The studies involving human participants were reviewed and approved by the Institutional Ethics Committees of the National Defense Medical College and Nagoya University. All protocols were in accordance with the Declaration of Helsinki, and written informed consent was obtained from each participant in the present study.

A total of 4,521 Japanese men were recruited from health examination participants in Shizuoka and Daiko areas of the Japan Multi-Institutional Collaborative Cohort Study (Hamajima, 2007; Asai et al., 2009). The characteristics of the participants are shown in Table 1. Of note, under normal physiological conditions, uric acid (molecular weight of 168.1) exists in the blood mainly as urate (the anion form of uric acid, molecular weight of 167.1); on the other hand, in clinical testing method, this parameter is measured and converted to concentration of “uric acid (mg/dl)” in the serum, as described previously (Nakatochi et al., 2021). Nevertheless, based on the G-CAN (The Gout, Hyperuricemia, and Crystal-Associated Disease Network) nomenclature for gout (Bursill et al., 2019), we herein used the term of “serum urate,” instead of serum uric acid, for mentioning the circulating form.

Quantitative trait locus (QTL) analysis of serum urate was performed for all participants. Among them, for 1,518 men with available urinary data, we performed subsequent QTL analysis of FE_UA. Based on the results of blood and urine tests, FE_UA was calculated as follows [urinary urate (mg/dl) × serum creatinine (mg/dl)]/[serum urate (mg/dl) × urinary creatinine (mg/dl)] × 100 (%).

Genomic DNA was extracted from whole peripheral blood cells of the 4,521 Japanese male participants according to methods described in our previous study (Sakiyama et al., 2021). To genotype rs117371763 (c.1129C>T: p.R377C) of the OAT10/SLC22A13 gene, we employed the TaqMan method (Thermo Fisher Scientific, Kanagawa, Japan) using a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific), as described in our previous study (Higashino et al., 2020). Also, like in the previous study, to confirm the accuracy of the genotyping results, >100 samples were also subjected to direct sequencing with the following primers: 5′-TGG​TGT​GTG​TTG​GCA​GAG-3′ and 5′-GGT​CCC​ATC​CAC​TGG​AAC-3′. DNA sequence analysis was performed with a 3130xl Genetic Analyzer (Thermo Fisher Scientific). Genotype distributions were as follows: 1129C/C, 4,128 subjects; 1129C/T, 384 subjects; 1129T/T, 9 subjects. Among them, there were 1,369, 143, and 6 subjects with available urinary data, respectively.

The critical materials and resources used in this study were summarized in Supplementary Table S1. Dotinurad was kindly provided by Fuji Yakuhin (Saitama, Japan) under a material transfer agreement. All other chemicals used were commercially available and of analytical grade. In this study, we evaluated the following urate-lowering drugs and their active metabolites: urate synthesis inhibitors (allopurinol, oxypurinol, febuxostat, and topiroxostat), uricosuric agents (benzbromarone, 6-hydroxybenzbromarone, dotinurad, lesinurad, and probenecid), and other drugs that decrease serum urate levels with an action mechanism different from their expected medicinal effects (fenofibrate and losartan). Detailed information is summarized in Table 2.

An anti-human organic anion transporter 10 (OAT10) polyclonal antibody was raised in rabbits according to methods described in our previous study (Takada et al., 2015). In brief, the rabbit polyclonal antibody was generated against two KLH-coupled human OAT10 peptides as follows: amino acids ^#523–536 (KLH)-C + PKSVPSEKETEAKG; amino acids ^#293–306, (KLH)-C + KAASVNRRKLSPEL, which were also used to determine the titer of antiserum. On day 77, whole blood was collected from an adequately-immunized rabbit. The prepared antiserum was further purified before use using an epitopes-conjugated affinity column.

