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Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids Selected Based on OAT1 Gene Expression: A Potential In Vitro Tool for Evaluating Human Renal Proximal Tubular Toxicity and Drug Disposition

Naoki Ishiguro, Etsushi Takahashi, Hiroshi Arakawa, Asami Saito, Fumihiko Kitagawa, Masayuki Kondo, Gaku Morinaga, Masahito Takatani, Ryo Takahashi, Takashi Kudo, Shin-ichi Mae, Moeno Kadoguchi, Daichi Higuchi, Yuya Nakazono, Ikumi Tamai, Kenji Osafune and Yoichi Jimbo
Drug Metabolism and Disposition September 2023, 51 (9) 1177-1187; DOI: https://doi.org/10.1124/dmd.122.001171

Abstract

The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition.

SIGNIFICANCE STATEMENT This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.

Introduction

The proximal tubule of the nephron—the primary site for the reabsorption and secretion of endogenous and exogenous substances—has a biotransformation capacity. Numerous enzymes are involved in the metabolism and clearance of endogenous and exogenous compounds in the kidney, including cytochrome P450 (CYP) and non-CYP enzymes, such as uridine-diphosphate-glucuronosyltransferases, esterases, glutathione-S-transferases, and sulfotransferases (Bajaj et al., 2018). Transporters expressed on the basal membrane transfer xenobiotics from the systemic circulation to proximal tubular cells. Subsequently xenobiotics are secreted into urine via transporters expressed on the brush border membrane of the proximal tubular cells or vice versa to be reabsorbed from the filtrate.

Recently, the roles of tubular apical and basolateral transporters in eliminating and reabsorbing xenobiotics and endogenous substrates were elucidated (Morrissey et al., 2013; Nigam et al., 2015; Miners et al., 2017). Examples of endogenous transporters include urate transporter 1 (URAT1; SLC22A12) and glucose transporter 9 (GLUT9; SLC2A9) as apical and basolateral urate transporters, respectively, SLC34A1/3 as an apical phosphate transporter (Segawa et al., 2015), sodium coupled monocarboxylate transporter 1/2 (SMCT1/2; SLC5A8/12) as an apical lactate transporter, sodium-glucose cotransporter 1/2 (SGLT1/2; SLC5A1/2), and GLUT2 (SLC2A2) as apical and basolateral glucose transporters, respectively, organic cation/carnitine transporter 1 (OCTN1; SLC22A4) as an apical ergothioneine transporter, OCTN2 (SLC22A5) as an apical carnitine transporter, and peptide transporter 1 (PEPT1; SLC15A1) as an apical peptide transporter. Regarding the drug transporters in the kidneys, basolateral organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) accept organic anions as substrates. OAT1 and OAT3 have overlapping substrate specificities; however, OAT1 recognizes organic anions with relatively smaller molecular weights than OAT3 (Hasegawa et al., 2002, 2003; Sweet et al., 2003; Deguchi et al., 2004; Eraly et al., 2006; Nozaki et al., 2007). The organic cation transporter 2 (OCT2) on the basolateral side and the multidrug and toxin extrusion 1/2-K (MATE1/2-K) at the brush border of proximal tubular cells play important roles in the renal secretion of organic cations (Hillgren et al., 2013). Regulatory bodies such as the US Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency recommend that pharmaceutical industries evaluate the interactions between candidate drugs and drug transporters such as OAT1, OAT3, OCT2, MATE1, and MATE2-K.

Typically, nephrotoxicity, which is detected only in the late stages of drug development, accounts for 2% of drug attrition in preclinical studies and 19% in phase 3 trials (Jang et al., 2013). These problems are associated with increased risks for patients and subjects enrolled in clinical trials as well as substantial costs for the health care system and the pharmaceutical industry. A major problem is the lack of preclinical models that offer high predictability. The predictability of animal models is compromised by interspecies variability. Drug-drug interactions in the kidney and the effects of the drug-induced tubular injury have been investigated using various systems, such as kidney slices, cell isolation methods, cultures of established kidney cell lines, and human kidney epithelial cell lines (Tiong et al., 2014). Nakanishi et al., (2011) reported that mRNA expression of multiple solute carrier (SLC) transporters in rat proximal tubular cells decreased dramatically when cultured using the two-dimensional (2D) method. Several cell systems, such as LLC-PK1 cell line, maintain the transport function of organic cations; however, only a limited number of cell systems have sufficient OAT transporter functions (Van der Hauwaert et al., 2014). To overcome the low OAT functionality in cell systems, recently Nieskens et al., (2016) developed conditionally immortalized human proximal tubule cells with OAT1 or OAT3 protein expression by transfecting cDNA. However, cell models of RPTEC with sufficient drug transporter functions, including OAT1/3 and OCT2, have not yet been established.

In this study, we hypothesized that the expression of other genes and proteins could be improved along with changing the OAT1 gene expression. To test the hypothesis, we developed a three-dimensional (3D) spheroid culture method for commercial RPTECs by monitoring OAT1 mRNA expression as a selection marker and characterized mRNA and protein expression, transporter functions, and cellular toxicity in 3D RPTEC spheroids; we discuss the usefulness of 3D RPTEC spheroids for toxicity and pharmacokinetic analyses during drug development.

Materials and Methods

Chemicals and Reagents

The RPTECs were purchased from Lonza (Walkersville, MD, USA). Unlabeled cimetidine, digoxin, and furosemide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Metformin hydrochloride, probenecid, and cisplatin were purchased from Fujifilm Wako (Osaka, Japan). Pyrimethamine was purchased from MP Biomedicals (Irvine, CA, USA), and zosuquidar was purchased from Cellagen Technology (San Diego, CA, USA). Adefovir was purchased from Cayman Chemical Company (Ann Arbor, MI). [3H]adefovir was purchased from Moravek Inc. (Brea, CA, USA). All other chemicals and reagents were commercially available and of analytical grade.

Human Renal Cortex

Frozen human renal cortex was purchased from Reprocell Japan (Yokohama, Japan). Information on the tissue donors is presented in Table 1.

View this table:
TABLE 1

Donor information of human kidneys and RPTECs

Cell Culture of RPTEC

RPTEC Maintenance Culture

RPTECs (Lonza, Item No. CC-2553) were used in this study. RPTECs (passage 3) were maintained in renal epithelial cell growth medium (REGM; Lonza, Walkersville, MD, USA) supplemented with 0.5% FBS at 37°C in a humidified atmosphere with 5% CO2. The culture medium was replaced every 2 days until the cells reached confluence.

