatment group.

Figure 3. H₂S inhibits H₂O₂-induced elevation of PI(3,4,5)P₃ near the apical compartment of A6 cells.

Left images show confocal microscopy XY sections of apical, lateral, and basal membranes of A6 cells as indicated. Right images show XZ sections of A6 cells. (A) Control; (B) the cells were treated with 3 mM H₂O₂ for 30 min; (C) the cells were treated with 0.1 mM NaHS for 30 min; (D) the cells were co-treated with 3 mM H₂O₂+0.1 mM NaHS for 30 min. (E) Summarized mean fluorescent intensities measured in and in the vicinity of the apical membrane from three independent experiments, which represent the levels of PI(3,4,5)P₃ near the apical region of the membrane. **P<0.01 compared with control group, ^## P<0.01 compared with H₂O₂ treatment group.

Figure 4. H₂O₂ oxidizes PTEN and H₂S protects PTEN to be oxidized by H₂O₂.

All experiments were assessed in the presence of 50 µM 2,2′-dipyridyl. (A) and (B) Western blot analysis demonstrates that the total PTEN protein expression was not altered by treatment of A6 cells with 1 mM, 3 mM and 5 mM H₂O₂. (C) and (D) Western blot analysis shows that the magnitude of reduced (active) PTEN was dramatically decreased upon treatment of A6 cells with 0.3 mM, 1 mM and 3 mM H₂O₂ for 30 min; the intensity of the bands corresponding to reduced PTEN in the top panels of (C) was determined and presented as a percentage of the sum of the intensities of the bands corresponding to the oxidized and reduced proteins. (E) and (F) show that H₂S protected 3 mM H₂O₂ induced PTEN oxidation (n = 3 in each group) **P<0.01 compared with control group; P<0.05 compared with 0.3 mM H₂O₂ treatment group; ^## P<0.01 compared with H₂O₂ treatment group.

Figure 5. Inhibition of PTEN by BPV_(pic) results in accumulation of PI(3,4,5)P₃ near the apical compartment of A6 cells.

The experiments were performed under different conditions as follows: (A) control; (B) the cells were treated with 3 mM H₂O₂ for 30 min; (C) the cells were treated with 30 nM BPV_(pic) for 30 min; (D) the cells were co-treated with 30 nM BPV_(pic) +3 mM for 30 min. (E) Summarized mean fluorescent intensities measured as ascribed in Figure 3 from three independent experiments, which represent the level of PI(3,4,5)P₃ in the apical membrane. **P<0.01 compared with control group, ^## P<0.01 compared with BPV_(pic) treatment group.

Figure 6. Inhibition of PTEN leads to an aberrant activation of ENaC in A6 cells.

Representative single-channel ENaC trace recorded from an A6 cell before and after addition of 30 nM BPV_(pic) and 3 mM H₂O₂ to the basolateral bath. Two breaks between the traces indicate the 20 min omitted recording periods. (B) Summarized Po of ENaC before and post application of different reagents. n = 5 paired experiments. **P<0.01 compared with control group, ^# P<0.05 compared with BPV_(pic) treatment group.