Immunohistochemical analysis using the anti-OAT10 antibody was conducted according to our previous study (Chiba et al., 2015). The analyzed tissue was the normal part of the human kidney obtained from a male patient with renal cell carcinoma. In brief, with 4-μm paraffin sections of the kidney, antigen retrieval was conducted by microwave oven-heating in a retrieval solution (0.01 M citrate buffer, pH 6.0) for 10 min; followed by inactivation of endogenous peroxidase activity with hydrogen peroxide (3% in methanol) treatment for 10 min, and further washing with Tris-buffered saline containing 0.05% Tween 20 (TBST). After blocking with 5% goat serum in TBS for 10 min, the sections were incubated with the anti-OAT10 antibody (1:50 dilution in TBS containing 1% bovine serum albumin, BSA) at 4°C overnight; subsequently the sections were treated with the Dako EnVision™ Kit/horseradish peroxidase (HRP) (K1491; Dako, Kyoto, Japan) as a secondary antibody, followed by 3,3′-diaminobenzidine (Dako) treatment for the detection of the immune reactions. Nuclei were counterstained with Mayer’s hematoxylin. For antigen absorption test, to block specific staining, the anti-OAT10 antibody was pre-incubated with the OAT10 peptides, which were used at a concentration of 100 μg/ml, before staining procedure. After visualization, the specimens were imaged using an upright microscope (Eclipse 50i; Nikon, Tokyo, Japan) equipped with a Nikon Digital Sight DS-5M camera (Nikon).

The full-length human OAT10/SLC22A13 wild-type (WT) (NCBI accession; NM_004256) open reading frame (ORF) in pEGFP-N1 plasmid was constructed in our previous study (Higashino et al., 2020). The full-length human URAT1/SLC22A12 WT (NCBI accession; NM_144585.3) ORF in pEGFP-C1 was derived from our previous study (Toyoda et al., 2020a). Prior to further experiments described below, each plasmid containing the transporter and control empty vectors were obtained in the same lot using a PureLink™ HiPure Plasmid Filter Midiprep Kit (Thermo Fisher Scientific).

Human embryonic kidney 293 (HEK293)-derived 293A cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 1% penicillin-streptomycin (Nacalai Tesque), 2 mM L-glutamine (Nacalai Tesque), and 1 × Non-Essential Amino Acid (Life Technologies, Tokyo, Japan) at 37°C in a humidified atmosphere of 5% (v/v) CO₂ in air; we confirmed that 293A cells used were negative for mycoplasma contamination using MycoAlert™ mycoplasma detection kit (Lonza, Basel, Switzerland). Madin-Darby canine kidney II (MDCKII) cells (a well-used polarized renal cell line to investigate the cellular localization of transporter proteins—apical or basal membranes) were also maintained in a similar way.

For the in vitro transport assay, 24 h after the seeding of the cells (0.92 × 10⁵ cells/cm²) onto 12-well cell culture plates, each vector plasmid was transfected into 293A cells with a forward transfection approach using polyethyleneimine “MAX” (PEI-MAX) (Polysciences, Warrington, PA, United States) as described previously (Toyoda et al., 2020a). All experiments were carried out with 293A cells at passages 12–20. Of note, such mammalian cell lines have been well-used to evaluate urate transport activity of target protein (Miner et al., 2016; Toyoda et al., 2020b; Higashino et al., 2020).

For confocal microscopy, each vector plasmid was transfected into MDCKII cells with a reverse transfection approach using PEI-MAX as described previously (Toyoda et al., 2016). In brief, MDCKII cells were collected after trypsinization, followed by centrifugation at 1,000 × g for 5 min. The cell pellet was re-suspended in fresh DMEM, and the resulting suspension was mixed with plasmid/PEI-MAX mixture (2 μg of plasmid/10 μl of PEI-MAX for 1 × 10⁶ cells of MDCKII cells). Then, the MDCKII cells were re-seeded onto cell culture plates at a concentration of 1 × 10⁵ cells/cm². For whole cell lysate preparations, MDCKII cells were re-seeded onto 6-well cell culture plates at the same concentration. All experiments were carried out using MDCKII cells at passages 7–10.