2D RPTEC

RPTECs (passage 3) were washed with PBS and treated with Accutase (Innovative Cell Technologies, San Diego, CA, USA) for a few minutes at room temperature. Cell suspensions were seeded onto a cell culture plate (six-well plate; Greiner AG) at a cell number of 3 × 105 cells/well. RPTECs (passage 4) were maintained in REGM supplemented with 0.5% FBS and culture until confluence. The culture medium was replaced every 2 days.

3D Culture

RPTECs were washed with PBS and treated with Accutase (Innovative Cell Technologies) for a few minutes at room temperature. The cells were suspended at 1 × 104/mL in REGM. The cell suspension was plated into the wells of a V-bottom ultra-low attachment 96-well plate (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan). In each well, the cell suspension formed spherical cellular aggregates of RPTEC (3D RPTEC; 1,000 cells/well). 3D RPTEC spheroids were maintained at 37°C in a humidified atmosphere with 5% CO2 in REGM, and the medium was replaced every 2 to 3 days.

Gene Expression Analysis

2D RPTECs or 3D RPTEC spheroids were directly lysed with the RLT lysis buffer and total RNA was isolated using an RNeasy Mini Kit (Qiagen Inc., Germantown, MD, USA) according to the manufacturer’s instructions. The total RNA from the biopsy-embedded optimal cutting temperature compound (donor 1) was extracted using the RNeasy Mini Kit from Aproscience (Tokushima, Japan). Total RNA from snap-frozen autopsies (donors 2–5) was obtained from Reprocell. RNA quality was assessed using an Agilent TapeStation to determine the 28S/18S rRNA ratio. The cDNA was synthesized from the total RNA using QuantiTect Whole Transcriptome Kit (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction was performed using TB green Premix EX Taq II (Takara Bio, Shiga, Japan) and primer sets (OAT1: Forward 5′-CTGTATCCCACAATGATCCG-3′, Reverse 5′- GGCAGTCATGCTCACCAG-3′; GAPDH: Forward 5′- TTGACGCTGGGGCTGGCATT-3′, Reverse 5′- GTGCTCTTGCTGGGGCTGGT-3′) on a Thermal Cycler Dice Real-Time System Single (Takara Bio). Expression levels were calculated using the comparative -ΔΔCt method and normalized to GAPDH as an endogenous control in the same sample. For microarray analysis, RNAs with an RNA integrity number > 6.0, were hybridized to the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA) at the Takara Bio according to manufacturer’s protocol. Expression data were analyzed using GeneSpring software (Agilent Technologies, Santa Clara, CA, USA).

Preparation of Total Lysates and Plasma Membrane Fractions

Frozen human renal cortices were washed with ice-cold PBS, then homogenized by BioMasher II (Nippi Inc., Tokyo, Japan) in buffer A (0.15 M KCl, 0.1 M phosphate buffer pH 7.4, and 0.5 mM phenylmethylsulfonyl fluoride) before total lysate preparation. 2D RPTECs, 3D RPTEC spheroids, and HEK293 cells were washed with ice-cold PBS and suspended in buffer A before total lysate preparation. Total lysates were prepared by ultrasonication for a total of 1.5 minutes (10 second × 9 times) using Bioruptor (BM Equipment, Tokyo, Japan). Plasma membrane fractions were further enriched from the total lysate as described previously (Schaefer et al., 2018). Briefly, total lysates were centrifuged at 10,700 × g for 20 minutes at 4°C, and post-nuclear supernatants were collected and sedimented twice at 100,000 × g for 60 minutes at 4°C. The resulting microsomal pellet was suspended in buffer B (0.25 M sucrose, 20 mM Tris-HCl, pH 7.4), layered on top of a 38% (w/v) sucrose solution and ultracentrifuged at 100,000 × g for 40 minutes at 4°C. The turbid interface fraction was recovered, suspended in buffer B, and sedimented at 100,000 × g for 60 minutes at 4°C. The plasma membrane fraction was obtained by suspending the pellet in MPEX PTS reagent B (GL Science, Tokyo, Japan). Protein concentration of total lysate and plasma membrane fraction was determined using the CBQCA Protein Quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA).

Sample Preparation for Tandem Mass Tag-Based Proteomics

Proteins in total lysates and plasma membrane fractions for liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics analysis were prepared using the single-pot solid-phase-enhanced sample preparation method (Sielaff et al., 2017; Hughes et al., 2019) and labeled with the TMTpro reagent (Thermo Fisher Scientific) as previously described (Ohtsu et al., 2022). In brief, 9 μg of samples were dissolved in 100 mM triethylammonium bicarbonate (Thermo Fisher Scientific) and 150 mM NaCl solution with MPEX PTS reagent B (GL Science) and then reduced with tris-(2-carboxyethyl) phosphine (Thermo Fisher Scientific) followed by alkylation with iodoacetamide (Thermo Fisher Scientific). After protein purification using Sera-Mag SpeedBeads Carboxylate-Modified Magnetic Particles (SP3 beads) (GE Healthcare, Chicago, IL, USA), the samples were digested with trypsin/LysC mix (Promega, Madison, WI, USA). The total concentration of the purified peptides was determined using a Fluorometric Peptide Assay Kit (Thermo Fisher Scientific). Then the peptides were labeled with TMTpro reagent, fractionated (into 12 fractions) with Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific), and dried.

Nano-LC-MS/MS of Tandom Mass Tag-Labeled Peptides

After the reconstitution with 0.1% formic acid/2% acetonitrile (eluent A), an aliquot of the peptide mixtures (2 μg/10 μL) was injected into the LC-MS/MS system consisting of an UltiMate 3000 RSLCnano LC system coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific). The peptides were loaded on a PepMap100 C18, 2 cm × 100 μm i.d., 5 µm trap column (Thermo Fisher Scientific) and subsequently separated on Aurora Series UHPLC emitter columns (25 cm × 75 μm i.d., 1.6 μm; IonOpticks, Melbourne, Australia) and heated to 45°C at a flow rate of 400 nL/min using a gradient of 2% to 24% eluent B (0.1% formic acid/100% acetonitrile) for 240 minutes, 24% to 32% eluent B for 30 minute, and 32% to 95% eluent B for 10 minutes followed by 95% eluent B wash step for 10 minutes. The mass spectrometer was operated in multinotch synchronous precursor selection mode.

