Variable sensitivity to diethylene glycol poisoning is related to differences in the uptake transporter for the toxic metabolite diglycolic acid.

INTRODUCTION/CONTEXT: Poisonings with diethylene glycol are characterized by acute kidney injury and peripheral neuropathy. In animal studies on the toxicities of diethylene glycol and its metabolite diglycolic acid, remarkable differences in susceptibility to acute kidney injury were observed in identically-dosed rats. In those studies, only about 60% showed acute kidney injury, yet all rats with acute kidney injury showed marked diglycolic acid accumulation in tissues, while no diglycolic acid accumulated in rats without injury. Diglycolic acid is taken into renal cells via sodium-dependent dicarboxylate transporters. When sodium-dependent dicarboxylate transporter-1 is inhibited or knocked down in human kidney cells, diglycolic acid uptake and toxicity are reduced. We hypothesize that the variation in sensitivity to tissue diglycolic acid retention and to diethylene glycol/diglycolic acid toxicity is explained by differential expression of sodium-dependent dicarboxylate transporter-1 in rat kidneys. METHODS: Using kidney tissue from previous studies, we performed rt-PCR analysis of sodium-dependent dicarboxylate transporter-1 mRNA. In those studies, Wistar-Han rats were either gavage with diethylene glycol 6 g/kg every 12 h for 7 days or with single doses of diglycolic acid 300 mg/kg. Kidney tissue was harvested after euthanasia and preserved in formalin. Tissue slices were homogenized and RNA was isolated using an RNAstorm FFPE RNA Isolation Kit. The expression of sodium-dependent dicarboxylate transporter-1 mRNA was compared between groups that showed diglycolic acid accumulation and acute renal injury with those that showed no diglycolic acid accumulation or toxicity. RESULTS: Significantly higher expression of sodium-dependent dicarboxylate transporter-1 mRNA was present in the kidneys of rats with acute kidney injury and diglycolic acid accumulation compared to those in rats that had no diglycolic acid in their kidneys and no acute kidney injury. DISCUSSION: The likelihood of acute kidney injury after dosing of rats with diethylene glycol or diglycolic acid is linked with an enhanced ability to take up diglycolic acid into renal cells via the sodium-dependent dicarboxylate transporter-1. The variability in diethylene glycol toxicity in humans, as reported in epidemiological studies, may also be linked with differences in tissue uptake of diglycolic acid. CONCLUSIONS: Animals with acute kidney injury after exposure to diethylene glycol or diglycolic acid had higher sodium-dependent dicarboxylate transporter-1 expression and greater diglycolic acid accumulation in renal tissues than animals without acute kidney injury.

Lack of efflux of diglycolic acid from proximal tubule cells leads to its accumulation and to toxicity of diethylene glycol.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of adulterated pharmaceuticals, leading to proximal tubular necrosis and acute kidney injury. Diglycolic acid (DGA), one of the primary metabolites, accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to Krebs cycle intermediates such as succinate. Previous studies have shown that DGA is taken into kidney cells via the succinate-related dicarboxylate transporters. These studies have assessed whether the DGA that is taken up by primary cultures of human proximal tubule (HPT) cells is effluxed. In addition, a possible mechanism for efflux, via organic anion transporters (OATs) that exchange external organic anions for dicarboxylates inside the cell, was assessed using transformed cell lines that actively express OAT activities. When HPT cells were cultured on membrane inserts, then loaded with DGA and treated with the OAT4/5 substrate estrone sulfate or the OAT1/3 substrate para-aminohippurate, no DGA efflux was seen. A repeat of this experiment utilizing RPTEC/TERT1 cells with overexpressed OAT1 and OAT3 had similar results. In these cells, but not in HPT cells, co-incubation with succinate increased the uptake of PAH, confirming the presence of OAT activity in the RPTEC/TERT1 cells. Thus, despite OATs stimulation in cells with OAT activity, there was little to no efflux of DGA from the cells. This study concluded that DGA is poorly transported out of cells and that stimulation of OAT transporters is not a viable target for reducing DGA accumulation in cells.