After preparation of whole cell lysates with cell lysis buffer A [50 mM Tris/HCl (pH 7.4), 1 mM dithiothreitol, 1% (v/v) Triton X-100, and cOmplete™, EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland)], to examine the N-linked glycosylation status of OAT10 and URAT1 proteins, whole cell lysate samples were treated with Peptide N-glycosidase F (PNGase F) (New England Biolabs, Ipswich, MA, United States) as described previously (Higashino et al., 2020). The protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) with bovine serum albumin (BSA) as a standard according to the manufacturer’s protocol. The samples were separated using SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Bedford, MA, United States) by electroblotting at 15 V for 60 min. Blots were probed with appropriate antibodies (Supplementary Table S1) according to previous studies (Toyoda et al., 2020a; Higashino et al., 2020), and the signals were visualized by using chemiluminescence and detected using a multi-imaging analyzer Fusion Solo 4™ system (Vilber Lourmat, Eberhardzell, Germany).

Confocal laser scanning microscopic observation was conducted as described previously, with some minor modifications (Toyoda et al., 2016). In brief, 72 h after the transfection, MDCKII cells were fixed with ice-cold methanol for 10 min at room temperature. After washing three times with PBS (−) containing 1% BSA (BSA-PBS), the cells were incubated with a mouse anti-gp135 monoclonal antibody (3F2/D8; the Developmental Studies Hybridoma Bank, Iowa City, IA, United States) diluted 100-fold in BSA-PBS for 1 h at room temperature, followed by incubation with a goat anti-mouse IgG-Alexa Fluor 546-conjugate (A-11003; Thermo Fisher Scientific), diluted 200-fold in BSA-PBS, for 1 h at room temperature in the dark. Subsequently, cells were subjected to TO-PRO-3 Iodide (Molecular Probes, Eugene, OR, United States) staining. After the visualization of the nuclei, the cells were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, United States). To analyze the localization of EGFP-fused OAT10 or EGFP-fused URAT1 proteins, fluorescence was detected using an FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).

With OAT10, urate uptake assay using OAT10-expressing 293A cells was conducted according to our previous study (Higashino et al., 2020). In brief, 48 h after the plasmid transfection, the cells were washed twice with a transport buffer (Ringer solution: 130 mM NaCl, 4 mM KCl, 1 mM Na₂HPO₄, 1 mM MgSO₄, 1 mM CaCl₂, 20 mM HEPES, 18 mM D-glucose, at pH 6.4 unless otherwise indicated) and pre-incubated in Ringer solution for 10 min at 37°C. The cells were further incubated for 1 min in pre-warmed fresh Ringer solution containing 10 μM [8–¹⁴C]-urate unless otherwise indicated. To examine the OAT10-inhibitory effects of the target compounds, Ringer solution either without (i.e., with only vehicle control, 0.1% dimethyl sulfoxide, DMSO) or with the individual compounds at the indicated concentrations was used. Subsequently, the cells were washed three times with ice-cold Ringer solution, lysed with 500 μl of 0.2 M NaOH, and neutralized with 100 μl of 1 M HCl. We then measured the radioactivity in the lysate using a liquid scintillator (Tri-Carb 3110TR; PerkinElmer, Waltham, MA, United States). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit, as described above. The urate transport activity was calculated as the incorporated clearance (μL/mg protein/min) [incorporated level of urate (DPM/mg protein/min)/urate level in the incubation mixture (DPM/μL)]. The OAT10-mediated urate transport activity was calculated by subtracting the urate transport activity of mock cells from that of OAT10-expressing cells. To examine the OAT10-inhibitory effects of the target compounds, their effects on the urate transport activity of mock cells were also examined.

With URAT1, urate uptake assay using URAT1-expressing 293A cells was conducted with a Cl⁻-free transport buffer [Cl⁻-free Hanks’ Balanced Salt Solution (HBSS): 125 mM Na-gluconate, 4.8 mM K-gluconate, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.3 mM Ca-gluconate, 25 mM HEPES, 5.6 mM D-glucose, at pH 6.4 unless otherwise indicated] in a similar way according to our previous studies (Miyata et al., 2016; Toyoda et al., 2020a). In this case, after pre-incubation in Cl⁻-free HBSS for 15 min at 37°C, the cells were further incubated for 20 s in pre-warmed fresh Cl⁻-free HBSS containing 10 μM [8–¹⁴C]-urate either without (i.e., with only vehicle control, 0.1% DMSO) or with the individual compounds at the indicated concentrations. Of note, imposing a Cl⁻ gradient by removal of external Cl⁻ results in the acceleration of URAT1-mediated urate uptake into the cell (Enomoto et al., 2002), bringing a relative reduction in the background levels for urate transport activities. Given this advantage for conducting in vitro assays, the Cl⁻-free condition has been used for functional investigations on URAT1 in various studies (Anzai et al., 2004; Miyata et al., 2016; Bao et al., 2019; Toyoda et al., 2020a). In this point, we had confirmed that the Cl⁻-free HBSS is a better choice for inhibitory tests against URAT1-mediated urate transport than Ringer solution (Supplementary Figure S1).

