A high-throughput respirometric assay for mitochondrial biogenesis and toxicity
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.
References (28)
- et al.
Signaling of mitochondrial biogenesis following oxidant injury
J. Biol. Chem.
(2007) - et al.
PGC-1α over-expression promotes recovery from mitochondrial dysfunction and cell injury
Biochem. Biophys. Res. Commun.
(2007) - et al.
Understanding mechanisms of toxicity: insights from drug discovery research
Toxicol. Appl. Pharmacol.
(2008) - et al.
Advances in measuring cellular bioenergetics using extracellular flux
Drug Discov. Today
(2008) - et al.
Measuring mitochondrial bioenergetics in INS-1E insulinoma cells
Methods Enzymol.
(2009) - et al.
Validation of putative genomic biomarkers of nephrotoxicity in rats
Toxicology
(2008) Measurement of oxygen consumption
- et al.
The significance of mitochondrial toxicity testing in drug development
Drug Discov. Today
(2007) Biotransformation and renal processing of nephrotoxic agents
Arch. Toxicol. Suppl.
(1996)- et al.
Nephrotoxicity testing in vitro: what we know and what we need to know
Environ. Health Perspect.
(1998)
The not so “mighty chondrion”: emergence of renal diseases due to mitochondrial dysfunction
Nephron Physiol.
Isoflavones promote mitochondrial biogenesis
J. Pharmacol. Exp. Ther.
SRT1720 induces mitochondrial biogenesis and rescues mitochondrial function after oxidant injury in renal proximal tubule cells
J. Pharmacol. Exp. Ther.
Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells
Am. J. Physiol.
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