Role of Plasma Membrane Dicarboxylate Transporters in the Uptake and Toxicity of Diglycolic Acid, a Metabolite of Diethylene Glycol, in Human Proximal Tubule Cells.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of pharmaceuticals mistakenly adulterated with DEG, typically leading to proximal tubular necrosis and acute kidney injury. The metabolite, diglycolic acid (DGA) accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to metabolites like succinate. These studies have assessed the mechanism for cellular accumulation of DGA, specifically whether DGA is taken into primary cultures of human proximal tubule (HPT) cells via sodium dicarboxylate transporters (NaDC-1 or NaDC-3) like those responsible for succinate uptake. When HPT cells were cultured on membrane inserts, sodium-dependent succinate uptake was observed from both apical and basolateral directions. Pretreatment with the NaDC-1 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) markedly reduced apical uptakes of both succinate and DGA. Basolateral uptake of both succinate and DGA were decreased similarly following combined treatment with ACA and the NaDC-3 inhibitor 2,3-dimethylsuccinate. When the cells were pretreated with siRNA to knockdown NaDC-1 function, apical uptake of succinate and toxicity of apically applied DGA were reduced, while the reduction in basolateral succinate uptake and basolateral DGA toxicity was marginal with NaDC-3 knockdown. DGA reduced apical uptake of succinate but not basolateral uptake. This study confirmed that primary HPT cells retain sodium dicarboxylate transport functionality and that DGA was taken up by these transporters. This study identified NaDC-1 as a likely and NaDC-3 as a possible molecular target to reduce uptake of this toxic metabolite by the kidney.

In-vivo evidence of nephrotoxicity and altered hepatic function in rats following administration of diglycolic acid, a metabolite of diethylene glycol.

CONTEXT: Diglycolic acid (DGA) is one of the two primary metabolites of diethylene glycol (DEG). DEG is an industrial solvent that has been implicated in mass poisonings resulting from product misuse in the United States and worldwide, with the hallmark toxicity being acute kidney injury, hepatotoxicity, encephalopathy and peripheral neuropathy. Our laboratory has generated in-vitro evidence suggesting that DGA is the metabolite responsible for the proximal tubule necrosis and decreased kidney function observed following DEG ingestion. Furthermore, we have shown that DGA specifically accumulates in kidney tissues (100× higher than peak blood concentrations) following DEG administration. OBJECTIVE: To examine renal and hepatic accumulation and dysfunction following direct administration of DGA in-vivo. We hypothesize that administration of DGA will result in renal and hepatic DGA accumulation, as well as proximal tubular necrosis and liver injury. MATERIALS AND METHODS: Adult male Wistar rats were divided into three groups dosed with 0, 100 or 300 mg/kg DGA via single oral gavage. Urine was collected every 6-12 h and blood, kidneys and liver were removed upon sacrifice at 48 h post-dosing for analysis. RESULTS: DGA accumulated significantly in both kidney and liver tissue only at 300 mg DGA/kg. DGA concentrations in the kidneys and liver correlated with renal and hepatic injury, respectively. Histopathological and clinical chemistry analysis revealed that DGA-treated animals exhibited moderate liver fatty accumulation and marked renal injury, again only at 300 mg/kg. DISCUSSION: DGA-induced kidney injury demonstrated a steep dose response threshold, where severe damage occurred only in animals given 300 mg/kg DGA, while no toxicity was observed at 100 mg/kg. CONCLUSION: These results provide evidence for in-vivo toxicity following direct administration of DGA, a metabolite of DEG. The steep dose-response threshold for toxicity suggests mechanistically that there is likely a saturable step that results in DGA accumulation in target organs.

Nucleoside reverse transcriptase inhibitors induce a mitophagy-associated endothelial cytotoxicity that is reversed by coenzyme Q10 cotreatment.

Cardiovascular complications have been documented in HIV-1 infected populations, and antiretroviral therapy may play a role. Nucleoside reverse transcriptase inhibitors (NRTIs) are antiretrovirals known to induce mitochondrial damage in endothelial cells, culminating in endothelial dysfunction, an initiating event in atherogenesis. Though the mechanism for NRTI-induced endothelial toxicity is not yet clear, our prior work suggested that a mitochondrial oxidative stress may be involved. To further delineate the mechanism of toxicity, endothelial cells were treated with NRTIs of varying subclasses, and the level of reactive oxygen species (ROS) and mitochondrial function were assessed. To test whether rescue of mitochondrial electron transport attenuated NRTI-induced endothelial cytotoxicity, in some cases, cells were cotreated with the electron transport cofactor coenzyme Q10 (Q10). At 4-6h, NRTIs increased levels of ROS but decreased the activities of electron transport chain complexes I-IV, levels of ATP and the NAD/NADH ratio. Moreover, nitric oxide levels were decreased, whereas endothelin-1 release was increased. Q10 abolished NRTI-induced mitochondria injury and effects on endothelial agonist production. Interestingly, in cells treated with NRTIs only, markers for mitochondrial toxicity returned to baseline levels by 18-24h, suggesting a compensatory mechanism for clearing damaged mitochondria. Using confocal microscopy, with confirmation utilizing the autophagy and mitophagy markers LC-3 and Nix, respectively, we observed autophagy of mitochondria at 8-10h after treatment. Q10 prevented NRTI-mediated increase in LC-3. These findings suggest that NRTI-induced mitophagy may be involved in NRTI-induced endothelial dysfunction and that this damage likely results from oxidant injury. Further, Q10 supplementation could potentially prevent NRTI-induced endothelial dysfunction.