Hanigan and Devarajan: Molecular mechanisms of cisplatin nephrotoxicity of this regulatory pathway at least in vitro. It is well known that both cisplatin-induced DNA damage and cisplatin-induced oxidant stress are potent activators of p53 (Muller et al, 1998; Chandel et al, 2000), and that p53 can in turn activate both Bax as well as the Fas-FADD axis (Miyashita and Reed 1995; Burns and El-Deiry 1999). It is therefore likely that this regulatory mechanism may play a crucial role in cisplatin-induced apoptosis. It is well known that one of the responses of the normally quiescent renal tubular epithelial cell to damage induced by cisplatin includes entry into the cell cycle with subsequent cell proliferation, which presumably represents a reparative event (Megyesi et al, 1995; Sano et al, 2000). However, cisplatin also results in DNA damage (Zamble and Lippard 1995), and uncontrolled proliferation of these cells would be expected to result in apoptotic and/or necrotic cell death.Fortunately, renal epithelial cells have evolved mechanisms to prevent further progression of the cell cycle, allowing time and opportunity for their DNA to be repaired and the cell to then complete the regeneration and replacement process (Megyesi et al, 1998; Megyesi et al, 2002). Candidate proteins that contribute to the cell cycle arrest required for DNA repair include p21 and 14-33". Several studies have now documented that cisplatininduced nephrotoxicity is associated with upregulation of p21 mRNA (Megyesi et al, 1996; Huang et al, 2001) and protein (Megyesi et al, 1998; Miyaji et al, 2001; Megyesi et al, 2002). While it is well known that the p53 gene is a potent regulator of p21 (El-Deiry et al, 1993), induction of p21 in cisplatin nephrotoxicity appears to be p53dependent as well as -independent (Megyesi et al, 1996). Mice lacking p21 develop normally, but respond to cisplatin with a more severe nephrotoxic injury, including a more rapid onset of renal failure, uncoordinated progression into S-phase of the cell cycle, and increased apoptosis (Megyesi et al, 1998). Recent work has suggested a role for another cell cycle inhibitor, 14-3-3". Following cisplatin exposure, there is a marked induction of 14-3-3" mRNA and protein in the kidney tubular cells both in vivo and in vitro (Megyesi et al, 2002). Both p21 and 14-3-3" are known to be induced following DNAdamaging injury, at least in part via a p53-dependent mechanism (Hermeking et al, 1997). Both are overexpressed in terminally differentiating epithelia, both are required for proper coordination of the cell cycle, and the absence of either of these factors can accelerate apoptosis (Megyesi et al, 2002). It is recognized that apoptosis represents a default pathway in most cells, and can be activated by a relative deficiency of a variety of "survival factors" (Raff 1992). One example of a survival factor for kidney tubular cells following cisplatin injury is hepatocyte growth factor (HGF). Kidney mRNA levels for HGF are rapidly induced by ischemic or nephrotoxic injury (Liu et al, 1998), and administration of exogenous HGF ameliorates the renal dysfunction induced by cisplatin in vivo by enhancing tubular repair processes (Kawaida et al, 1994). It has recently been shown by a variety of assays that forced over-expression of HGF in cultured renal tubular cells partially inhibited the apoptotic response to cisplatin
incubation (Liu et al, 1998). Whether HGF exerts its beneficial effects in vivo also by protecting tubular cells from cisplatin-induced apoptotic death is not known.
XII. Role of oxidative stress and mitochondrial dysfunction The mechanisms by which cisplatin activates the myriad of apoptotic pathways outlined above remain unclear. However, a role for cisplatin-induced oxidative stress may provide an attractive hypothesis (Baliga et al, 1997). Several studies have now documented the importance of reactive oxygen metabolites (ROM) in cisplatin-induced renal cell apoptosis (Ueda et al, 2000). It is well known that mitochondria continuously produce ROM such as superoxide (Richter et al, 1995). Mitochondria also continuously scavenge ROM via the action of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase, and glutathione S-transferase (Richter et al, 1995). Cisplatin is known to accumulate in mitochondria of renal epithelial cells (Singh 1989; Gemba and Fukuishi 1991). Several investigators have demonstrated that cisplatin induces ROS in renal epithelial cells primarily by decreasing the activity of antioxidant enzymes and by depleting intracellular concentrations of GSH (Sadzuka et al, 1992; Kruidering et al, 1997; Husain et al, 1998; Huang et al, 2001). A large number of studies have now accumulated documenting the beneficial effects of a variety of antioxidants in cisplatin-induced nephrotoxicity. Agents such as superoxide dismutase, dimethylthiourea, and GSH have been shown to reduce the degree of renal failure and tubular cell damage when administered simultaneously with cisplatin in rats (McGinness et al, 1978; Sadzuka et al, 1992; Matsushima et al, 1998). Antioxidants such as GSH, superoxide dismutase, catalase, deferoxamine, probucol, and heme oxygenase-1 specifically provide partial protection against cisplatin-induced apoptosis in cultured renal epithelial cells (Lieberthal et al, 1996; Okuda et al, 2000; Shiraishi et al, 2000). Furthermore, significant attenuation of cisplatin-induced apoptosis and renal failure in animal models have resulted from maneuvers such as treatment with the hydroxyl radical scavenger DMTU (Zhou et al, 1999), targeted proximal tubular delivery of superoxide dismutase (Nishikawa et al, 2001), and pre-treatment with L-carnitine (Chang et al, 2002). Reactive oxygen molecules can trigger several of the apoptotic mechanisms activated by cisplatin (Figure 3). For example, ROM can induce Fas (Bauer et al, 1998), activate p53 (Chandel et al, 2000), alter mitochondrial permeability (Kruidering et al, 1997; Nowak 2002), release cytochrome c into the cytosol (Reed 1997), and even directly activate caspases (Higuchi et al, 1998). However, one recent study has suggested that at least in cultured proximal tubular cells, the primary cause of cell death following cisplatin exposure is not ROM formation per se (Kruidering et al, 1996). Rather, cisplatin-induced mitochondrial dysfunction with consequent induction of cell death pathways appeared to be the underlying mechanism. Several studies have
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