Immunofluorescence Microscopy

3D RPTEC spheroids were fixed in methanol for 10 minutes at −20°C. Then the spheroids were washed twice with PBS and incubated in a blocking solution (0.1% Triton X-100 and 5% donkey serum in PBS) for 60 minutes and then with rabbit polyclonal anti-P-gp antibody (Abcam, ab129450) and goat polyclonal anti-Na+/K+-ATPase antibody, N-15 (SantaCruz, SC-16041) at 4°C overnight. Immunoreactions were visualized by cell incubation with Anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific) for P-gp and Anti-goat Alexa Flour 647 (Thermo Fisher Scientific) for Na+/K+-ATPase. Finally, the 3D-RPTEC spheroids were mounted with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) and observed using a Zeiss LSM 800 (Carl Zeiss AG, Oberkochen, Germany) and Zeiss C-Apochromat 63 × 1.2 W Correction Ring water immersion objective. All images were analyzed using the LSM 800 software.

Transport Experiments

Approximately 200 wells of 3D RPTEC spheroids cultured for 12 to 14 days were collected in one tube. The collected spheroids were washed with an assay buffer (pH 7.4), consisting Hanks’ balanced salt solution supplemented with 15 mM HEPES, by repeating the following cycle twice: 1) addition of the assay buffer, 2) centrifugation at 100 × g for 3 minutes, and 3) aspiration of the supernatant. The pellet of spheroids was resuspended in the assay buffer and then equilibrated for 15 minutes at 37°C. After centrifugation (100 × g, 3 minutes) and removal of the supernatant, uptake was initiated by adding an assay buffer containing either a transporter cocktail (10 μM metformin, 10 μM furosemide, 10 μM digoxin) or [3H]adefovir (80.6 nM) in the presence or absence of each transporter inhibitor. After the designated incubation time, the spheroids were centrifuged at 500 × g for 1 minute. The concentrations of compounds in the assay were determined using the supernatant. Uptake was terminated by removing the supernatant, followed by the addition of ice-cold assay buffer (3 mL), centrifugation at 500 × g for 1 minute, and removal of the supernatant. The cells were solubilized in sodium hydroxide for approximately 1 hour at room temperature. The lysates were neutralized with hydrochloric acid. The concentration of the drug in cell lysates was measured either by LC-MS analysis or with a liquid scintillation counter (AccuFLEX LSC-7200, Hitachi, Tokyo, Japan) after mixing with a scintillation cocktail (Clear-sol I, Nacalai Tasque, Inc., Kyoto, Japan). Protein concentrations were determined using the Lowry method (Lowry et al., 1951) or BCA Protein Assay Kit (Fujifilm-Wako Chemicals, Osaka, Japan) with bovine serum albumin as the protein standard.

LC-MS/MS Analysis for Drugs

Cell lysate (20 μL) was mixed with 60 μL of internal standard solution (acetonitrile with 0.1% formic acid containing 200 nmol/L hesperetin) followed by centrifuging at 10,000 × g for 5 minutes. The supernatant (45 μL) was mixed with 10 mmol/L ammonium formate (45 μL). After the sample preparation, an aliquot of the samples (5 μL) was injected into the LC-MS/MS system consisting of an ExionLCTM AD LC system coupled to a QTRAP5500 mass spectrometer (AB SCIEX, Framingham, MA, USA). The samples were separated on Kinetex column, 2.6 µm, F5, 100 Å, 2.1mm I.D.×100 mm (Phenomenex, Torrance, CA, USA) heated to 40°C with a constant flow rate of 0.4 mL/min of mobile phases (A) 5 mmol/L ammonium formate and (B) acetonitrile and a gradient elution program. The program started with 10% eluent B and increased to 95% at 2.6 minutes followed by a wash step with 95% eluent B for 1 minute before equilibration for 1.4 minutes with 10% eluent B. The total LC run time was 5 minutes. Metformin, furosemide, and digoxin were monitored using multiple reaction monitoring. The ionization conditions and m/z transitions are listed in Supplemental Table 1. The limits of detection for metformin, furosemide, and digoxin were 1 nmol/L. The mean accuracy of the intra- and inter-run quality control samples were within 80% to 120% (data not shown).

In Vitro ATP Assay

3D RPTEC spheroids (on days 10–12) in a V-bottom ultra-low attachment 96-well plate (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan) were exposed with respective concentrations of cisplatin and adefovir in REGM supplemented with 0.5% FBS. The exposure medium was replaced on days 3 and 5. Intracellular ATP levels were assessed using CellTiter-Glo 3D Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, 100 µL of the reagent was plated into each well and incubated for 30 minutes at room temperature. Bioluminescence was measured using a Wallac ARVO MX 1420 luminometer (Perkin Elmer, Waltham, MA, USA). The ATP concentration was determined using an ATP standard curve.

Data Analysis

Uptake Experiment Data Analysis

Uptake clearance was calculated by normalizing the amount of compound inside the cells to that in the initial solution and the protein concentration in each tube using the following equation:

Embedded Image

where Uptake CL is the uptake clearance (μL/designated time point/mg), Xcell is the amount of compound in the cells (pmol/designated time/tube), and Cbuffer is the concentration of the compound in the initial solution (pmol/μL). Uptake CL was normalized to the amount of total cellular protein (mg/tube).

