Elsevier

Analytical Biochemistry

Volume 404, Issue 1, 1 September 2010, Pages 75-81
Analytical Biochemistry

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

https://doi.org/10.1016/j.ab.2010.04.040Get rights and content

Abstract

Mitochondria are a common target of toxicity for drugs and other chemicals and result in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality, and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in multiwell plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl2, and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone)-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTCs. The merger of the RPTC model and multiwell respirometry results in a single high-throughput assay to measure mitochondrial biogenesis and toxicity and nephrotoxic potential.

Section snippets

Materials

Female New Zealand white rabbits (1.5–2.0 kg) were purchased from Myrtle’s Rabbitry (Thompson Station, TN, USA). The basal medium was a 50:50 mixture of Dulbecco’s modified Eagle’s essential medium and Ham’s F12 nutrient mix without phenol red supplemented with 15 mM NaHCO3, 0.2 mM glycine, and 6 mM sodium lactate. The medium was adjusted to pH 7.4 while gassing with 95% O2/5% CO2 and was diluted to 295 mosmol/kg H2O before filter sterilization. The isolation medium was the basal medium supplemented

Results

RPTCs cultured under standard conditions (stationary with 17 mM glucose) had basal OCRs that were more than 100-fold lower than those for RPTCs cultured under optimized conditions (shaking with lactate and no glucose) (Fig. 1A). Inhibition of the F1F0–ATPase with oligomycin is a measure of the fraction of the OCR that is coupled to ATP production. In “healthy” cells, in tissues, and in humans, the oligomycin-induced decrease in the OCR is typically approximately 70% of basal [14], [15]. The RPTC

Discussion

Scientists in the pharmaceutical industry are highly cognizant of the potential for adverse drug effects due to agents with mitochondrial liabilities. Indeed, roughly half of the drugs with US Food and Drug Administration “black box” warnings for cardiotoxicity or hepatotoxicity in 2007 had documented mitochondrial liabilities [25]. Unfortunately, nephrotoxicity has not received the same level of attention despite the frequency of loss in renal function due to adverse drug effects and

Acknowledgments

This study was supported by the National Institutes of Health/National Institute of General Medical Sciences (GM 084147). Nathan Perron provided the IN Cell images.

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