Cisplatin-Induced Apoptosis Is Enhanced by Hypoxia and by Inhibition of Mitochondria in Renal Collecting Duct Cells

TOXICOLOGICAL SCIENCES 85, 735–742 (2005) doi:10.1093/toxsci/kfi117 Advance Access publication February 16, 2005 Cisplatin-Induced Apoptosis Is Enhanced by Hypoxia and by Inhibition of Mitochondria in Renal Collecting Duct Cells Gerald Schwerdt,1 Ruth Freudinger, Claudia Schuster, Florian Weber, Oliver Thews, and Michael Gekle Physiologisches Institut, Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany Cisplatin is a widely used chemotherapeutic agent. Here we show that cisplatin induces apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells) in a dosedependent manner. Additionally, we studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1Fo-ATP synthase or by uncoupling. The role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATPsynthase, cisplatin-induced apoptosis was markedly enhanced. Mitochondrial blockade led to enhanced production of lactic acid. Also, anoxia potentiated the cisplatin-induced caspase-3 activation. Neither intra- nor extracellular pH had an influence on caspase-3 activation at low cisplatin concentrations. Acidic conditions (pH 6.8) potentiated the caspase-3 activation when high (100 mM) cisplatin concentrations were used. We demonstrate that intact mitochondria are important to prevent cisplatin-induced apoptosis in MDCK-C7 cells and that acidic conditions can aggravate the toxic effects of cisplatin. Key Words: cisplatin; hypoxia; collecting duct; apoptosis; mitochondria. Cisplatin is a potent and frequently used anticancer drug, which has nephrotoxic side effects (Safirstein et al., 1986). Either the proximal tubule or the collecting duct segments of the nephron are impaired by cisplatin even though by obviously different mechanisms (Brady et al., 1993; Kroning et al., 1999). However, in both segments, proximal tubule and collecting duct, mitochondria seem to play a role in the cytotoxic action of cisplatin. Cisplatin causes apoptosis in proximal tubule cells by caspase-3-dependent and -independent mechanisms (Cummings et al., 2002; Schwerdt et al., 2003). Also, in mouse collecting duct cells, cisplatin induces apoptosis (Lee et al., 2001). Cisplatin-induced cell death is believed to be mediated by its interaction with nuclear DNA (Eastman, 1999; Pinto and Lippard, 1985). Additionally, mitochondria are thought to be a major target of cisplatin and mitochondrial DNA is heavily damaged by cisplatin (Olivero et al., 1995; Murata et al., 1990) leading to mitochondrial loss of energy production, release of 1 To whom correspondence should be addressed. Fax: þ49 931 312741. E-mail: . a mitochondrial serin protease (Cilenti et al., 2005) with subsequent cell death. Apoptosis is a well-described sort of cell death induced by a variety of substances. The processes in an apoptotic cell are well characterized and several reports describe that mitochondria play a crucial role (Kroemer et al., 1997; Kroemer and Reed, 2000). Release of cytochrome C and other proteins from mitochondria (Liu et al., 1996; Patterson et al., 2000) often induces a series of events which finally leads to activation of caspase-3, followed by DNA ladder formation and cell death. Besides different substances, inhibition of mitochondrial respiration was shown to lead to apoptosis induction (Wolvetang et al., 1994). Additionally, inhibition of mitochondrial respiration forces the cell to increase the anaerobic glycolysis pathway to guarantee the ATP supply of the cell. In this case, the cell produces increased amounts of lactic acid which acidify either the cell interior and/or the surrounding media. The collecting duct cell can experience an acidic microenvironment both physiologically and pathophysiologically: physiologically, in the collecting duct the pH underlies considerable variations from alkaline conditions down to pH 4.5 (Hamm and Alpern, 1992; Sabatini and Kurtzman, 1989) and pathopysiologically hypoxic conditions with lowered extracellular pH (down to pH 6.0; Vaupel et al., 1989) at the basolateral side of the cells may occur. Furthermore, pH maintenance plays an important role in tumor genesis. It is well known that cancer cells produce acidification of the extracellular compartment. This is due to increased use of anaerobic, lactic acid producing glycolysis, which is the main source of energy production of cancerous cells. At the same time the mitochondria-mediated oxidative phosphorylation is diminished (Gatenby and Gawlinski, 2003; Warburg, 1956). These observations underline the importance of mitochondria and extracellular pH on cell survival. Additionally, in collecting duct cells acidic apical pH leads to increased cellular apical uptake, increased transepithelial reabsorptive transport, and increased apoptosis rates induced by other nephrotoxic substances, e.g., ochratoxin A (Dahlmann et al., 1998; Schwerdt et al., 1997, 2004; Zingerle et al., 1997). Intact mitochondria are thus a prerequisite for a cell to fulfill its responsibilities in an organism. Therefore, to investigate the role of mitochondria in cisplatin-induced apoptosis, we studied the effects of inhibition of mitochondria or of hypoxia on Toxicological Sciences vol. 85 no. 1 Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: 736 SCHWERDT ET AL. cisplatin-induced apoptosis in epithelial collecting duct cells. Additionally, the effects of extracellular pH and the role of the intracellular pH on cell survival after cisplatin administration of MDCK-C7 cells were studied. MATERIALS AND METHODS Cell culture. Madin-Darby canine kidney cells, clone C7 (MDCK-C7 cells), which resemble the principal cells of the collecting duct (Gekle et al., 1994) were seeded in plastic culture dishes (9.62 cm2 culture area) in 1 ml of minimum essential medium (MEM) with Earle’s salts, nonessential amino acids, and L-glutamine (Biochrom KG, Berlin, Germany), and cultured under standard culture conditions (37°C, 5% CO2). MEM was supplemented with 10% fetal calf serum (Biochrom KG) and 24 mM NaHCO3. Media were changed three times a week and cells were subcultivated once a week. Cells were exposed to cisplatin or other substances for 24 h if not indicated otherwise. Hypoxic conditions were maintained by incubation at 37°C in a water-saturated atmosphere containing 95% nitrogen and 5% CO2. Measured fractional oxygen content was below 0.01. Caspase-3 activity assay. Before incubation with cisplatin or other substances, cells were incubated in serum-free medium for 24 h in Petri dishes (9.62 cm2 culture area, 104 ll media/cm2). Caspase-3 activity was measured according to the manufacturer’s instructi (...truncated)