Proteomics Data Analysis

Proteins were identified and quantified using Proteome Discoverer (version 2.4; Thermo Fisher Scientific). Briefly, the MS/MS spectra were searched against UniProtKB/Swiss-Prot human data downloaded in March 2020 using the SEQUEST HT search engine. Two missed cleavages were allowed, along with carbamidomethylation of cysteine as a fixed modification. Variable modifications included oxidation of methionine, deamidation of asparagine/glutamine, acetylation/methionine-loss/methionine-loss + acetylation of the protein N-terminus, and phosphorylation of serine, tyrosine, and threonine. Mass tolerance for precursor and fragment ions was 10 ppm and 0.6 Da, respectively, and false discovery rates at the peptide and protein levels were less than 0.01. Relative protein abundances were calculated from the sum of the peptide ion abundances of unique and razor peptides, which are peptides unique to a protein group and shared peptides in a protein group with more identified peptides, respectively. The peptide ion abundances were quantified using tandom mass tag (TMT) reporter ion signal-to-noise ratios in MS3 scans, with a coisolation threshold of 50 and an average reporter S/N threshold of 10. To normalize the quantitative data across TMT channels, the total peptide amount for each TMT channel was calculated and corrected for all abundance values in all other channels using a constant factor per channel. The normalized peptide abundances were then summed to calculate the protein abundances. To bridge the quantitative values across the two TMT16-plex experiments, all protein abundance values were scaled to control the channel average, which was set for a common sample over two-batch experiments. The calculated protein abundances were used for further analysis.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) or MS-Excel 2010 (Microsoft Corporation, USA). Data were presented as mean ± SD for at least three independent experiments. Unpaired two-tailed Student’s t test was used to compare datasets from independent groups of cells. Multiple comparisons were performed using one-way ANOVA followed by Dunnett’s test. Data were considered statistically significant at < 0.05. The 3D RPTEC spheroids/human cortex ratios of the adjusted geometric means of mRNA expression [geometric mean ratio (GMR)] and their two-sided 90% confidence intervals were computed using an ANOVA on a logarithmic scale. For exploratory purposes, equivalences were evaluated when the GMR point estimate was fivefold (≤ 20% or ≥ 500%), threefold (≤ 30% or ≥ 300%), and twofold (≤ 50% or ≥ 200%).

Data Availability

The microarray dataset generated in the current study is available from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE216153). Raw MS data and result files were deposited in the ProteomeXchange Consortium (http://www.proteomexchange.org/; PXID, PXD037535) via the jPOST partner repository (https://jpostdb.org; jpost ID, JPST001891) (Okuda et al., 2017).

Results

OAT1 mRNA Expression in 3D RPTEC Spheroids

When RPTECs were cultured in spherical cellular aggregates at a seeding cell number of 1,000 cells/well, OAT1 mRNA increased in accordance with the culture period (Fig. 1A). OAT1 mRNA showed the highest level at a seeding cell number of 1,000 cells/well and decreased at a seeding cell number greater than 1,000 cells/well (Fig. 1B). The size of the spheroid showed seeding cell number dependence; the size at a seeding cell number of 1,000 cells/well was approximately 271±19 µm (n = 10; data not shown). Based on these results, a seeding cell number of 1,000 cells/well was chosen for further studies.

Fig. 1.
Fig. 1.

OAT1 mRNA expression in 3D RPTEC spheroids at various culture days (A) and various seeding densities (B). (A) RPTEC (lot: 664995) were cultured in spheroidal aggregate for 0, 2, 4, 6, 8, and 13 days at a cell number of 1,000 cells/spheroid. (B) RPTECs (lot: 664995) were seeded at a cell number of 500, 1,000, 2,500, 5,000, 10,000, 25,000, and 50,000 cells/spheroid and cultured for 12 days. mRNA expression of OAT1 was determined on day 12. Bar represents the average ± SD of three replications on three different days.

mRNA Expression in 2D RPTECs and 3D RPTEC Spheroids in Comparison with Human Renal Cortical Tissues

The mRNA expression of the renal transporters, OCT2, OAT1, OAT3, MATE1, and MATE2-K recommended by regulatory authorities (US Food and Drug Administration, 2020; European Medicines Agency 2012; Ministry of Health, Labor and Welfare, 2018) in the 3D spheroid condition was compared with that in RPTECs cultured under 2D conditions. In 2D RPTECs even on day 12, the mRNA expression of the transporters was less than 27% of the average mRNA expression in human kidney cortical tissues from five donors (Fig. 2, A and C). With the exception of OAT3, gene expression in 3D RPTEC spheroids increased and was within the variability range of that of human kidney cortices (Fig. 2, B and C).

Fig. 2.
Fig. 2.

mRNA expression of drug transporters in 2D RPTECs (A), 3D RPTEC spheroids (B), and human cortical tissues (C) and comparison of mRNA expression profiles of enzymes (D), SLC transporters (E), and ABC transporters (F) in 2D RPTECs and 3D RPTEC spheroids with those in human cortical tissues. (A) 2D RPTECs (lot: 664995) were cultured for 2 and 12 days. (B) 3D RPTEC (lot: 664995) was cultured in spheroidal aggregates for 5, 12, 20, and 32 days. (C) mRNA expression of typical drug transporters in five donors of human kidney cortical tissues. Bar represents the mean ± SD of five different donors. (D–F) the number represents protein (%) to the total number of genes. 2D, 2D RPTECs; 3D, 3D RPTEC spheroids.

Additionally, the mRNAs of 3,194 enzymes, 334 SLC transporters, and 39 ATP-binding cassette (ABC) transporters were compared among 2D RPTECs, 3D RPTEC spheroids, and human kidney cortices (Fig. 2, D–F). Approximately 90% of the mRNA of the detected 3,194 enzymes in 2D and 3D RPTECs at all culture periods was within the fivefold difference of the average mRNA expression in human kidney cortices, although more enzymes showed similar mRNA expression in 3D RPTECs at culture periods other than day 5. Approximately 80% of the detected ABC transporters when cultured in 2D on days 2 and 12 and in 3D on day 5 showed a difference within fivefold of the average mRNA expression in the human kidney cortices. When RPTECs were cultured under 3D spheroid conditions for more than 5 days, the percent of ABC transporters showing a difference within fivefold increased to 90%. In 2D RPTECs, 73% and 25% of the detected SLC transporters on day 2 showed a difference within fivefold and less than 20% of the average mRNA expression in human kidney cortices, respectively, and these values changed to 83% and 16%, respectively, on day 12. In 3D RPTEC spheroids, 82% and 17% of the detected SLC transporters on day 5 showed a difference within fivefold and less than 20% of the average mRNA expression in human kidney cortices, respectively, and these values changed to 83% and 16%, respectively, on day 12. The mRNA expression patterns of SLC transporters did not change largely beyond day 12. Next, we compared the mRNA expression profiles of 3D RPTEC spheroids from three different donors (days 12–14) with those of human kidney cortices from five different donors. The equivalence between 3D RPTEC spheroids and the human kidney cortex was investigated using GMR and their 90% confidence intervals by applying two-, three-, and fivefold differences (Table 2), because the coefficient of variation × 100 (CV%) of mRNA expression of the majority of the detected genes (16,985) in human kidney cortices was > 20 CV% (Supplemental Table 4). When a fivefold difference was used as a benchmark, 92% of all genes, 87% of enzymes, 77% of SLC transporters, and 44% of ABC transporters were judged to be statistically equivalent to those in the human kidney cortex. The percentage of genes decreased when low-fold differences (two- and threefold) were applied.

