Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells

Compounds

Bongkrekic acid (BKA; #B6179), carboxyatractyloside potassium salt (CATR; #C4992), antimycin A (# A8674), oligomycin (#O4876), rotenone (#R8875) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; #C2920) were all purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Suramin sodium (#SC-200833) was from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and CD437 (#HY-100532) was obtained from MedChem Express (Monmouth Junction, NJ, USA). [14C]-ADP (1 µCi, #NEC559050UC) was purchased from Perkin Elmer (Waltham, MA, USA).

Cell culture

Human conditionally immortalized proximal tubule epithelial cells expressing organic anion transporter 1 (ciPTEC-OAT1, RRID:CVCL_LI01) were obtained and cultured as previously described (Nieskens et al. 2016; Wilmer et al. 2010). Proliferating cells were cultured at 33 °C and 5% (v/v) CO2 in 1:1 (v/v) Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 with 17.5 mM glucose, 0.5 mM sodium pyruvate, 2.5 mM glutamine and without phenol red (DMEM Ham’s F-12, Life Technologies, Paisley, UK), supplemented with 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 36 ng/mL hydrocortisone, 10 ng/mL human epidermal growth factor (EGF), 40 pg/mL trio-iodothyrine (all purchased from Sigma-Aldrich) and 10% (v/v) fetal bovine serum (FBS, Greiner Bio-One, Alpen a/d Rijn, The Netherlands), referred to as PTEC complete medium, which was refreshed every 2–3 days. Experimental cells proliferated for one day at 33 °C and 5% (v/v) CO2, followed by maturation at 37 °C and 5% (v/v) CO2 in PTEC complete medium for seven days to differentiate into an epithelial monolayer. Cells in experiment varied in passage numbers from 56 to 78.

Single guide RNA design

Single guide RNAs (sgRNAs) targeting exon 1 and 2 of the Homo sapiens gene AAC3 (SLC25A6) were designed for clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) using CHOPCHOP and crispor.tefor.net (hg38/GRCh38 was used as reference genome). sgRNAs were selected based on predicted efficiency score and absence of off-targets with > 3 mismatches (Table 1). The oligonucleotide pair for each sgRNA was phosphorylated and annealed using T4 Polynucleotide Kinase (EK0031, Thermofisher Scientific) in a thermocycler at 37 °C for 30 min, followed by 5 min at 95 °C and 5 °C/min decrease to 25 °C, according to the manufacturer’s instructions. Plasmid pX333-GFP (kindly provided by Eric Verschuren, Dept. of Physiology, Radboudumc, Addgene, #64,073) was linearized at 37 °C for 1 h using BbsI (R0539, New England Biolabs, Ipswich, MA, USA), or BsaI (R0535, New England Biolabs) restriction enzymes. Ligation of annealed oligonucleotides into the linearized pX333-GFP plasmid was performed using T4 ligase (New England Biolabs) overnight at 16 °C, following instructions by the manufacturer. Heat shock at 42 °C transformed the ligation mix into TOP10F competent cells. The next day, colonies with pX333 containing sgRNAs were inoculated and plasmids were isolated using GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich) according to manufacturer’s instructions. As the plasmid enables dual expression of sgRNAs, sequencing resulted in selection of plasmids containing both sgRNAs (forward primer exon 1/2 sgRNA, see Table 1). Insertion of T2A-eGFP after NLS-Cas9-NLS, allowed GFP-positive FACS sorting after transfection. Prior to transfection ciPTEC-OAT1 exons 1–4 were sequenced to confirm wild-type sequences (Table 1).

Table 1 Primers for CRISPR/Cas9 gene editingCRISPR/Cas9-mediated genome editing

750,000 ciPTEC-OAT1 cells/well were seeded into a 6-wells plate and proliferated for 24 h at 33 °C and 5% v/v CO2. Cells were transfected with 2.5 μg of the isolated plasmid containing the sgRNAs using Mirus TransIT-X2 or LT1 reagent (MirusBio, Madison, WI, USA, 1:2 plasmid:reagent ratio (v/v)) in serum-free pTEC complete medium according to the manufacturer’s instructions. GFP-positive cells were single cell sorted into 96-wells plates by fluorescence-activated cell sorting (FACS, Aria Flow cytometer, BD Biosciences, San Jose, CA, USA) 40 h post-transfection and single cell clones were grown at 33 °C and 5% v/v CO2 in pTEC CM. Upon ~ 90% confluency, genomic DNA was isolated using prepGEM™ Universal DNA extraction kit (MicroGEM, Aotearoa, New Zealand) according to manufacturer’s protocol. Genome editing efficiency was assessed by T7 endonuclease mismatch detection assay, as described by the manufacturer. As inefficient targeting of exon 2 by sgRNA occurred, this region was not considered further. The targeted region in AAC3 exon 1 was amplified using primer pairs (Table 1) and phire hot start II DNA polymerase (F-122S, Thermofisher Scientific) in a thermocycler (98 °C for 1 min and 40 cycles of 98 °C for 5 s, 68 °C for 5 s, and 72 °C for 15 s followed by a final elongation step of 72 °C for 1 min). PCR products were ExoSAP-IT (78,200, Applied Biosystems, Bleijswijk, The Netherlands), Sanger sequenced (primers see Table 1) and analyzed using SnapGene® Viewer (version 4.3.10, GSL Biotech LLC, San Diego, CA, USA) or Chromas Lite (version 2.1.1, Technelysium Pty Ltd, Australia). Both AAC3−/− clones used in this study were generated independently.

Real-time quantitative PCR

mRNA was isolated from matured ciPTEC-OAT1 cells using the RNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using Molony-Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen, 28025013), as described in the manufacturer’s protocol. Gene expression levels of solute carrier (SLC) family members SLC25A4 (ADP/ATP carrier 1: AAC1), SLC25A5 (AAC2), SLC25A6 (AAC3) and SLC25A31 (AAC4) were measured by real-time quantitative PCR (RT-qPCR) using a CFX96-Touch Real-Time PCR system (BioRad Laboratories, Veenendaal, The Netherlands) and analyzed using BioRad CFX Manager (version 3.1). GAPDH levels were used as reference. Relative gene expression was determined by GAPDH subtraction, followed by calculation of 2−ΔCt. TaqMan Universal PCR Master Mix (Life Technologies, 4304437) and gene specific primer–probe sets (SLC25A4: Hs00154037_m1, SLC25A5: Hs00854499_g1, SLC25A6: Hs00745067_s1, SLC25A31: Hs00935856_m1 and GAPDH: Hs99999905_m1) were purchased from Applied Biosystems (Thermofisher Scientific). Non-template samples served as control.

Western blot

To identify mitochondrial protein abundance of AAC1, AAC2 and AAC3 in ciPTEC-OAT1 wild type and AAC3−/−, Western blotting was performed. AAC4 was excluded from further analysis due to tissue-specific expression in the testis and absence of mRNA expression levels in our cell model. Cells were seeded at 63,000 cells/cm2 in T175 cell culture flasks and matured as described before. Mature cells were pelleted and lysed in RIPA buffer (50 mM Tris–HCl, 50 mM NaCl, 1% Triton X-100, 5 mM Na2-EDTA, 10 mM Na4P2O7.10H2O, 50 mM NaF, protease inhibitor and phosphatase inhibitor, pH 7.4 and 100 µg/ml DNase) on ice for 30 min, followed by centrifugation for 10 min at 4 °C and 6000 g. Protein was determined using the Protein Assay Dye Reagent Concentrate (500–0006, BioRad) according to the manufacturer’s instructions. Samples were incubated for 5 min at 95 °C in Laemmli sample buffer, after which 30 μg protein was loaded on a 4–15% (w/v) Mini-PROTEAN® TGX stain-free pre-cast SDS-PAGE gel (456-8024S, BioRad) for electrophoresis. Samples ran for 30 min at 50 V, followed by 45 min at 150 V and were transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 μm) using the Trans-Blot® Turbo™ Transfer system (1704275, RTA transfer kit, BioRad) according to the manufacturer’s instructions. After transfer, the blot was blocked in Odyssey blocking buffer (927-40000, LI-COR Biosciences), and incubated overnight in Odyssey blocking buffer containing 0.1% (v/v) TWEEN and primary antibodies against AAC1 (ab102032, 1:500 (v/v) Abcam, Cambridge, UK), AAC2 (ab118125, 1:1000 (v/v), Abcam), AAC3 (ab154007, 1:2000 (v/v), Abcam) or COX5A (ab110262, 1:1000 (v/v), Abcam), as loading control at 4 °C, followed by incubation with corresponding secondary antibodies Alexa Fluor® 800 goat-anti-rabbit (926-32211, 1:10,000, LI-COR Biosciences) or Alexa Fluor® 680 goat-anti-mouse (926-68070 1:10,000, LI-COR Bioscience) in Odyssey blocking buffer containing 0.1% (v/v) TWEEN at room temperature for 1 h. Fluorescence was visualized using the Odyssey CLx scanner (Li-Cor Biosciences, USA).

Functional ADP and ATP transport assessment

Cells were cultured and matured as described above. Mature cells were pelleted and resuspended in ice cold 10 mM Tris–HCl, pH 7.6, for isolation of mitochondrial enriched fractions. Cell suspension was mechanically homogenized (eight times) using a Potter–Elvehjem homogenizer on ice. To increase protein yield, a maximum of 3 million cells was homogenized at once. The homogenate was transferred to ice cold 1.5 M sucrose solution, mixed and centrifuged for 10 min at 2 °C and 600g. To obtain mitochondrial-enriched protein fractions, supernatant was spun down for 10 min at 2 °C and 14,000g, followed by resuspension of pellet in 10 mM Tris–HCl, pH 7.6 complemented with cOmplete™ Mini EDTA-free protease inhibitor, PhosSTOP™ and 0.1 mg/mL RNase-free DNase I (Roche, Basel, Switzerland). Mitochondrial protein in the isolated fraction was determined after three freeze/thaw cycles in liquid nitrogen, to break mitochondrial membranes, using Pierce™ BCA protein assay kit (Thermofisher Scientific) according to the manufacturer’s instructions. In order to evaluate AAC-dependent ADP import, 25 µg intact isolated mitochondria of wild type and AAC3−/− ciPTEC were incubated in ADP import buffer, consisting of 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 5 mM succinate, 3 mM KH2PO4, 1.5 mM MgCl2, 1 mM EGTA and 5 µM rotenone, pH 7.2, supplemented with 100 µM AAC inhibitor bongkrekic acid (BKA; B6179, Sigma-Aldrich), or MilliQ on ice in a multiscreen 96-wells HV filter plate (0.45 µM, MSHVN4B, Merck SKU). After 10 min, 1 µM [14C]-ADP (~ 1 µCi; # NEC559050UC, Perkin Elmer) was added for 15 min on ice, after which samples were vacuum aspirated and washed three times in ADP import buffer. Samples were resuspended in scintillation solvent (Perkin Elmer) and [14C]-ADP counts were quantified (Hidex 600SL, Turku, Finland). AAC-dependent ATP transport was measured by means of bioluminescence, using the ATP bioluminescence assay kit CLS II (#11699695001, Roche), according to the manufacturer’s instructions. In short, 25 µg intact isolated mitochondria were incubated with 100 µM BKA or MilliQ, for 10 min on ice. For the first measurement, ADP reaction buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 3 mM KH2PO4, 1.5 mM MgCl2, 1 mM EGTA, 1 mM malate and 1 mM pyruvate, pH 7.6 and kept at 28 °C) was added to a white/clear bottom 96-wells plate with 100 µM BKA or MilliQ and luciferase reagent (1:1, v/v). For the second measurement, pre-incubated mitochondria or MilliQ was added, followed by addition of 25 µM ADP in a third measurement. Bioluminescence signal was immediately measured for 0.1 s after each addition and 0.5 s shaking, on a Victor X3 multiplate reader (Perkin Elmer). Conditions without mitochondria, ADP or BKA served as control. All samples were analyzed at least in triplicate in three independent experiments.

