Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively), compared to 19 nM for the wild-type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8,420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. Significance Statement Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting OATs. This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 was found to mediate hydrogen bonding.

Role of Organic Anion Transporter NPT4 in Renal Handling of Uremic Toxin 3-indoxyl Sulfate.

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3-/- mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3-/- mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3-/- mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3-/- mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.

Prediction and causal inference of hyperuricemia using gut microbiota.

Hyperuricemia (HUA) is a symptom of high blood uric acid (UA) levels, which causes disorders such as gout and renal urinary calculus. Prolonged HUA is often associated with hypertension, atherosclerosis, diabetes mellitus, and chronic kidney disease. Studies have shown that gut microbiota (GM) affect these chronic diseases. This study aimed to determine the relationship between HUA and GM. The microbiome of 224 men and 254 women aged 40 years was analyzed through next-generation sequencing and machine learning. We obtained GM data through 16S rRNA-based sequencing of the fecal samples, finding that alpha-diversity by Shannon index was significantly low in the HUA group. Linear discriminant effect size analysis detected a high abundance of the genera Collinsella and Faecalibacterium in the HUA and non-HUA groups. Based on light gradient boosting machine learning, we propose that HUA can be predicted with high AUC using four clinical characteristics and the relative abundance of nine bacterial genera, including Collinsella and Dorea. In addition, analysis of causal relationships using a direct linear non-Gaussian acyclic model indicated a positive effect of the relative abundance of the genus Collinsella on blood UA levels. Our results suggest abundant Collinsella in the gut can increase blood UA levels.

Drug-drug conjugates of MEK and Akt inhibitors for RAS-mutant cancers.

Controlling RAS mutant cancer progression remains a significant challenge in developing anticancer drugs. Whereas Ras G12C-covalent binders have received clinical approval, the emergence of further mutations, along with the activation of Ras-related proteins and signals, has led to resistance to Ras binders. To discover novel compounds to overcome this bottleneck, we focused on the concurrent and sustained blocking of two major signaling pathways downstream of Ras. To this end, we synthesized 25 drug-drug conjugates (DDCs) by combining the MEK inhibitor trametinib with Akt inhibitors using seven types of linkers with structural diversity. The DDCs were evaluated for their cell permeability/accumulation and ability to inhibit proliferation in RAS-mutant cell lines. A representative DDC was further evaluated for its effects on signaling proteins, induction of apoptosis-related proteins, and the stability of hepatic metabolic enzymes. These in vitro studies identified a series of DDCs, especially those containing a furan-based linker, with promising properties as agents for treating RAS-mutant cancers. Additionally, in vivo experiments in mice using the two selected DDCs revealed prolonged half-lives and anticancer efficacies comparable to those of trametinib. The PK profiles of trametinib and the Akt inhibitor were unified through the DDC formation. The DDCs developed in this study have potential as drug candidates for the broad inhibition of RAS-mutant cancers.

Dynamic, IPSC-derived hepatic tissue tri-culture system for the evaluation of liver physiologyin vitro.

Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derivedin vitrosystems that can compare functionally with currently used pharmacological and toxicological applications.

Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides.

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.

Uric acid in health and disease: From physiological functions to pathogenic mechanisms.

Owing to renal reabsorption and the loss of uricase activity, uric acid (UA) is strictly maintained at a higher physiological level in humans than in other mammals, which provides a survival advantage during evolution but increases susceptibility to certain diseases such as gout. Although monosodium urate (MSU) crystal precipitation has been detected in different tissues of patients as a trigger for disease, the pathological role of soluble UA remains controversial due to the lack of causality in the clinical setting. Abnormal elevation or reduction of UA levels has been linked to some of pathological status, also known as U-shaped association, implying that the physiological levels of UA regulated by multiple enzymes and transporters are crucial for the maintenance of health. In addition, the protective potential of UA has also been proposed in aging and some diseases. Therefore, the role of UA as a double-edged sword in humans is determined by its physiological or non-physiological levels. In this review, we summarize biosynthesis, membrane transport, and physiological functions of UA. Then, we discuss the pathological involvement of hyperuricemia and hypouricemia as well as the underlying mechanisms by which UA at abnormal levels regulates the onset and progression of diseases. Finally, pharmacological strategies for urate-lowering therapy (ULT) are introduced, and current challenges in UA study and future perspectives are also described.

Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

Mechanobiological stimulation in organ-on-a-chip systems reduces hepatic drug metabolic capacity in favor of regenerative specialization.

Hepatic physiology depends on the liver's complex structural composition which among others, provides high oxygen supply rates, locally differential oxygen tension, endothelial paracrine signaling, as well as residual hemodynamic shear stress to resident hepatocytes. While functional improvements were shown by implementing these factors into hepatic culture systems, direct cause-effect relationships are often not well characterized-obfuscating their individual contribution in more complex microphysiological systems. By comparing increasingly complex hepatic in vitro culture systems that gradually implement these parameters, we investigate the influence of the cellular microenvironment to overall hepatic functionality in pharmacological applications. Here, hepatocytes were modulated in terms of oxygen tension and supplementation, endothelial coculture, and exposure to fluid shear stress delineated from oxygen influx. Results from transcriptomic and metabolomic evaluation indicate that particularly oxygen supply rates are critical to enhance cellular functionality-with cellular drug metabolism remaining comparable to physiological conditions after prolonged static culture. Endothelial signaling was found to be a major contributor to differential phenotype formation known as metabolic zonation, indicated by WNT pathway activity. Lastly, oxygen-delineated shear stress was identified to direct cellular fate towards increased hepatic plasticity and regenerative phenotypes at the cost of drug metabolic functionality - in line with regenerative effects observed in vivo. With these results, we provide a systematic evaluation of critical parameters and their impact in hepatic systems. Given their adherence to physiological effects in vivo, this highlights the importance of their implementation in biomimetic devices, such as organ-on-a-chip systems. Considering recent advances in basic liver biology, direct translation of physiological structures into in vitro models is a promising strategy to expand the capabilities of pharmacological models.

A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.

Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Low blood level of tumour suppressor miR-5193 as a target of immunotherapy to PD-L1 in gastric cancer.

BACKGROUND: Recent studies have identified that low levels of some tumour suppressor microRNAs (miRNAs) in the blood contribute to tumour progression and poor outcomes in various cancers. However, no study has proved these miRNAs are associated with cancer immune mechanisms. METHODS: From a systematic review of the NCBI and miRNA databases, four tumour suppressor miRNA candidates were selected (miR-5193, miR-4443, miR-520h, miR-496) that putatively target programmed cell death ligand 1 (PD-L1). RESULTS: Test-scale and large-scale analyses revealed that plasma levels of miR-5193 were significantly lower in gastric cancer (GC) patients than in healthy volunteers (HVs). Low plasma levels of miR-5193 were associated with advanced pathological stages and were an independent prognostic factor. Overexpression of miR-5193 in GC cells suppressed PD-L1 on the surface of GC cells, even with IFN-γ stimulation. In the coculture model of GC cells and T cells stimulated by anti-CD3/anti-CD28 beads, overexpression of miR-5193 increased anti-tumour activity of T cells by suppressing PD-L1 expression. Subcutaneous injection of miR-5193 also significantly enhanced the tumour-killing activity and trafficking of T cells in mice. CONCLUSIONS: Low blood levels of miR-5193 are associated with GC progression and poor outcomes and could be a target of nucleic acid immunotherapy in GC patients.

Application Site of Transdermal Scopolamine Influences Efficacy and Drug Concentration in Salivary Glands in Rats.

Transdermal scopolamine applied to the postauricular area is used to treat drooling. We investigated the duration of action of scopolamine ointment and the effect of the application site on drug efficacy and concentration in the salivary glands of rats. Scopolamine ointment was applied to the skin over the salivary glands (SSG) and back (SB). Saliva volume was measured after intraperitoneal administration of pilocarpine. Blood and salivary glands were collected after scopolamine ointment application, and scopolamine concentrations in the plasma and salivary glands were measured. Saliva volume after application in the SSG group was significantly lower at all time points than in the non-treated group, and the change in saliva volume in the SSG group was greater than that in the SB group at all time points. This suggests that applying scopolamine ointment to the SSG strongly suppresses salivary secretion. Scopolamine concentration in the salivary glands of the SSG group was significantly higher at 9 h. The change in the efficacy of scopolamine ointment depending on the application site was due to the difference in transfer to the salivary glands. Transdermal administration of scopolamine to the skin over the salivary glands may have high efficiency in treating drooling.

Differences of clinical features and outcomes between male and female elderly patients in gastric cancer.

Although the average life span differs between males and females, little is known about differences in clinical features and short and long-term outcomes between elderly male and female gastric cancer patients. This study was designed to clarify these issues to identify the possibility for sex-based treatment strategies in elderly gastric cancer patients. This study included 295 consecutive elderly gastric cancer patients (75 years or older) who underwent curative gastrectomy between 1997 and 2016. We defined postoperative complications as Clavien-Dindo classification grade II or higher. Comorbidities were present in 67% of all patients. Males tended to have more comorbidities than females (P = 0.077). Male patients had significantly more upper gastric cancers (P = 0.001), a higher incidence of postoperative complications (P = 0.045), and poorer prognoses than females (P = 0.003). Multivariate analysis revealed that being male was an independent risk factor for postoperative complications (Odds ratio 2.5, P = 0.045) and a poor prognostic factor (Hazard ratio 1.81, P = 0.008). Patients who underwent limited surgery without postoperative complications tended to have a better prognosis than patients receiving standard surgery with postoperative complications (3-year overall survival: 78% vs. 55%, P = 0.156). Male was an independent risk factor for postoperative complications and an independent poor prognostic factor in elderly gastric cancer patients. To avoid postoperative complications, the limited surgery might be justified for high-risk elderly male patients.

Lateral Force and EMG Activity in Wide- and Narrow-Grip Bench Press in Various Conditions.

The purpose of this study was to investigate the lateral force and contribution of shoulder horizontal adductor and elbow extensor muscles activity during wide- and narrow-grip bench press (BP) in various conditions, such as resistance-trained/non-trained, concentric/eccentric, and muscle fatigue/non-fatigue. We measured the lateral force on the bar and the electromyographic (EMG) muscle activity of pectoralis major (PM) and triceps brachii (TB) during 10 RM BP with wide grip (81 cm) and narrow grip (40 cm) in seven resistance-trained men and seven non-trained men. The all-reps average of the lateral-to-vertical force ratio both in resistance-trained and non-trained subjects was about 30% outward for the wide grip and about 10% inward for the narrow grip. The EMG contribution ratio PM/TB shows no significant differences between narrow and wide grip in all evaluated conditions except in non-trained subjects' muscle fatigue eccentric phase. Both resistance-trained and non-trained subjects did not push the bar straight upward, and the EMG PM/TB was almost unchanged by hand width. The direction adjustment of the force on the bar that achieves almost the same muscle activity degree of the shoulder and elbow joints might be optimal BP kinetics.

Renal Pharmacokinetic Adaptation to Cholestasis Causes Increased Nephrotoxic Drug Accumulation by Mrp6 Downregulation in Mice.

In hepatic dysfunction, renal pharmacokinetic adaptation can be observed, although information on the changes in drug exposure and the interorgan regulation of membrane transporters in kidney in liver diseases is limited. This study aimed to clarify the effects of renal exposure to nephrotoxic drugs during cholestasis induced by bile duct ligation (BDL). Among the 11 nephrotoxic drugs examined, the tissue accumulation of imatinib and cisplatin in kidney slices obtained from mice 2 weeks after BDL operation was higher than that in sham-operated mice. The uptake of imatinib in the kidney slices of BDL mice was slightly higher, whereas its efflux from the slices was largely decreased compared to that in sham-operated mice. Proteomic analysis revealed a reduction in renal expression of the efflux transporter multidrug resistance-associated protein 6 (Mrp6/Abcc6) in BDL mice, and both imatinib and cisplatin were identified as Mrp6 substrates. Survival probability after cisplatin administration was reduced in BDL mice. In conclusion, the present study demonstrated that BDL-induced cholestasis leads to the downregulation of the renal basolateral efflux transporter Mrp6, resulting in drug accumulation in renal cells and promoting drug-induced renal injury.