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TABLE 2

Equivalence between 3D RPTEC spheroids (n = 3) and human kidney cortices (n = 5) based on mRNA expression

Protein Expression in 2D RPTECs and 3D RPTEC Spheroids in Comparison with Human Renal Cortical Tissues

Protein expression in the total lysate (TL) of 2D RPTECs, 3D RPTEC spheroids, and human kidney cortices was investigated using label-based LC-MS/MS proteomics to examine whether the results based on mRNA, often used as a surrogate for protein expression, were observed in protein expression. A total of 4,848 proteins were quantified, and approximately 90% of the detected proteins showed a 0.2- to 5-fold difference in the average protein expression in human kidney cortices; the same percentage was observed in the detected 1,642 enzymes and 21 ABC transporters (Table 3). In contrast, 70.5% and 25.9% of the 139 SLC transporters detected in 2D RPTECs showed a difference within fivefold in average protein expression and less than 20% in the human kidney cortices, respectively. When RPTECs were cultured in 3D spheroid conditions, the percentage of proteins showing a 0.2- to 5-fold difference improved to 78.4%. The impact of the 3D spheroidal culture condition, selected by monitoring OAT1 gene expression, was further assessed (Supplemental Tables 2 and 3). When a fivefold difference was used as the criterion for assessing the similarity between human kidney cortices and RPTECs, a total of 203 proteins showed similar protein expression to that in human kidney cortex compared to 2D RPTECs. In contrast, 85 proteins showed diversified expression in 3D RPTEC spheroids compared to 2D RPTECs. The protein expression of more than 20 transporters has been investigated using the total membrane fraction of human kidney cortices from 41 different donors by the LC-MS based quantitative targeted proteomics method (Prasad et al., 2016). However, it was difficult to compare the absolute expression levels between the two studies because TL and a different proteomics method were used in our study. However, it was possible to evaluate the appropriateness of the human cortical samples used in our study by comparing the variability in the protein expression of transporters between the two studies. The CV% of protein expression of the 11 transporters (OAT1, OAT3, OAT4, OCT2, OCTN1, OCTN2, SGLT2, P-gp, ABCC2, ABCC4, and MATE1) was analyzed in both studies. Except for OCTN1 and MRP2, the transporters showed similar CV% within a 1.6-fold difference. The CV% for OCTN1 and MRP4 differed by 1.8 and 5.0, respectively. Since five to six outliers were observed in 41 samples for OCTN1 and MRP4 in the study by Prasad et al. (2016), and showed larger variability, the CV% in our study is considered to be similar if outliers are omitted from the results of Prasad et al. (2016), indicating that the samples used in our study were valid (Fig. 3, B–D).

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TABLE 3

Difference of the protein expression profiles of 2D RPTECs and 3D RPTEC spheroids (cultured for 12 days) compared with human cortical tissues (N = 5)

Fig. 3.
Fig. 3.

Protein expression of kidney markers (A), SLC transporters (B and C), and ABC transporters (D). Protein abundance was expressed as percentage of the human kidney cortex 1. The protein abundance of human kidney cortices represents the mean ± SD of five different donors. Human cortex, human kidney cortex.

Protein Expression of Kidney Markers and Selected SLC and ABC Transporters

RPTECs are renal epithelial cells isolated from human proximal tubules. However, they may dedifferentiate during the culture process, and the original function of proximal tubular cells may not be maintained. The properties of the RPTECs were assessed based on the protein expression of podocalyxin and nephrin 1 as representative glomerular markers, uromodulin and SLC12A1 as representative distal tubule markers, and aquaporin 1 (AQP1) and cadherin 6 as representative markers of the proximal tubules (Tsujimoto et al., 2020). The expression of glomerular and distal tubule markers was considerably lower in both 2D and 3D RPTEC spheroids than in human renal cortices, which contain both glomeruli and distal tubules. In contract, AQP1 expression in 2D RPTECs was as low as 26% of that in human renal cortices, whereas AQP1 and cadherin 6 were expressed at the same level in 3D RPTEC spheroids as in human renal cortices (Fig. 3A). This observation, based on the proteomics results, was further confirmed by the mRNA expression of additional markers (Supplemental Fig. 1), although there were some exceptions (such as CUBN for proximal tubules, LCN2 for distal tubules, and CD2AP for podocytes).

OAT1 protein expression, used as the selection marker of culture condition, in 2D RTPECs and 3D RPTEC spheroids was 33% and approximately 65% of the average protein expression in the human renal cortices, respectively (Fig. 3B), and the protein expression was almost the same as that of mRNA. As is often the case with the downregulation of proteins occurring in cells with passages of several primary cells under conventional culture conditions, in 2D RPTECs, the expression of GLUT2, SGLT1, SGLT2, URAT1, OCTN1, OAT3, and OAT4 was less than 30% of the respective average protein expression in the human renal cortices (Fig. 3B). In the 3D RPTEC spheroids, out of the seven SLC transporters, proteins of GLUT2 and OCTN1 were increased to more than 30% of the average protein expression in the human renal cortices (Fig. 3B). The protein expression of the five selected ABC transporters in 3D RPTEC spheroids ranged from 70% to 306%, compared with the respective average protein expression in human renal cortices and 2D RPTECs, which ranged from 47% to 396% (Fig. 3D). No significant change in protein expression was observed in proteins other than PEPT1, even when the culture period of 3D RPTEC spheroids was extended from 12 days to 20 days (Fig. 3A-D). A heat map of the protein expression profiles of all quantified enzymes and SLC/ABC transporters is shown in Supplemental Fig. 2. In addition to protein expression in the TL, which represents total protein expression in cells, protein expression in the plasma membrane (PM) fraction, an indicator of functional transporters in the PM, was also investigated in 3D RPTEC spheroids (Fig. 4). The PM/TL ratios of cytosolic proteins were below 0.7, and those of Na+/K+-ATPase and P-gp, which were confirmed to be expressed mainly in the PM of the 3D RPTEC spheroids (Fig. 8A and B), were 7.54 and 7.09, respectively, indicating that the preparation of the PM fraction was carried out without any remarkable contamination. All ABC transporters and some SLC transporters such as PEPT1, GLUT9, OATP4C1, MATE1, OAT4, OCTN2, and OAT1 showed PM/TL ratios similar to those of Na+/K+-ATPase and P-gp but were far different from those of cytosolic proteins, suggesting that these transporters are mainly expressed in the PM. However, the PM/TL ratios for GLUT2, SGLT1, SGLT2, URAT1, OCTN1, OCT2, OAT3, and MATE2-K ranged from 1.28 to 4.00. SGLT1, SGLT2, URAT1, and OAT3 showed very low protein expression in both the TL (Fig. 3) and PM, indicating that these transporters were not abundantly expressed in the 3D RPTEC spheroids. GLUT2 and OCT2 protein expression in the PM was low, although that in the TL was high, indicating that GLUT2 and OCT2 proteins are expressed not only in the PM but also in other parts of the 3D RPTEC spheroids.

