HYDROGEN-PEROXIDE INCREASES THE INTRACELLULAR CALCIUM ACTIVITY IN RATMESANGIAL CELLS IN PRIMARY CULTURE

Oxygen radicals are known to be mediators of renal injury under severa l pathophysiological conditions. We have examined the effect of hydrog en peroxide (H2O2) on intracellular calcium activity ([Ca2+](i)) in me sangial cells in primary culture. Mesangial cells were loaded with 1 m u mol/liter fura-2, and kept in a Ringer-like solution. Fura-2 fluores cence was measured in an inverted microscope at 37 degrees C. Angioten sin II (0.1 nmol/liter) and ATP (0.1 mu mol/liter) induced a rapid tra nsient increase of [Ca2+](i), which was followed by a sustained platea u (N = 37 and N = 24). In contrast, the addition of H2O2 (0.01 to 10 m mol/liter, N = 157) caused a time- and concentration-dependent slow in crease of [Ca2+](i), which reached a stable [Ca2+](i) plateau after 3 to 10 minutes (ED(50): 100 mu mol/liter). After the removal of H2O2 [C a2+](i) decreased partially and reached a stable value approximately 9 0% above the resting [Ca2+](i) value. Addition of 100 mu mol/liter H2O 2 to an extracellular Ca2+-free solution resulted either in no rise of [Ca2+](i) in some experiments (N = 7), or [Ca2+](i) oscillations in o thers (N = 10). In the presence of H2O2 (> 25 mu mol/liter), the angio tensin II or ATP mediated increases in [Ca2+](i) were almost completel y inhibited (N = 15 and N = 10). The cations Ni2+ and La3+ and the Ca2 +-antagonist verapamil (10 mu mol/liter) did not inhibit the H2O2 medi ated increase of [Ca2+](i), (N = 6 to 9). Flufenamate (100 mu mol/lite r), an inhibitor of non-selective cation channels inhibited the H2O2 i nduced increase of [Ca2+](i) by 63 +/- 11% (N = 7). Preincubation of t he cells with a disulphide reducing agent (dithiothreitol, 500 mu mol/ liter, N = 5) or an iron-chelator (deferoxamine, 100 mu mol/liter, N = 5) attenuated the H2O2 mediated effect by 95 +/- 15% and 74 +/- 6%, r espectively. The H2O2 mediated [Ca2+](i) increase was completely inhib ited when mesangial cells were preincubated with 1 mu mol/liter U-8383 6E, an inhibitor of lipid peroxidation (N = 7), and inhibited by 84 +/ - 6% when the cells were pretreated with 1 mmol/liter pyruvate (N = 5) . The data indicate that H2O2: (i) increases [Ca2+](i) in mesangial ce lls by a mechanism distinct from angiotensin II or ATP and(ii) that it inhibits the [Ca2+](i) response to both agonists.