To calculate the half maximal inhibitory concentration (IC₅₀) values of the target compounds against urate transport by OAT10 or URAT1, the urate transport activities were measured in the presence of each compound at several concentrations. The OAT10- or URAT1-mediated transport activities were expressed as a percentage of the control (100%). Based on the calculated values, fitting curves were obtained according to the following formula using the least-squares method with Excel 2019 (Microsoft, Redmond, WA, United States) as described previously (Toyoda et al., 2020a): Predicted   value [ % ] = 100 − ( E max × C n EC 50 n + C n ) where, E_max is the maximum effect, EC₅₀ is the half-maximal effective concentration, C is the concentration of the test compound, and n is the sigmoid-fit factor. Finally, based on these results, the IC₅₀ was calculated.

With clinical analyses, statistical analyses were performed using SPSS v.22.0J (IBM Japan, Tokyo, Japan). Univariate linear regression analysis was used for quantitative trait locus analyses. With functional analyses, all statistical analyses were performed using Excel 2019 with Statcel4 add-in software (OMS publishing, Saitama, Japan). To determine the kinetic parameters of the OAT10-mediated urate transport—Michaelis-Menten constant (K _m) and maximal velocity (V _max), the Michaelis–Menten model was fitted to the experimental values of the urate transport rates, and the urate concentrations by non-linear regression curve fitting using GraphPad Prism 8 (GraphPad Software, San Diego, CA, United States). Different statistical tests were used for different experiments, as described in the table captions of figure legends which include the number of biological replicates (n). In the case of a single pair of quantitative data, after comparing the variances of a set of data using an F-test, an unpaired Student’s t-test was performed. Statistical significance was defined in terms of p values less than 0.05 or 0.01.

To investigate the effect of a genetic dysfunction of OAT10 on renal urate secretion, we conducted clinico-genetic analyses. Regarding the functionally null variant (OAT10 c.1129C>T), only a few subjects were homozygotes (1129T/T); the others were heterozygotes (1129C/T) or wild-types (1129C/C). Thus, we compared the following parameters related to renal urate handling between the latter two groups (OAT10 1129C/T vs. 1129C/C) (Figure 1). Serum urate levels were significantly lower (Figure 1A; Supplementary Table S2) and the values of FE_UA were significantly higher (Figure 1B; Supplementary Table S3) in heterozygous carriers of OAT10 c.1129T (C377) than those in non-carriers. This inverse association suggested that, like URAT1, OAT10 could be involved in urate re-uptake from urine. Additionally, the value of coefficient for the effect allele with the urate-lowering effect was −0.135 mg/dl/allele (Supplementary Table S2) which was not so large but could support the physiological importance of OAT10 as discussed later (see Discussion). Also, similar results were obtained when we combined the homozygotes with the heterozygotes (Supplementary Tables S4, S5). Moreover, immunohistochemistry revealed the luminal expression of OAT10 in the proximal tubules in the cortex of the human kidney (Figure 2), but the expression of OAT10 was hardly detected in the medulla, which is a similar pattern to that of URAT1 (Enomoto et al., 2002). The specificity of the anti-OAT10 antibody was verified by antigen absorption test (Supplementary Figure S2). Also, this luminal expression of OAT10 in the proximal tubules is consistent with the data in the Human Protein Atlas (Uhlén et al., 2015) (https://www.proteinatlas.org/ENSG00000172940-SLC22A13/tissue/kidney#img). Besides, when expressed in polarized MDCKII cells, OAT10 was matured as a glycoprotein (Figure 3A) and localized on the apical membrane, similar to URAT1 (Figure 3B). These results indicate that OAT10 prefers to be on the apical side of renal proximal tubule cells in the kidney, the position required for the urate re-uptake from urine into the cell. These findings are consistent with a result of immunoblotting addressing renal expression of rat Oat10 in a previous study (Bahn et al., 2008). Given these clinical and experimental data, OAT10 would be a renal urate re-absorber in humans that influences serum urate levels and net amount of renal urate excretion.