Fig. 4.
Fig. 4.

Protein expression ratio of SLC transporters (A), ABC transporters (B), and cytosolic proteins in PM and TL fractions of 3D RPTEC spheroids. The PM/TL ratio was calculated from the protein abundance (percentage of human kidney cortex 1 in the TL) of each protein in the PM and TL. RPTECs (lot. 664995) were cultured in 3D spheroid culture for 12 to 14 days. The PM/TL ratio of Na+/K+-ATPase was 7.54. The PM/TL ratios of proteins detected are shown in Supplemental Table 5. ACAT2, acetyl-CoA aceyltransferase 2; ALDH2, aldehyde dehydrogenase 2; CBR1, carbonyl reductase 1; G6PD, glucose 6 phosphate dehydrogenase; SHMT1, serine hydroxymethyltransferase 1.

Effect of Cisplatin and Adefovir on Intracellular ATP Levels of 3D RPTECs

Cisplatin and adefovir are substrates of OCT2/MATE1 and OAT1, respectively (de Jongh et al., 2003; Izzedine et al., 2005). We investigated whether cisplatin and adefovir showed difference in toxicity between 2D RPTECs and 3D RPTEC spheroids and exhibited transporter-dependent toxicity in 3D RPTEC spheroids using intracellular ATP levels as an endpoint; because the dose-response to cefalotin and cisplatin on intracellular ATP levels showed a very similar response to those on lactate dehydrogenase release, which is often used to evaluate cell viability (Supplemental Fig. 3). Cisplatin showed a similar dose-response to intracellular ATP levels in 2D RPTECs and 3D RPTEC spheroids (Fig. 5), whereas the in vitro toxicity of adefovir was weaker in 2D RPTECs than in 3D RPTEC spheroids (Fig. 5). Cisplatin showed a concentration-dependent decrease of intracellular ATP levels and the addition of 1,000 µM cimetidine—an OCT and MATEs inhibitor—significantly suppressed the decrease in ATP compared with that in the absence of cimetidine, while 1,000 µM probenecid—an OAT inhibitor—did not cause a change (Fig. 6A). Adefovir reduced the intracellular ATP levels in a concentration-dependent manner. The decrease of intracellular ATP levels was not weakened by the addition of 1,000 µM cimetidine, while the addition of 1,000 µM probenecid almost completely diminished the adefovir-dependent decrease of intracellular ATP levels (Fig. 6B).

Fig. 5.
Fig. 5.

Effect of cisplatin (A) and adefovir (B) on cellular ATP contents in 2D RPTECs and 3D RPTEC spheroids. 2D RPTECs and 3D RPTEC spheroids (lot: 664995) were exposed to cisplatin (0, 1, 3, 10, 30, and 100 µM) or adefovir (0, 1, 3, 10, 30, and 100 µM) for 7 days. Each bar represents the mean ± SD of four replications. * Statistically significant compared with the control, p < 0.05, Dunnett’s test.

Fig. 6.
Fig. 6.

Effect of cisplatin (A) and adefovir (B) on cellular ATP contents in the absence and presence of the OCT inhibitor cimetidine and the OAT inhibitor probenecid. 3D-RPTECs (lot: 664995) were exposed to cisplatin (0, 3, 10, and 30 µM) or adefovir (0, 10, 100, and 1,000 µM) for 3 days in the absence or presence of cimetidine (1,000 µM) or probenecid (1,000 µM). Each bar represents mean ± SD of four replications. * Statistically significant compared with the control, p < 0.05, Dunnett’s test.

Transport Function in 3D RPTEC Spheroids Using Metformin, Furosemide, Adefovir, and Digoxin as Probe Substrates

The transporter function in the 3D RPTEC spheroids was evaluated using the following substrates: metformin for OCT2/MATEs, furosemide and adefovir for OAT1, and digoxin for P-gp (Uwai et al., 2007; Ebner et al., 2015) (Fig. 7). The uptake of metformin by 3D RPTEC spheroids was decreased by the OCT/MATEs inhibitor cimetidine but only very weakly decreased by the OAT inhibitor probenecid. Probenecid did not significantly decrease the uptake of furosemide or adefovir, nor did it increase the uptake of digoxin. The P-gp inhibitor zosuquidar did not show a statistically significant increase in digoxin uptake, whereas cimetidine significantly increased digoxin uptake.

Fig. 7.
Fig. 7.

Uptake of metformin (A), furosemide (B), adefovir (C), and digoxin (D) in 3D RPTEC spheroids in the presence and absence of typical inhibitors. Inhibitor concentrations of probenecid, cimetidine and zosuquidar were 100/1,000 µM (A, B, and D/C), 4,000 µM, and 1 µM, respectively. * Statistically significant, t test, RPTECs (lot: 664995) were used.

P-gp and Na+/K+ ATPase Localization in 3D RPTEC Spheroids

P-gp and Na+/K+-ATPase are expressed on the apical and basolateral membrane sides of proximal renal tubular cells, respectively (Rostgaard and Møller, 1980; Launay-Vacher et al., 2006). The structure of 3D RPTEC spheroids was investigated using confocal laser scanning microscopy with the respective antibodies. One 3D RPTEC spheroid was approximately 200 to 300 µm in size, and the apical membrane marker P-gp was mainly detected on the outside of the 3D RPTEC spheroids and in the non-cell-to-cell contact area (Fig. 8A, green). P-gp was not highly expressed at the center of 3D RPTEC spheroids. In contrast, Na+/K+-ATPase, a marker of basolateral membranes, was mainly expressed inside 3D RPTEC spheroids and in the area where the cells were in contact with each other (Fig. 8B, red).

Fig. 8.
Fig. 8.