Measuring cellular metabolic activity

Effects of compounds on cellular metabolic activity were evaluated using the colorimetric tetrazolium salt-based MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Mosmann 1983). ciPTEC-OAT1 were seeded at a density of 63,000 cells/cm2 in transparent/clear flat bottom 96-wells plates, proliferated for 1 day at 33 °C and 5% (v/v) CO2 in PTEC complete medium, followed by 7 days maturation at 37 °C, 5% (v/v) CO2 to differentiate into an epithelial monolayer. Matured ciPTEC-OAT1 were exposed to known AAC inhibitors BKA, CATR, CD437 and suramin for 24 h at 37 °C and 5% (v/v) CO2. Relative drug concentrations applied were 0.01, 0.0316, 0.1, 0.316, 10, 31.6, 100, 316 and 1000 μM (dissolved in MilliQ or DMSO). Subsequently, cellular metabolic activity was assessed. In short, compound exposed cells were washed three times with serum-free PTEC complete medium (SFM) and incubated with 0.5 mg/mL MTT in SFM for 3 h at 37 °C and 5% (v/v) CO2. After dissolving formazan crystals in DMSO for 2 h on a microplate shaker, absorption was measured at 560 nm and subtracted from background at 670 nm using Benchmark (Bio-Rad, Veenendaal, The Netherlands). DMSO concentrations did not exceed 0.1% (v/v). Obtained results were normalized to vehicle (0.1% DMSO) exposed ciPTEC-OAT1.

Cellular respiration and real-time ATP production rate measurements

To investigate cellular metabolism in both genetically and chemically inhibited cells, cellular oxygen consumption rates were measured using extracellular flux analyses (Seahorse XF96 Agilent, Santa Clara, CA, USA). 10,000 cells/well were seeded in 96-wells Seahorse XF96 cell culture microplates and matured as described above. Routinely culturing cells in high glucose conditions can inhibit mitochondrial function, known as the Crabtree effect, previously described for rapidly proliferating cancer cells (e.g., HepG2), thymocytes and human primary muscle cells (Aguer et al. 2011; Marroquin et al. 2007; Rossignol et al. 2004). Although a predominant oxidative phenotype for ciPTEC was previously suspected (Vriend et al. 2019), we attempted to stimulate mitochondrial respiration by replacing the glucose in the medium with galactose. While the glycolytic metabolism of glucose yields two net ATP molecules, pyruvate production via metabolism of galactose yields no ATP, and consequently increases reliance on oxidative phosphorylation for energy production (Marroquin et al. 2007). The effects of AAC interactors on mitochondrial respiration were evaluated in glucose-free DMEM (Sigma, D5030) complemented with 10 mM galactose, 0.64 mM MgCl2, 14.3 mM NaHCO3, 15 mM HEPES, 0.5 mM sodium pyruvate, and 2.5 mM glutamine, supplemented with ciPTEC growth factors mentioned above, pH 7.4 and 100 µM AAC inhibitors BKA, CATR, CD437, suramin or 0.1% DMSO for 12 h at 37 °C and 5% (v/v) CO2. Galactose-rich (10 mM) medium containing AAC inhibitors was replaced one hour before initiation of the experiment by NaHCO3-free DMEM containing 10 mM galactose, 0.5 mM sodium pyruvate, 31.7 mM NaCl, 5 mM HEPES and 2.5 mM glutamine, pH 7.4 and incubated at 37 °C without CO2. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured following subsequent addition of 2.5 µM oligomycin (Oli), 1 µM FCCP and 1 µM/2.5 µM rotenone (Rot)/antimycin A (AA). The effect of each addition was measured during 3 measurement cycles (each consisting of 3 min mixing and 3 min recording). For evaluation of ATP production rates, the buffer factor of the 10 mM galactose DMEM medium containing ciPTEC growth factors was empirically determined by titrating buffer pH with 5 mM HCl steps according to the manufacturer’s protocol. Non-mitochondrial respiration (after addition of Rot/AA) was subtracted from all OCR values. Basal (routine) OCR was determined in the absence of inhibitors at T = 14 min (point 3), while T = 54 min (i.e. in the presence of Oli and FCCP) was used as a measure of maximal respiration (point 9). Spare respiratory capacity was determined by subtracting routine OCR values from the maximal OCR values. Average data was obtained in N = 4 experiments (biological replicates), each consisting of four experimental replicates. Wave Desktop Software (Agilent) was used for data analysis. Measured OCR and ECAR rates were corrected for cell count. To this end, nuclei were stained with 20 µg/mL Hoechst 33,342 (Life Technologies) for 30 min at 37 °C, after which fluorescence was imaged using a 10 × objective Becton Dickinson (BD) Pathway 855 microscope (BD Bioscience, Breda, The Netherlands) using emission/excitation maxima of 380/435 nm.

