Original article
Differential roles of hydrogen peroxide and hydroxyl radical in cisplatin-induced cell death in renal proximal tubular epithelial cells☆

https://doi.org/10.1016/S0022-2143(03)00111-2Get rights and content

Abstract

Reactive oxygen species (ROS) have been suggested as important mediators of cisplatin-induced acute renal failure in vivo. However, our previous studies have shown that cisplatin-induced cell death in vitro could not be prevented by scavengers of hydrogen peroxide and hydroxyl radical in rabbit renal cortical slices. This discrepancy may be attributed to differential roles of ROS in necrotic and apoptotic cell death. We therefore examined, in this study, the roles of ROS in necrosis and apoptosis induced by cisplatin in primary cultured rabbit proximal tubule. Cisplatin induced necrosis at high concentrations over a few hours and apoptosis at much lower concentrations over longer periods. Necrosis induced by high concentration of cisplatin was prevented by a cell-permeable superoxide scavenger (tiron), hydrogen peroxide scavengers (catalase and pyruvate), and antioxidants (Trolox and deferoxamine), whereas hydroxyl radical scavengers (dimethythiourea and thiourea) did not affect the cisplatin-induced necrosis. However, apoptosis induced by lower concentration of cisplatin was partially prevented by tiron and hydroxyl radical scavengers but not by hydrogen peroxide scavengers and antioxidants. Cisplatin-induced apoptosis was mediated by the signaling pathway that is associated with cytochrome c release from mitochondria and caspase-3 activation. These effects were prevented by tiron and dimethylthiourea but not by catalase. Dimethylthiourea produced a significant protection against cisplatin-induced acute renal failure, and the effect was associated with an inhibition of apoptosis. These results suggest that hydrogen peroxide is involved in the cisplatin-induced necrosis, whereas hydroxyl radical is responsible for the cisplatin-induced apoptosis. The protective effects of hydroxyl radical scavengers are associated with an inhibition of cytochrome c release and caspase activation.

Section snippets

Primary culture of rabbit proximal tubules

Proximal tubules were isolated from rabbit kidney and prepared for cultures as described previously.23 In brief, adult male New Zealand white rabbits were killed by means of cervical dislocation. Their kidneys were removed immediately, cleaned of fat and debris, and washed with sterile antibiotic-supplemented medium. The kidneys were perfused with PBS (pH 7.4) through the renal artery and subsequently with DMEM/F12 (Sigma-Aldrich, St Louis, Mo) containing 0.5% (wt/vol) iron oxide until the

Induction of necrotic and apoptotic cell death in cells treated with cisplatin

Because previous studies10, 11, 17 of renal tubular epithelial cells have shown that cisplatin causes necrotic cell death at high concentrations and apoptosis at lower concentrations, we exposed cells to high (2 mmol/L) and low (50 μmol/L) concentrations of cisplatin for various periods in this study. Exposure of cells to 50 μmol/L cisplatin resulted in gradual loss of cell viability (about 55% after 36 hours), whereas in 2 mmol/L cisplatin, 98% of the cells lost viability in 24 hours (Fig 1).

Discussion

Many in vivo and in vitro studies have shown that oxidative stress may play an important role in the pathogenesis of cisplatin nephrotoxicity.15, 16, 27, 28 Cisplatin generates superoxide in a cell-free system29 and hydrogen peroxide in renal proximal tubular cells.30 However, the chemical nature of ROS responsible for cisplatin nephrotoxicity remains unidentified.

We demonstrated that cisplatin causes necrosis or apoptosis, depending on dosage and exposure time, in primary cultured renal

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    ☆

    Supported by grant R01-2002-000-00460-0 (2002) from the Basic Research Program of the Korean Science and Engineering Foundation and Pusan National University Research Grant.

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