Functional characterization of human organic anion transporter 10 (OAT10/SLC22A13) as an orotate transporter

Orotate, a nutritional compound typically utilized as an intermediate in pyrimidine synthesis, has been suggested to undergo renal reabsorption. However, the detailed mechanisms involved in the process remain unclear, with only urate transporter 1 (URAT1/SLC22A12) being indicated as a transporter involved in its tubular uptake. As an attempt to identify transporters involved in that to help clarify the mechanisms, we examined a possibility that organic anion transporter 10 (OAT10/SLC22A13), which is present at the brush border membrane in renal tubular epithelial cells, could transport orotate. The operation of human OAT10 for orotate transport was demonstrated indeed and analyzed in detail in Madin-Darby canine kidney II cells introduced with this transporter by stable transfection. Orotate transport by OAT10 was found to be kinetically saturable with a biphasic characteristic and dependent on Cl⁻. These are unique characteristics previously unknown in its operation for the other substrates. Orotate transport by OAT10 was, on the other hand, inhibited by several anionic compounds known as OAT10 inhibitors. Finally, the rat ortholog of OAT10 was found not to be able to transport orotate, indicating animal species differences in that function. Thus, human OAT10 has been demonstrated to operate for orotate transport with unique characteristics.     

Targeting uptake transporters for cancer imaging and treatment

Cancer cells reprogram their gene expression to promote growth, survival, proliferation, and invasiveness. The unique expression of certain uptake transporters in cancers and their innate function to concentrate small molecular substrates in cells make them ideal targets for selective delivering imaging and therapeutic agents into cancer cells. In this review, we focus on several solute carrier (SLC) transporters known to be involved in transporting clinically used radiopharmaceutical agents into cancer cells, including the sodium/iodine symporter (NIS), norepinephrine transporter (NET), glucose transporter 1 (GLUT1), and monocarboxylate transporters (MCTs). The molecular and functional characteristics of these transporters are reviewed with special emphasis on their specific expressions in cancers and interaction with imaging or theranostic agents [e.g., I-123, I-131, ¹²³I-iobenguane (mIBG), ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and ¹³C pyruvate]. Current clinical applications and research areas of these transporters in cancer diagnosis and treatment are discussed. Finally, we offer our views on emerging opportunities and challenges in targeting transporters for cancer imaging and treatment. By analyzing the few clinically successful examples, we hope much interest can be garnered in cancer research towards uptake transporters and their potential applications in cancer diagnosis and treatment.     

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.     

Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease

Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.     

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na⁺-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na⁺-dependent and electrogenic. The Na⁺-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na⁺-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na⁺-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.     

Impaired Transport Activity of Human Organic Anion Transporters (OATs) and Organic Anion Transporting Polypeptides (OATPs) by Wnt Inhibitors

The Wnt/β-catenin signaling pathway is dysregulated in diseases and Wnt inhibitors like PRI-724 are in clinical development. This study evaluated the regulatory actions of PRI-724 and other Wnt inhibitors on the transport activity of human renal Organic anion transporters (OATs) and Organic anion transporting polypeptides (OATPs). The substrate uptake by OAT4 and OATP2B1 was markedly decreased by PRI-724 (V_max/K_m: ～26% and ～17% of corresponding control), with less pronounced decreases in OAT1, OAT3 and OAT1A2. PRI-724 decreased the plasma membrane expression of inhibited OATs/OATPs but didn't affect their total cellular expression. Two model Wnt inhibitors - FH535 and 21H7 - were also tested in comparative studies. Like PRI-724, they also strongly decreased the activities and membrane expression of multiple OATs/OATPs. In contrast, FH535 didn't affect the substrate uptake by organic cation transporters. In control studies, the EGFR inhibitor lapatinib did not inhibit the function of some OATs/OATPs. Together these findings suggest that Wnt inhibitors selectively modulate the function of multiple organic anions transporters, so their clinical use may have unanticipated effects on drug entry into cells. These findings are pertinent to current clinical trials that have been designed to understand the safety and efficacy of new Wnt inhibitor drugs.     

A Novel Fluorescence-Based Method to Evaluate Ileal Apical Sodium-Dependent Bile Acid Transporter ASBT

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 μM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 μM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 μM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na⁺ free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na⁺-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.     

Mathematical modeling analysis of hepatic uric acid disposition using human sandwich-cultured hepatocytes

Uric acid is biosynthesized from purine by xanthine oxidase (XO) mainly in the liver and is excreted into urine and feces. Although several transporters responsible for renal and intestinal handling of uric acid have been reported, information on hepatic transporters is limited. In the present study, we studied quantitative contribution of transporters for hepatic handling of uric acid by mathematical modeling analysis in human sandwich-cultured hepatocytes (hSCH). Stable isotope-labeled hypoxanthine, hypoxanthine-¹³C2,¹⁵N (HX), was incubated with hSCH and formed ¹³C2,¹⁵N-labeled xanthine (XA) and uric acid (UA) were measured by LC-MS/MS time dependently. Rate constants for metabolism and efflux and uptake transport across sinusoidal and bile canalicular membranes of HX, XA and UA were estimated in the presence of inhibitors of XO and uric acid transporters. An XO inhibitor allopurinol significantly decreased metabolisms of HX and XA. Efflux into bile canalicular lumen was negligible and sinusoidal efflux was considered main efflux pathway of formed UA. Transporter inhibition study highlighted that GLUT9 strongly and MRP4 intermediately contribute to the sinusoidal efflux of UA with minor contribution of NPT1/4. Modeling analysis developed in the present study should be useful for quantitative prediction of uric acid disposition in liver.     

