Developmental renal tubular toxicity: A hypothetical framework and experimental approaches

Background: The antineoplastic agent ifosfamide is capable of causing renal damage in children. It is not known why children younger than 3 years experience more nephrotoxicity, regardless of the concurrent administration of the synthetic thiol compound sodium 2-mercaptoethane sulfonate (mesna). The ability of mesna to form stable nontoxic thioether compounds occurs at the expense of intracellular glutathione, which when decreased may predispose kidney cells to localized oxidative stress, resulting in renal damage. We present a hypothetical framework that assumes that the renal tubular cell is capable of producing nephrotoxic metabolites, and that this happens more often among young children, either because of higher cytochrome P450 (CYP) activity or lower levels of antioxidants. Objective: The aim of this study was to show whether the prodrug ifosfamide is cytotoxic to renal proximal tubule cells once they are depleted of glutathione. Methods: Human kidney microsomes and CYP 3A4, 3A5, and 3A7 Supersomes™ were incubated with 1 mM ifosfamide and the results analyzed by means of gas chromatography using thermionic nitrogen-specific detection (GC/TSD). Microsomes were separated by means of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10% gels, transferred to nitrocellulose, and immunoblotted with CYP 3A4 and 3A5 antigen. Finally, porcine renal proximal tubular cell monolayer (LLC-PK-1) was used to determine whether ifosfamide is cytotoxic to renal tubule cells once the cells have been depleted of glutathione with 250 μM buthionine sulfoxime (BSO). Results: CYP 3A4 and 3A5 were found to be present in human kidney microsomes. The ability of kidney microsomes to metabolize ifosfamide to its 2-de-chloroethylifosfamide and 3-dechloroethylifosfamide metabolites was confirmed. Furthermore, pretreatment of LLC-PK-1 cells for 24 hours with 250 μM of the glutathione-depleting substance BSO led to a significant decrease in the number of viable cells when cells were incubated with either 100 μM or 1 mM ifosfamide for either 72 or 96 hours. BSO pretreatment for 24 hours followed by ifosfamide treatment for 24 or 48 hours did not significantly decrease the number of viable LLC-PK-1 cells. Conclusions: The human kidney possesses the necessary enzymatic machinery (CYP 3A4 and 3A5) to metabolize ifosfamide to nephrotoxic metabolites. Decreasing the intracellular levels of glutathione in renal proximal tubule cells subjects them to damage that results in a decrease in the number of viable cells. Additional studies on the ontogeny of these processes are in progress.