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. 2010 Sep 1;404(1):75-81.
doi: 10.1016/j.ab.2010.04.040. Epub 2010 May 11.

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Craig C Beeson  1 , Gyda C Beeson, Rick G Schnellmann
Affiliations

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Craig C Beeson et al. Anal Biochem. .

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, HgCl(2), 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.

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Figures

Fig. 1
Fig. 1. Assessment of RPTC mitochondrial metabolism using the Seahorse Biosciences XF instrument
Primary rabbit RPTC were plated and differentiated using either the traditional glucose containing media with no shaking (standard) or with optimized media with lactate and shaking and their basal OCR rates and OCR rates in the presence of oligomycin (5 μM) followed by FCCP 1 μM) (panel A), or with ouabain (0.1 mM) (panel B). Data are means ± standard deviations for 4-5 wells measured in a single experiment.
Fig. 1
Fig. 1. Assessment of RPTC mitochondrial metabolism using the Seahorse Biosciences XF instrument
Primary rabbit RPTC were plated and differentiated using either the traditional glucose containing media with no shaking (standard) or with optimized media with lactate and shaking and their basal OCR rates and OCR rates in the presence of oligomycin (5 μM) followed by FCCP 1 μM) (panel A), or with ouabain (0.1 mM) (panel B). Data are means ± standard deviations for 4-5 wells measured in a single experiment.
Fig. 2
Fig. 2. Assessment of toxicity in RPTC using the Seahorse Biosciences XF instrument
Shown are basal and uncoupled (1 μM FCCP) OCR measured for RPTC treated for 24 h with cisplatin, gentamicin, and HgCl2. The basal rates for treated wells, and the uncoupled rates for all wells, were normalized to the basal rates of vehicle control wells (Control). Bars are the mean ± SEM for n = 6 and the asterisk represent statistical difference from control basal or FCCP uncoupled at p<0.05.
Fig. 2
Fig. 2. Assessment of toxicity in RPTC using the Seahorse Biosciences XF instrument
Shown are basal and uncoupled (1 μM FCCP) OCR measured for RPTC treated for 24 h with cisplatin, gentamicin, and HgCl2. The basal rates for treated wells, and the uncoupled rates for all wells, were normalized to the basal rates of vehicle control wells (Control). Bars are the mean ± SEM for n = 6 and the asterisk represent statistical difference from control basal or FCCP uncoupled at p<0.05.
Fig. 2
Fig. 2. Assessment of toxicity in RPTC using the Seahorse Biosciences XF instrument
Shown are basal and uncoupled (1 μM FCCP) OCR measured for RPTC treated for 24 h with cisplatin, gentamicin, and HgCl2. The basal rates for treated wells, and the uncoupled rates for all wells, were normalized to the basal rates of vehicle control wells (Control). Bars are the mean ± SEM for n = 6 and the asterisk represent statistical difference from control basal or FCCP uncoupled at p<0.05.
Fig. 3
Fig. 3. Viability of RPTC 24 h post-treatment
Cells treated with toxicants for 24 h were analyzed on the XF instrument, washed with PBS, and then stained with ethidium bromide and acridine orange and visualized via epifluorescence microscopy. Shown are the representative images for RPTC treated with (A) vehicle control, (B) 10 μM gentamicin, (C) 10 μM cisplatin, (D) 3 μM HgCl2.
Fig. 3
Fig. 3. Viability of RPTC 24 h post-treatment
Cells treated with toxicants for 24 h were analyzed on the XF instrument, washed with PBS, and then stained with ethidium bromide and acridine orange and visualized via epifluorescence microscopy. Shown are the representative images for RPTC treated with (A) vehicle control, (B) 10 μM gentamicin, (C) 10 μM cisplatin, (D) 3 μM HgCl2.
Fig. 3
Fig. 3. Viability of RPTC 24 h post-treatment
Cells treated with toxicants for 24 h were analyzed on the XF instrument, washed with PBS, and then stained with ethidium bromide and acridine orange and visualized via epifluorescence microscopy. Shown are the representative images for RPTC treated with (A) vehicle control, (B) 10 μM gentamicin, (C) 10 μM cisplatin, (D) 3 μM HgCl2.
Fig. 3
Fig. 3. Viability of RPTC 24 h post-treatment
Cells treated with toxicants for 24 h were analyzed on the XF instrument, washed with PBS, and then stained with ethidium bromide and acridine orange and visualized via epifluorescence microscopy. Shown are the representative images for RPTC treated with (A) vehicle control, (B) 10 μM gentamicin, (C) 10 μM cisplatin, (D) 3 μM HgCl2.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
Fig. 4
Fig. 4. Respirometric measurement of mitochondrial biogenesis
Shown are the basal and uncoupled (1 μM FCCP) OCR for primary rabbit RPTC treated with agents known to induce mitochondrial biogenesis. The OCR rates in nmol/min mg protein for treated cells were normalized to the rates of the basal, untreated wells for the given experiment. Bars are the mean ± SEM for n = 6 and the asterisk represents statistically significant difference from control basal or FCCP uncoupled at p<0.05.
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