Citrate synthase activity

Mitochondrial mass was determined by citrate synthase activity in matured ciPTEC-OAT1 wild type and AAC3−/−, as previously described (Srere 1969). In short, cells were seeded and matured in transparent/clear flat bottom 96-wells plates as described above. Cells were lysed by 0.33% (v/v) Triton X-100 in 10 mM Tris–HCl, pH 7.6, followed by three freeze–thaw cycles in liquid nitrogen. Absorption was measured spectrophotometrically at 412 nm after addition of five volumes of 1 mM 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, D8130, Sigma) with 10% (v/v) Triton X-100 in MilliQ, and 300 µM acetyl coenzyme A (A2181, Sigma), in a 25 °C prewarmed microplate reader (BioRad) using a kinetic protocol of medium mixing for 10 s with 20 s interval, followed by twenty cycles of 15 s interval without mixing. Total activity was measured after addition of 10 µL 10 mM oxaloacetic acid (A4126, Sigma) using the same kinetic protocol. Data analysis was performed in GraphPad Prism 9.0.0. For each measurement, slopes of at least five time points were defined, subtracted, and plotted.

Mitochondrial morphology and function by TMRM

Cells were seeded and matured as described above in black/clear bottom flat 96-wells plates, after which mitochondrial morphology and membrane potential were assessed microscopically. Cells were washed once in HBSS, subsequently fluorescently stained with 25 nM tetramethylrhodamine methyl ester (TMRM) and co-stained with 0.75 µg/mL Hoechst 33342 in HBSS for 30 min at 37 °C, followed by image acquisition (40 × objective) and 12 × 12 montage using Becton Dickinson (BD) Pathway 855 microscope (BD Bioscience, Breda, The Netherlands), as described before (Iannetti et al. 2016). Acquired images were processed and quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA), as described previously (Iannetti et al. 2016). Average data was obtained in N = 3 experiments, consisting of 20 replicates each, and 8 cells/well were analyzed.

Statistical analysis

Statistical data analysis was performed using GraphPad Prism 9.0.0 (GraphPad Software Inc., San Diego, CA). All data were normalized to wild type ciPTEC-OAT1 and presented as mean ± SEM of at least three independent experiments (N = 3), performed with five or six experimental replicates as indicated in the figure legends, unless stated otherwise. One-way ANOVA with Dunnett’s post hoc test or two-way ANOVA with Bonferroni post hoc test to correct for multiple comparison were used to evaluate differences between cell lines or responses, as indicated. Concentration-dependent effect of AAC inhibitors on cellular metabolic activity were fitted using nonlinear regression analysis.

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