Hydrogen peroxide increases the intracellular calcium activity in rat mesangial cells in primary culture

Abstract

Hydrogen peroxide increases the intracellular calcium activity in rat mesangial cells in primary culture. Oxygen radicals are; known to be mediators of renal injury under several pathophysiological conditions. We have examined the effect of hydrogen peroxide (H2O2) on intracellular calcium activity ([ICa2+](i)) in mesangial cells in primary culture. Mesangial cells were loaded with 1 μmol/liter fura-2, and kept in a Ringer-like solution. Fura-2 fluorescence was measured in an inverted microscope at 37°C. Angiotensin II (0.1 nmol/liter) and ATP (0.1 μmol/liter) induced a rapid transient increase of [Ca2+] (i), which was followed by a sustained plateau (N = 37 and N = 24). In contrast, the addition of H2O2 (0.01 to 10 mmol/liter, N = 157) caused a time- and concentration-dependent slow increase of [Ca2+](i) which reached a stable [Ca2+](i) plateau after 3 to 10 minutes (ED50: 100 μmol/liter). After the removal of H2O2 [Ca2+], decreased partially and reached a stable value approximately 90% above the resting [Ca2+](i) value. Addition of 100 μmol/liter H2O2 to an extracellular Ca2+-free solution resulted either in no rise of [Ca2+](i) in some experiments (N = 7), or [Ca2+](i) oscillations in others (N = 10). In the presence of H2O2 (>25 μmol/liter), the angiotensin II or ATP mediated increases in [Ca2+](i) were almost completely inhibited (N = 15 and N = 10). The cations Ni2+ and La3+ and the Ca2+-antagonist verapamil (10 μmol/liter) did not inhibit the H2O2 mediated increase of [Ca2+](i) (N = 6 to 9). Flufenamate (100 μmol/liter), an inhibitor of non-selective cation channels inhibited the H2O2 induced increase of [Ca2+](i) by 63 ± 11% (N = 7). Preincubation of the cells with a disulphide reducing agent (dithiothreitol, 500 μmol/liter, N = 5) or an iron-chelator (deferoxamine, 100 μmol/liter, N = 5) attenuated the H2O2 mediated effect by 95 ± 15% and 74 ± 6%, respectively. The H2O2 mediated [Ca2+], increase was completely inhibited when mesangial cells were preincubated with 1 μmol/liter U-83836E, 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 cells by a mechanism distinct from angiotensin II or ATP and (ii) that it inhibits the [Ca2+](i) response to both agonists.