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.

Impact of Direction of Transport on the Evaluation of Substrate Recognition of Mouse Multidrug and Toxin Extrusion Protein 1.

Multidrug and toxin extrusion protein (MATE/SLC47A) secretes metabolites and xenobiotics into the urine in the proximal tubules of the kidney. Uptake assays have been commonly used for evaluating MATE-mediated transport of new chemical entities in drug development. The purpose of this study was to examine the relationship between in vitro uptake activities by MATEs and the impact of MATE-mediated transport in in vivo renal secretion. In vitro uptake in mouse Mate1 (mMate1)-expressing human embryonic kidney 293 (HEK293) cells and several in vivo parameters from mMate1 knockout and wild-type mice were compared using nine cationic compounds (almotriptan, naratriptan, talinolol, sumatriptan, alogliptin, sitagliptin, rivaroxaban, saxagliptin, and vildagliptin). Compounds that showed statistically significant decrease in secretory clearances with respect to kidney concentrations (CLR,kidney) in mMate1 knockout mice were categorized as in vivo substrates in this study. A good correlation (R2 = 0.637) was observed between the in vitro uptake ratio and the in vivo ratio of CLR,kidney of mMate1 knockout mice and wild-type mice. This study supported the rationale of using an uptake assay to determine whether investigational compounds are the substrate of MATEs and to predict drug-drug interaction risk via renal secretion by MATE from the viewpoint of drug development in pharmaceutical companies. SIGNIFICANCE STATEMENT: We revealed that substrates judged by in vitro experiments using mouse multidrug and toxin extrusion (mMate)1-expressing cells were excreted in urine via mMate1 in vivo, and a good correlation (R2 = 0.637) was observed between in vitro uptake ratio and in vivo ratio of secretory clearance with respect to the kidney concentrations (CLR,kidney) of mMate1 knockout and wild-type mice. This study supported the rationale of using an uptake assay to predict potential human MATE1-mediated drug-drug interaction as a victim.

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research.

The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.

Impact of Direction of Transport on the Evaluation of Inhibition Potencies of Multidrug and Toxin Extrusion Protein 1 Inhibitors.

Multidrug and toxin extrusion (MATE) transporters are expressed on the luminal membrane of renal proximal tubule cells and extrude their substrates into the luminal side of the tubules. Inhibition of MATE1 can reduce renal secretory clearance of its substrate drugs and lead to drug-drug interactions (DDIs). To address whether IC50 values of MATE1 inhibitors with regard to their extracellular concentrations are affected by the direction of MATE1-mediated transport, we established an efflux assay of 1-methyl-4-phenylpyridinium (MPP+) and metformin using the human embryonic kidney 293 model transiently expressing human MATE1. The efflux rate was defined by reduction of the cellular amount of MPP+ and metformin for 0.25 minutes shortly after the removal of extracellular MPP+ and metformin. Inhibition potencies of 12 inhibitors toward MATE1-mediated transport were determined in both uptake and efflux assays. When MPP+ was used as a substrate, 8 out of 12 inhibitors showed comparable IC50 values between assays (<4-fold). IC50 values from the efflux assays were higher for cimetidine (9.9-fold), trimethoprim (10-fold), famotidine (6.4-fold), and cephalexin (>3.8-fold). When metformin was used as a substrate, IC50 values of the tested inhibitors when evaluated using uptake and efflux assays were within 4-fold of each other, with the exception of cephalexin (>4.7-fold). IC50 values obtained from the uptake assay using metformin showed smaller IC50 values than those from the efflux assay. Therefore, the uptake assay is recommended to determine IC50 values for the DDI predictions. SIGNIFICANCE STATEMENT: In this study, a new method to evaluate IC50 values of extracellular added inhibitors utilizing an efflux assay was established. IC50 values were not largely different between uptake and efflux directions but were smaller for uptake. This study supports the rationale for a commonly accepted uptake assay with metformin as an in vitro probe substrate for multidrug and toxin extrusion 1-mediated drug-drug interaction risk assessment in drug development.

Impact of Experimental Conditions on the Evaluation of Interactions between Multidrug and Toxin Extrusion Proteins and Candidate Drugs.

Multidrug and toxin extrusion transporters (MATEs) have a determining influence on the pharmacokinetic profiles of many drugs and are involved in several clinical drug-drug interactions (DDIs). Cellular uptake assays with recombinant cells expressing human MATE1 or MATE2-K are widely used to investigate MATE-mediated transport for DDI assessment; however, the experimental conditions and used test substrates vary among laboratories. We therefore initially examined the impact of three assay conditions that have been applied for MATE substrate and inhibitor profiling in the literature. One of the tested conditions resulted in significantly higher uptake rates of the three test substrates, [(14)C]metformin, [(3)H]thiamine, and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)), but IC50 values of four tested MATE inhibitors varied only slightly among the three conditions (<2.5-fold difference). Subsequently, we investigated the uptake characteristics of the five MATE substrates: [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), [(3)H]estrone-3-sulfate (E3S), and rhodamine 123, as well as the impact of the used test substrate on the inhibition profiles of 10 MATE inhibitors at one selected assay condition. [(3)H]E3S showed atypical uptake characteristics compared with those observed with the other four substrates. IC50 values of the tested inhibitors were in a similar range (<4-fold difference) when [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), or [(3)H]E3S were used as substrates but were considerably higher with rhodamine 123 (9.8-fold and 4.1-fold differences compared with [(14)C]metformin with MATE1 and MATE2-K, respectively). This study demonstrated for the first time that the impact of assay conditions on IC50 determination is negligible, that kinetic characteristics differ among used test substrates, and that substrate-dependent inhibition exists for MATE1 and MATE2-K, giving valuable insight into the assessment of clinically relevant MATE-mediated DDIs in vitro.