Potentiation of the Uricosuric Effect of Dotinurad by Trans-Inhibition of the Uric Acid Reabsorptive Transporter 1.

Urate transporter 1 (URAT1) is a transporter responsible for uric acid (UA) reabsorption by renal proximal tubules and a pharmacological target of uricosuric agents. Probenecid and benzbromarone have been used as uricosuric agents, while dotinurad was recently approved in Japan. Notably, the in vitro IC 50 of dotinurad on URAT1 is not strong enough to explain its in vivo uricosuric effect estimated based on clinical unbound plasma concentrations, suggesting the presence of mechanisms other than competition with UA uptake at the extracellular domain of URAT1 (cis-inhibition). In this study, trans-inhibition was hypothesized as the mechanism underlying URAT1 inhibition by dotinurad, wherein intracellularly accumulated dotinurad inactivates URAT1. In URAT1-expressing Madin-Darby Canine Kidney-II cells and Xenopus oocytes, pre-incubation with dotinurad potentiated the inhibitory effect more than co-incubation alone, but this effect was not observed with benzbromarone or probenecid. Under co-incubation, dotinurad inhibited UA uptake in a competitive manner (cis-inhibition). When we pre-injected dotinurad directly into oocytes and immediately measured [14C]UA uptake without coincubation (only trans-inhibition), dotinurad noncompetitively inhibited UA uptake. URAT1 is an exchange transporter for UA and monocarboxylates such as nicotinic acid (NA). Pre-injected dotinurad and extracellular UA attenuated and facilitated efflux of [3H]NA, respectively, whereas pre-injection of benzbromarone or probenecid did not affect it, suggesting that dotinurad exhibits trans-inhibition by attenuating URAT1-mediated efflux of monocarboxylates, which is a driving force for UA uptake by URAT1. Accordingly, dotinurad ameliorates URAT1-mediated UA reabsorption by both cis- and trans-inhibition, explaining its clinically stronger uricosuric effect than that estimated by the in vitro IC50 value. SIGNIFICANCE STATEMENT: The uricosuric agent dotinurad inhibits uric acid reabsorptive transporter (URAT) 1 with a clinical potency stronger than that estimated from IC 50 obtained by in vitro URAT1 inhibition. This in vivo-in vitro discrepancy was explained by the trans-inhibition effect of dotinurad on URAT1. Trans-inhibition was due to the attenuation of monocarboxylates efflux via URAT1, which is a driving force for URAT1-mediated exchange transport of uric acid. Overall, this is the first study to experimentally demonstrate trans-inhibition mechanism of URAT1.

Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion.

Biliary excretion is a major drug elimination pathway that affects their efficacy and safety. The currently available in vitro sandwich-cultured hepatocyte method is cumbersome because drugs accumulate in the closed bile canalicular lumen formed between hepatocytes and their amounts cannot be mealsured directly. This study proposes a hepatocyte culture model for the rapid evaluation of drug biliary excretion using permeation assays. When hepatocytes are cultured on a permeable support coated with the cell adhesion protein claudins, an open-form bile canalicular lumen is formed at the surface of the permeable support. Upon application to the basolateral (blood) side, drugs appear on the bile canalicular side. The biliary excretion clearance of several drugs, as estimated from the obtained permeabilities, correlates well with the reported in vivo biliary excretion clearance in humans. Thus, the established model is useful for applications in the efficient evaluation of biliary excretion during drug discovery and development.

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.

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.