Diglycolic acid, the toxic metabolite of diethylene glycol, chelates calcium and produces renal mitochondrial dysfunction in vitro

Abstract

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. Materials and methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5 mmol/L) than succinate respiration (100 mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.

Keywords:

• Calcium chelation
• diethylene glycol
• diglycolic acid
• mitochondrial toxicity

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Acknowledgements

The authors would like to thank Corie Robinson, Cody Dunning and the other members of the McMartin and Aw Labs for their assistance in the completion of this project.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Funding information

This work was supported by the Ethylene Glycol/Ethylene Oxide Panel of the American Chemistry Council and by the National Institutes of Health grant to Dr. Aw [DK-44510]. Financial support for Mr. Conrad was provided by the Louisiana State University Health Sciences Center-Shreveport Medical Student Research Program and the Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship.