INHIBITION OF RAT GLOMERULAR MESANGIAL CELL SODIUM/HYDROGEN EXCHANGE BY HYDROGEN PEROXIDE

1. pH_i regulation In glomerular mesangial cells (GMC) includes both Na⁺/H⁺ and Cl-/HCO₃^- exchange. As a fall in pH_i may protect against H₂O₂-mediated GMC damage during ischaemia-reperfusion, the involvement of these mechanisms in the GMC pH_i response to H₂O₂ was assessed using confluent GMC grown in RPMI medium with 20% fetal calf serum (10–15 passages). 2. Cells were loaded with BCECF-AM and pH_i evaluated using standard fluorometric-ratio techniques. In HEPES buffer, GMC exposure to H₂O₂ dose-dependently (25μmol/L-1 mmol/L) decreased pH_i over 10 rnin from 7.3 ± 0.1 to 6.7 ± 0.1 (at 100 μmol/L) partly due to rapid non-competitive inhibition of amiloride-sensltive Na⁺/H⁺ exchange. 3. BCECF fluorescence in free solution was unchanged by H₂O₂ whereas the pH_i decrease was abolished by nigericin/K⁺ clamp. Buffer capacity by direct titration was not changed by H₂O₂ and averaged 100 ± 9 nmol/2.6±10⁶ cells/pH unit. Similarly, zero-Na⁺/high-K⁺ buffer, used to minimize Passive H⁺ entry, did not prevent the fall in pH_i while GMC H⁺-formation/extrusion, assessed by the rate of extracellular acidification in low-capacity buffer (0.05 mmol/L), was rapidly inhibited. 4. In contrast, following only a brief 3 min exposure to 1 mmol/L H₂O₂, HCO₃-/CO₂ buffer potentiated the inhibition of Na⁺/ H⁺ exchange from 50 to 80% of control and reduced the acidification from pH_i 6.6 ± 0.1 to 7.15 ± 00.05. This effect was reversed (to pH_i 6.8 ± 0.07) by pretreatment with 200 μmol/L DIDS, an inhibitor of Cl-/HCO₃- exchange. 5. Thus, the decrease in GMC pH_i in Fespohsë to H₂O₂ In HEPES, partly mediated by inhibition of Na⁺/H⁺ exchange and a possible redistribution of intraceliular H⁺, is antagonized in HCO₃-/CO₂ through a DIDS-sensitive Cl-/HCO₃^- exchange mechanism. This may act to negate potentially protective effects of low pH_i and potentiate oxidative damage to membrane lipids, enzymes and intracellular organelles on reperfusion.