RPTC cultured under standard conditions (stationary with 17 mM glucose), had basal OCR that were > 100-fold lower than for RPTC cultured under optimized conditions (shaking, with lactate and no glucose) (). Inhibition of the F1
-ATPase with oligomycin is a measure of the fraction of the OCR that is coupled to ATP production. In “healthy” cells, in tissue, and in humans, the oligomycin-induced decrease in OCR is typically about 70% of basal [14
]. RPTC OCR decreased by approximately 30% in standard conditions and by approximately 80% in optimized conditions following 1 μM oligomycin addition (). As a further test of their differentiation and respiratory function, the RPTC were treated with ouabain, an inhibitor of the plasma membrane Na+
-ATPase, to determine the fraction of OCR that is coupled to maintenance of plasma membrane potential. Ouabain decreased OCR approximately 60% in RPTC cultured in optimized conditions while it had a minimal effect in RPTC cultured under standard conditions (). Approximately 60% of the respiration is typically inhibited by ouabain in these cells, and in isolated tubules [16
Assessment of RPTC mitochondrial metabolism using the Seahorse Biosciences XF instrument
Treatment of RPTC with FCCP uncouples the mitochondrial membrane potential to cause an increase in the OCR. For isolated mitochondria, the increase in OCR relative to the oligomycin treated rate is often referred to as the maximum oxidative phosphorylation capacity and it is an approximate measure of the Vmax
for the electron transport chain [14
]. Preliminary experiments determined that 5 μM FCCP produced the maximal OCR in RPTC cultured under optimized conditions. The uncoupled rates were not increased with triple the lactate concentration (18 mM), added glucose (5.5 mM), or added palmitate (0.25 mM, BSA carrier). As shown in , FCCP produced an approximately 10-fold increase in OCR in RPTC cultured under optimized conditions compared to oligomycin-OCR. The increase in OCR produced by FCCP in RPTC cultured under standard conditions was minimal.
To assess reproducibility, the experiment shown in was repeated for multiple primary cell preparations. The basal rates were measured for 90 s followed by a 2 min mix period, a 2 min wait period, and the cycle was repeated four times before the injection of a pharmacological agent. For any given plate, the well-to-well variation over 20 wells at the 4th basal rate measurement was typically 12-15% and the standard deviation for the average of the last three basal rates for any one well was about 7%. The greatest source or variance was found to be among different RPTC preparations as would be anticipated for primary cell cultures.
To evaluate preparation-dependent variances and to compare respiration rates to historical values, RPTC in each well were lysed and the protein concentration was measured with the bicinchoninic acid assay. The mean ± standard deviation of the basal and uncoupled OCR among 15 preparations were 14.4 + 3.5 and 34.2 ± 7.2, respectively, and the protein-normalized rates are comparable to previously reported values [9
To examine the ability of the XF-24 instrument to measure RPTC mitochondrial dysfunction, RPTC were cultured under optimized conditions and treated with nephrotoxicants or vehicle control (0.1% DMSO) for 24 h prior to assessment of OCR. After measuring basal rates, the cells were treated with FCCP to uncouple the mitochondria. To account for variations in OCR rates from different preparations, the basal rates for treated wells and the uncoupled rates (in nmol/min mg protein) were normalized to the basal rates of vehicle control wells. Separate experiments confirmed that 24 h pretreatment with 0.1% DMSO vehicle had no measurable effect on basal or uncoupled rates. It was found that the uncoupled rates, but not basal rates showed concentration-dependent decreases in RPTC treated with cisplatin, gentamicin, and HgCl2
(), nephrotoxicants known to cause mitochondrial damage [18
]. The uncoupled were rates decreased by as much as 40% with no significant decrease in basal. The FCCP uncoupled rates can be used as a stress response to uncover disrupted electron transport chain activity by toxicants, even though basal metabolism is not impaired [21
]. As a control, RPTC were treated with mitomycin C, an antineoplastic agent that does not cause measurable tubular damage in rats [20
]. RPTC exposed to 10 μM mitomycin C did not exhibit changes in either basal or uncoupled OCR (data not shown).
Assessment of toxicity in RPTC using the Seahorse Biosciences XF instrument
The viability of the toxicant-treated RPTC were assessed via plasma membrane integrity as measured from automated imaging of cells stained with propidium iodide to measure loss of plasma membrane integrity. Staining with Hoechst 33342 was used to identify all cell nuclei and pink nuclei were counted as dead and divided by all nuclei. The proportion of live cells was ~95% for untreated and vehicle control cells, and the cells treated with the nephrotoxicants, mitomycin C, and metformin also exhibited ~95% live cells. The integrity of the treated cells was also evident from the intact morphology of the monolayer as seen in the corresponding bright field images (). Thus, the XF-24 assay provides a sensitive measure of nephrotoxicant damage of mitochondrial respiratory capacity that is evident before impairment of basal metabolism or cell death.
Viability of RPTC 24 h post-treatment
We recently demonstrated that several classes of compounds produce mitochondrial biogenesis in RPTC and that FCCP-uncoupled OCR is a marker of mitochondrial biogenesis [5
]. To determine the ability of the XF-24 instrument to measure mitochondrial biogenesis, RPTC were cultured under optimized conditions, treated with agents known to produce mitochondrial biogenesis for 24 hr and basal and FCCP-uncoupled OCR determined. The AMP-kinase activator aminoimidazole carboxamide ribonucleotide (AICAR) has been reported to induce mitochondrial biogenesis [22
] and increased FCCP OCR at 300 and 500 μM and basal OCR increased at 500 μM with a maximal increase of approximately 40% (). Metformin, a biguanide anti-diabetic drug, has also been reported to induce mitochondrial biogenesis [22
] and it also increased basal and uncoupled rates to a similar extent as AICAR. The piperazine-thiazole SRT1720 was originally reported as a SIRT-1 activator and has been shown to induce mitochondrial biogenesis by Milne, et al.
] and by our laboratory [7
]. Treatment with SRT1720 also revealed concentration-dependent increases in basal and uncoupled rates with maximums of ~50%. The 5-hydroxytryptamine (5-HT) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) has been extensively studied in the primary RPTC where it has been shown to induce mitochondrial biogenesis via activation of PGC-1α [24
]. In the respirometric assay, treatment with 10 and 20 μM DOI produced up to 40% increases in both basal and uncoupled OCR. Similarly, treatment of RPTC with resveratrol or the isoflavone daidzein (10 μM), characterized previously as biogenesis agents in RPTC [6
], also increased basal and uncoupled respiration rates. These data confirm that the XF measurement of RPTC respiration is a sensitive, functional assay of mitochondrial biogenesis.
Respirometric measurement of mitochondrial biogenesis