Representative immunofluorescence images of P-gp (green; A), Na+-K+ ATPase (red; B), and nuclei (blue; C) in 3D RPTEC spheroids. P-gp, Na+/K+-ATPase, and nuclei were stained with rabbit polyclonal anti-P-gp antibody (visualized using anti-rabbit Alexa Fluor 488), goat polyclonal anti-Na+/K+-ATPase antibody (visualized using anti-goat Alexa Fluor 647), and 4',6-diamidino-2-phenylindole, respectively. Images were captured at the original magnification (x20). Scale bars, 20 µm. RPTECs (lot: 664995) were used.

Discussion

Various in vitro tools have been developed for toxicity and pharmacokinetic assessments in the kidney (Faria et al., 2019). However, few reports remain on in vitro tools with sufficient transporter functions, such as OAT, even in 3D (Nieskens et al., 2016; King et al., 2017). In this study, we aimed to develop and evaluate a simple culture method with high throughput for RPTECs that express important transporters for pharmacokinetic and toxicity assessments.

When protein expression was measured as an endpoint, 90% of the detected 4,848 proteins in 3D RPTEC spheroids were within the fivefold range of protein expression in the human renal cortices (Table 3). For SLC transporters, the percentage of transporters that were not within the fivefold range of human renal cortices was approximately 30% in the 2D RPTECs, which was approximately twofold higher than that of enzyme and ABC transporters (Table 3). The 3D spheroid culture improved SLC transporter expressions by approximately 8%. However, the expression of transporters, such as URAT1/OAT4 [important transporters for the renal reabsorption of uric acid (Sato et al., 2008)], and SGLT1/SGLT2 (important transporters for glucose reabsorption), was as low as that of OAT3. OAT1, OAT3, OAT4, URAT1, and SGLT2 have been reported to be positively modified by hepatocyte nuclear factor 1 (HNF1) and negatively modified by DNA methylation (Kikuchi et al., 2006, 2007; Saji et al., 2008; Jin et al., 2012; Takesue et al., 2018). HNF1α/β protein expression was not detected by the LC-MS/MS proteomics analysis, but mRNA expression in 2D RPTECs and 3D RPTEC spheroids was similar to those in human renal cortices (data not shown). This suggests that the low expression of these transporters is not due to the low expression of HNF1 but because of DNA methylation. Saji et al., (2008) reported that OAT1 and OAT3 were regulated in clusters. The results of the present study differ from those of Saji et al. (2008) in that the expression of OAT1, but not OAT3, was upregulated by 3D spheroid culture of RPTECs. Therefore, it is reasonable to consider that methylation of the post-promoter region, rather than of the promoter region, occurs in the 3D RPTEC spheroids. It is also possible that factors other than HNF1 and methylation are involved in the regulation of OAT1/3 expression. Further investigations are necessary to better understand the regulation of OAT1/3 expression in RPTECs.

Cisplatin decreases glomerular filtration rate and increases urinary albumin at clinical concentrations of approximately 10 µM unbound cisplatin (Erdlenbruch et al., 2001). Cisplatin showed a concentration-dependent decrease in intracellular ATP levels in 3D RPTEC spheroids, and a 50% reduction of intracellular ATP was obtained at 10 µM, which is the clinical plasma-free cisplatin concentration (Fig. 6A). Intracellular ATP levels in HK-2 cells, which do not express OCT2 or MATE (Sancho-Martínez et al., 2011; Van der Hauwaert et al., 2014), were reduced to about half at 30 to 100 µM cisplatin. To understand why almost the same concentration decreased intracellular ATP levels by half in HK-2 and 3D RPTEC spheroids although the expression of OCT/MATE transporters was different, two points should be taken into consideration. First, the contribution of passive diffusion of basic drugs is larger than that of acidic drugs because passive diffusion is affected by the internal negative membrane potential. Second, longer exposure times are generally used in in vitro toxicity studies than in in vitro transporter studies. The intracellular concentration of cisplatin is unlikely to differ greatly between 3D RPTEC spheroids and HK-2 cells, because the impact of the OCT2/MATE process is not large in in vitro toxicity studies.

Adefovir (120 mg/d) has been reported to cause nephrotoxicity in 20% to 30% of patients when administered at a clinic for 48 weeks (Kahn et al., 1999; Fisher et al., 2001). The intracellular ATP level in 3D RPTEC spheroids was reduced by adefovir in a concentration-dependent manner, with the amount of intracellular ATP reduced to about half of the control at concentrations of 100 to 1,000 µM (Fig. 6B). The addition of the OAT inhibitor probenecid completely suppressed the decrease in cellular ATP, suggesting that adefovir toxicity observed in 3D RPTEC spheroids is a function of the OAT transporter. The in vitro cytotoxicity of adefovir was also evaluated with RPTECs in which OAT1 was transfected using cell viability as an endpoint and the obtained IC50 value was 230 µM (Nieskens et al., 2016). The clinical unbound Cmax of adefovir was approximately 1 µM after 125 mg/d (Barditch-Crovo et al., 1997), and nephrotoxicity was seen in 20% to 30% of patients. The discrepancy may be explained by exposure period with adefovir is not sufficient because the clinical toxicity of adefovir has been observed 48 to 72 weeks post-dose in clinics, and the in vitro toxicity of cisplatin is reported to be enhanced 100-fold by increasing the duration of drug exposure from 24 hours to 14 days (Secker et al., 2019). This study revealed that 3D RPTEC spheroids maintained their morphology over 5 to 20 days and the gene expression did not significantly change from 12 to 32 days (Fig. 2), thus the toxic effects of long-term exposure can also be examined in 3D RPTEC spheroids.

