Cytotoxic effects of diglycolic acid, the nephrotoxic metabolite of diethylene glycol, and the mechanisms leading to proximal tubule cell dysfunction.

摘要

Diethylene glycol (DEG) is an organic solvent used in common consumer products allowing the increased risk for consumer exposure. DEG metabolism produces two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA). Studies were designed to assess the proximate DEG metabolite responsible for DEG-induced proximal tubule necrosis which leads to acute kidney injury, the hallmark of DEG poisoning. DGA, not DEG or 2-HEAA, induced human proximal tubule (HPT) cell necrosis in a concentration and time-dependent manner. DGA significantly decreases ATP production occurring prior to cell death. HPT cells pretreated with DGA show a 50% decrease in oxygen consumption, significant inhibition of succinate dehydrogenase activity, and a significant dissipation of the mitochondrial membrane potential (Deltapsi) suggesting the ability of DGA to interfere with mitochondrial function. DGA significantly increased reactive oxygen species (ROS) production, which was significantly reduced at 6 and 48 h in cells exposed to the antioxidant, trolox. Reduction in both ROS production and cell death was only observed in the 25 mmol/l DGA group suggesting that high concentration DGA-induced cytotoxicity is occurring independently of ROS production, likely by additional mechanisms. Uptake of DGA by HPT cells occurs over time and in a bidirectional fashion. The kinetic constants and specific transporter inhibition of DGA uptake exhibits characteristics which indicate a low affinity, low capacity transporter typical of how the basolateral sodium dicarboxylate-3 (NaDC-3) transporter would function with lower affinity substrates. In addition, only half of loaded DGA is effluxed from intracellular stores suggesting that a substantial portion remains intracellular, and could contribute to DGA-induced PT cell dysfunction. To address potential risk assessment issues, studies evaluated possible rat strain sensitivity to a high dose of DEG (10 g/kg) utilizing parameters of kidney and liver damage at high DEG doses both strains. Results indicate that both F-344 and Wistar rats appear to be equally sensitive in vivo, with F-344 isolated proximal tubule cells showing increased sensitivity to DGA-induced cell death in vitro. These results indicate that DGA produces proximal tubule cell dysfunction by specific inhibition of mitochondrial-mediated processes, and mimics substrates to gain PT cell intracellular access via transporter-mediated processes.