H2O2-INDUCED STIMULATION OF CL-/HCO3- EXCHANGER ACTIVITY IN SHR CELLS IS ASSOCIATED WITH OXIDATION OF EXCHANGER THIOL GROUPS: PP.29.176

Abstract

Objective: NaCl reabsorption in proximal tubule occurs through apical Cl-/HCO3- exchanger and NHE3. Increased reactive oxygen species production in the kidney decreases sodium excretion. Expression of Slc26a6 (primary candidate for apical Cl-/HCO3- exchange in the kidney) in proximal tubular epithelial (PTE) cells from SHR was 7-fold that in WKY PTE cells. The present study investigated the mechanism by which H2O2-induced stimulation of Cl-/HCO3- exchanger activity is enhanced in immortalized SHR cells as compared to WKY cells. Methods: Cells were loaded with BCECF-AM and exchanger activity was assayed as the initial rate of pHi recovery after an alkaline load. Results: The enhanced capacity of SHR cells to produce H2O2, comparatively with WKY cells, paralleled an increase in expression of Nox2 and p22phox and a decrease in expression of SOD2, SOD3, and catalase. In both cell lines, exogenous H2O2 stimulated, in a concentration-dependent manner, Cl-/HCO3- exchanger activity. This stimulation was higher in SHR than in WKY cells. The effect of H2O2 upon exchanger activity was blocked by SP600125 (JNK-inhibitor), but not by U0126 (ERK1/2-inhibitor) or SB203580 (p38-inhibitor) in WKY and SHR cells. DTDP, a thiol-selective oxidizing reagent (membrane permeable), stimulated in a concentration-dependent manner Cl-/HCO3- exchanger activity, in both cell lines. This stimulation was higher in SHR cells. Thimerosal, another thiol oxidizing reagent (poorly membrane permeable) had no effect on exchanger activity in WKY and SHR cells. The effects of H2O2 and DTDP upon the exchanger activity were blocked by DTT (a thiol-selective reducing reagent), in WKY and SHR cells. Conclusion: H2O2-induced stimulation of Cl-/HCO3- exchanger activity appears to be regulated by JNK in both cell lines. However, the enhanced sensitivity to H2O2-induced stimulation of Cl-/HCO3- exchanger activity in SHR cells may arise from a more marked oxidation of SHR cells exchanger's intracellular thiol groups. Additionally, decreased antioxidant defence in SHR cells may contribute to the enhanced sensitivity of H2O2-induced stimulation of Cl-/HCO3- exchanger activity in this cell line.