In addition to the OCT/MATE-dependent in vitro cytotoxicity of cisplatin in 3D RPTEC spheroids (Fig. 6), cimetidine reduced the uptake of metformin into 3D RPTEC (Fig. 7). This was consistent with the proteomics results showing that the expression levels of OCT2, MATE1, and MATE-2K in 3D RPTEC spheroids were within the range of variation in their expression levels in human renal cortices, and their expression was confirmed in the PM fraction (Figs. 3B and 4A). In contrast, no uptake of adefovir or furosemide by OAT1 was observed, although OAT1 protein expression was within the range of variation in protein expression in the human renal cortices, and the in vitro cytotoxicity of adefovir in 3D RPTEC spheroids was OAT-dependent. Immunostaining revealed that P-gp, a marker of apical membranes, was strongly expressed on the outer side of the 3D RPTEC spheroids and in areas where the RPTECs were not in contact with each other. However, P-gp expression decreased toward the center of the 3D RPTEC spheroids. Na+/K+- ATPase, a basolateral membrane marker, was strongly expressed in areas where RPTECs were in contact with each other, and its expression in the center of the 3D RPTEC spheroids was not as weak as that of P-gp (Fig. 8). The following two possibilities were considered as reasons why basolateral OAT1 activity was not detected in 3D RPTEC spheroids: 1) apical and basal transporter activities cannot be evaluated separately because both apical transporters are exposed to the buffer as well as in vitro kidney slice systems (Arakawa et al., 2017, 2019), and 2) a wash process in the in vitro transporter assay is not sufficient for transporters on the basolateral side because transporters on the basolateral side of RPTECs are expressed in the area where cells are in contact. Genetic polymorphisms can weaken the correlation between activity and protein expression. However, for OAT1, although single nucleotide polymorphisms (SNPs) with a complete loss of function (R454Q) has been reported, the allele frequency is extremely low (0.2%) (Fujita et al., 2005). Therefore, it is unlikely that the SNPs in OAT1 caused a discrepancy between OAT1 protein expression and the undetected OAT activity in the 3D RPTEC spheroids, although unidentified SNPs may be involved. Further efforts are necessary to precisely evaluate transporter activity in the future.

Reproducibility is an important factor in evaluating the usefulness of an in vitro system. The reproducibility of the 3D RPTEC spheroids was evaluated by comparing the CV% of mRNA expression in 3D RPTEC spheroids from three different lots of RPTECs at a culture period of 12 to 14 days. Furthermore, these CV% values were compared with those of human kidney cortex from five different donors (Supplemental Table 4). Of the 16,993 genes detected, 87% showed variation within 50 (CV%), which was also true for 3,193 enzymes, 334 SLC transporters, and 39 ABC transporters in the case of 3D RPTEC spheroids, whereas CV% for all genes, enzymes, SLC transporters, and ABC transporters in the human kidney cortex were 75%, 80%, 61%, and 80%, respectively. This indicates that the 3D culture method used in this study is a highly reproducible culture condition with a small variation in mRNA expression among the three different lots of RPTECs (from three different donors) compared with the variation in mRNA expression in the human kidney cortex from five different donors.

In summary, we have confirmed that a spheroidal culture condition, selected by monitoring OAT1 gene expression, improved the protein expression of other genes to be more similar to that in human kidney cortices, resulting in a similar protein expression level of 90% of more than 4,800 proteins detected over an extended period of up to 20 days in culture. This condition also enabled the evaluation of not only transporter-dependent cisplatin nephrotoxicity, which has been evaluated in 2D culture conditions and other cell systems, but also transporter-dependent adefovir nephrotoxicity more sensitively. In vitro 3D RPTEC spheroids could positively impact preclinical drug discovery/drug development, helping to prevent unwanted failures in late-stage drug development including clinical trials. However, the polarized expression of transporters has not yet been fully evaluated in the current system, and genetic polymorphisms may interfere the comparison of mRNA and protein expression between RPTECs and human kidney cortices. Moreover, the current system is a static culture, and the inclusion of laminar flow on the RPTEC could further enhance tubular epithelial cell function.

Acknowledgments

The excellent technical assistance of Kaori Makino, Tokuko Takatsuka, and Michiru Miyake in performing the in vitro experiments at Nippon Boehringer Ingelheim and of Akiko Matsui and Hijiri Fujioka in performing the mRNA data analysis are gratefully acknowledged. This study was supported by the Japan Agency for Medical Research and Development (AMED, grant number: JP20mk0101182h0001).

Authorship Contributions

Participated in research design: Ishiguro, E. Takahashi, Arakawa, Saito, Kitagawa, Kondo, Morinaga, M. Takatani, R. Takahashi, Kudo, Mae, Tamai, Osafune, Jimbo.

Conducted experiments: E. Takahashi, Saito, Kitagawa, Morinaga, M. Takatani, R. Takahashi, Kudo, Kadoguchi, Higuchi, Nakazone.

Performed data analysis: E. Takahashi, Saito, Kitagawa, Morinaga, M. Takatani, R. Takahashi, Kudo, Kadoguchi, Higuchi, Nakazono.

Wrote or contributed to the writing of the manuscript: Ishiguro, E. Takahashi, Saito, Kondo, Morinaga, Tamai, Osafune.

Footnotes

    • Received October 23, 2022.
    • Accepted June 21, 2023.

  • This research was supported by the Japan Agency for Medical Research and Development (AMED, under grant number: JP20mk0101182h0001), Nippon Boehringer Ingelheim Co., Ltd., and Nikkiso Co., Ltd.

  • None of the authors have any actual or perceived conflicts of interest with the contents of this article.

  • 1Co-first authors.

  • dx.doi.org/10.1124/dmd.122.001171.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

2D
two dimensional
3D
three-dimensional
ABC
ATP-binding cassette
AQP1
aquaporin 1
GLUT9
glucose transporter 9
GMR
geometric mean ratio
HNF1
hepatocyte nuclear factor 1
LC-MS/MS
liquid chromatography-tandem mass spectrometry
MATE
multidrug and toxin extrusion
OAT
organic anion transporter
OCT
organic cation transporter
OCT2
organic cation transporter 2
OCTN1
organic cation/carnitine transporter 1
PEPT1
peptide transporter 1
PM
plasma membrane
REGM
renal epithelial cell growth medium
RPTEC
renal proximal tubular epithelial cells
SGLT1/2
sodium-glucose cotransporter 1/2
SLC
solute carrier
SMCT1/2
sodium coupled monocarboxylate transporter 1/2
SNP
single nucleotide polymorphism
TL
total lysate
TMT
tandem mass tag
URAT1
urate transporter 1

This is an open access article distributed under the CC BY Attribution 4.0 International license.

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3D RPTEC Spheroids: An Improved In Vitro Tool

Naoki Ishiguro, Etsushi Takahashi, Hiroshi Arakawa, Asami Saito, Fumihiko Kitagawa, Masayuki Kondo, Gaku Morinaga, Masahito Takatani, Ryo Takahashi, Takashi Kudo, Shin-ichi Mae, Moeno Kadoguchi, Daichi Higuchi, Yuya Nakazono, Ikumi Tamai, Kenji Osafune and Yoichi Jimbo
Drug Metabolism and Disposition September 1, 2023, 51 (9) 1177-1187; DOI: https://doi.org/10.1124/dmd.122.001171
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