Cisplatin is a potent chemotherapy drug and a nephrotoxin. The studies funded by this grant have revealed that the enzyme gamma-glutamyl transpeptidase is essential for the nephrotoxicity of cisplatin, but induces resistance to cisplatin in rapidly growing tumor cells. These data have led to the discovery that cisplatin is metabolized to a nephrotoxin via a platinum-glutathione conjugate. This is the first demonstration that cisplatin can undergo enzymatic activation to a metabolite that is more toxic than the parent compound. Our hypothesis is that cisplatin kills the non-dividing cells in the kidney by a mechanism that is distinct from the mechanism by which it kills the dividing tumor cells. This renewal application has three specific aims. The first is to identify the glutathione-S-transferases (GSTs) that catalyze the formation of the nephrotoxic platinum-GSH conjugate of cisplatin. An HPLC assay will be used to identify and monitor the purification of GSTs that catalyze the formation of the nephrotoxic platinum-GSH conjugate. A combination of siRNA and transfection studies will be done to confirm the role of the GST in cisplatin nephrotoxicity. Expression of GSTs in tumors has been associated with resistance to cisplatin. The resistance may be due to their ability to serve as a binding protein. GSTs that bind cisplatin will also be identified. The second specific aim is to identify the renal enzyme that catalyzes the metabolism of the platinum-cysteine conjugate to a toxin. In vivo and in vitro studies have shown that a pyridoxal 5'- phosphate-dependent enzyme catalyzes this reaction. The third specific aim is to identify the mechanism by which the sodium stress response blocks cisplatin nephrotoxicity. Sodium chloride is used clinically to reduce cisplatin nephrotoxicity. The mechanism of this protective effect is not known. A cell culture system will be used to investigate the molecular basis of this response. Tumor cells will be analyzed to determine if the sodium stress response is constitutively expressed in cisplatin-resistant cells. Cisplatin is one of the most commonly used chemotherapy drugs. The goal of this research is to understand the molecular mechanisms by which cisplatin kills cells and the mechanism by which cells become resistant to cisplatin.