To gain insight into the molecular properties of OAT10 as a urate transporter, we performed in vitro functional analyses using transiently OAT10-expressing HEK-derived 293A cells. The measured amount of radiolabeled urate incorporated into the cells revealed that OAT10 was more active at lower pH, mimicking urine conditions (Figure 4A). A similar result was obtained in URAT1 (Supplementary Figure S1). On the other hand, in contrast to the case in URAT1, OAT10-mediated urate transport was remarkably decreased by excluding Cl⁻ from the Ringer solution (Figure 4B); the calculated OAT10-mediated urate transport activities in the Cl⁻-free condition were 6.1 ± 4.5% of those in the normal Ringer solution. This chloride-dependency is consistent with previous results obtained in an oocyte study (Mandal et al., 2017). Moreover, from time-course experiments we used uptake at 1 min to determine the initial rate of urate uptake by OAT10 (Figure 4C). Hence, uptake for 1 min at pH 6.4—a condition appropriate for the evaluation of OAT10-mediated urate transport—was examined in subsequent analyses. Under the experimentally maximum urate concentration (500 μM, which was due to the limited solubility of uric acid in the transport buffer), OAT10-mediated urate uptake was not completely saturated; thus, the estimated K _m and V _max for urate were 558 μM and 518 pmol/min per mg protein, respectively (Figure 4D). Considering that the K _m value of OAT10 was the second lowest among the already characterized human urate transporters working as importers next to URAT1 (K _m for urate, 371 μM) (Enomoto et al., 2002) (Table 3), OAT10 may have a role as a parallel backup of URAT1.

Although some drugs used for urate-lowering therapy inhibit URAT1 function, their effects on OAT10 remain unclear. Thus, we investigated the OAT10-inhibitory activities of urate-lowering drugs and their active metabolites (total 11), including urate synthesis inhibitors, uricosuric agents, fenofibrate, and losartan, five of which were compared with those against URAT1 (Figure 5).

First, we conducted inhibitory tests in the presence of each authentic compound at the maximum soluble levels in the transport buffer (Table 2). The results revealed that, contrary to all the urate synthesis inhibitors and fenofibrate, five compounds—benzbromarone, 6-hydroxybenzbromarone (an active metabolite of benzbromarone), dotinurad, lesinurad, and losartan—decreased the urate transport activity of OAT10 to < 20% that of the vehicle control (Figure 5A). Next, we compared their effects at 10 μM on the urate transport activity of OAT10 and URAT1 (Figure 5B), which demonstrated that i) benzbromarone, 6-hydroxybenzbromarone, and dotinurad were more specific to URAT1 than OAT10; ii) lesinurad inhibited OAT10 and URAT1 comparably; iii) losartan exhibited a stronger inhibitory effect on OAT10 than URAT1. These results were confirmed by further investigations of concentration-dependent inhibitory effects and calculated IC₅₀ values (Figure 5C). Given the proposed mechanism, lesinurad (Miner et al., 2016) and losartan (Lee and Terkeltaub, 2006) do and can inhibit URAT1 in the kidney, respectively; hence, they may also act as renal OAT10 inhibitors in clinical situations (Figure 6). Of note, given that lesinurad reportedly inhibits not only URAT1 but also other transporters such as OATP1B1, OCT1, OAT1 and OAT3 in vitro (Garg et al., 2018), lesinurad is not necessarily highly-specific to URAT1. Thus, the OAT10-inhibitory effect of lesinurad we identified here is not inconsistent with previous findings.