6-Hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor elevated in renal failure patients

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [³H]estrone-3-sulfate (E₁S), although some directly inhibited [³H]E₁S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [³H]E₁S uptake, even after the inhibitor was washed out before [³H]E₁S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [³H]E₁S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [³H]E₁S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.     

pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the K_m value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.     

Evaluation of transporter-mediated hepatobiliary transport of newly developed 18F-labeled pitavastatin derivative,PTV-F1, in rats by PET imaging

Quantitative evaluations of the functions of uptake and efflux transporters directly in vivo is desired to understand an efficient hepatobiliary transport of substrate drugs. Pitavastatin is a substrate of organic anion transporting polypeptides (OATPs) and canalicular efflux transporters; thus, it can be a suitable probe for positron-emission tomography (PET) imaging of hepatic transporter functions. To characterize the performance of [¹⁸F]PTV-F1, an analogue of pitavastatin, we investigated the impact of rifampicin (a typical OATP inhibitor) coadministration or Bcrp (breast cancer resistance protein) knockout on [¹⁸F]PTV-F1 hepatic uptake and efflux in rats by PET imaging. After intravenous administration, [¹⁸F]PTV-F1 selectively accumulated in the liver, and the radioactivity detected in plasma, liver, and bile mainly derived from the parent PTV-F1 during the PET study (∼40 min). Coadministration of rifampicin largely decreased the hepatic uptake of [¹⁸F]PTV-F1 by 73%. Because of its lower clearance in rats, [¹⁸F]PTV-F1 is more sensitive for monitoring changes in hepatic OATP1B function that other previously reported OATP1B PET probes. Rifampicin coadministration also significantly decreased the biliary excretion of radioactivity by 65%. Bcrp knockout did not show a significant impact on its biliary excretion.[¹⁸F]PTV-F1 enables quantitative analysis of the hepatobiliary transport system for organic anions.     

Cilastatin protects against imipenem-induced nephrotoxicity via inhibition of renal organic anion transporters (OATs)

Imipenem is a carbapenem antibiotic. However, Imipenem could not be marketed owing to its instability and nephrotoxicity until cilastatin, an inhibitor of renal dehydropeptidase-I (DHP-I), was developed. In present study, the potential roles of renal organic anion transporters (OATs) in alleviating the nephrotoxicity of imipenem by cilastatin were investigated in vitro and in rabbits. Our results indicated that imipenem and cilastatin were substrates of hOAT1 and hOAT3. Cilastatin inhibited hOAT1/3-mediated transport of imipenem with IC₅₀ values comparable to the clinical concentration, suggesting the potential to cause a clinical drug–drug interaction (DDI). Moreover, imipenem exhibited hOAT1/3-dependent cytotoxicity, which was alleviated by cilastatin and probenecid. Furthermore, cilastatin and probenecid ameliorated imipenem-induced rabbit acute kidney injury, and reduced the renal secretion of imipenem. Cilastatin and probenecid inhibited intracellular accumulation of imipenem and sequentially decreased the nephrocyte toxicity in rabbit primary proximal tubule cells. Renal OATs, besides DHP-I, was also the target of interaction between imipenem and cilastatin, and contributed to the nephrotoxicity of imipenem. This therefore gives in part the explanation about the mechanism by which cilastatin protected against imipenem-induced nephrotoxicity. Thus, OATs can potentially be used as a therapeutic target to avoid the renal adverse reaction of imipenem in clinic.     

Altered cisplatin pharmacokinetics during nonalcoholic steatohepatitis contributes to reduced nephrotoxicity

Disease-mediated alterations to drug disposition constitute a significant source of adverse drug reactions. Cisplatin (CDDP) elicits nephrotoxicity due to exposure in proximal tubule cells during renal secretion. Alterations to renal drug transporter expression have been discovered during nonalcoholic steatohepatitis (NASH), however, associated changes to substrate toxicity is unknown. To test this, a methionine- and choline-deficient diet-induced rat model was used to evaluate NASH-associated changes to CDDP pharmacokinetics, transporter expression, and toxicity. NASH rats administered CDDP (6 mg/kg, i.p.) displayed 20% less nephrotoxicity than healthy rats. Likewise, CDDP renal clearance decreased in NASH rats from 7.39 to 3.83 mL/min, renal secretion decreased from 6.23 to 2.80 mL/min, and renal CDDP accumulation decreased by 15%, relative to healthy rats. Renal copper transporter-1 expression decreased, and organic cation transporter-2 and ATPase copper transporting protein-7b increased slightly, reducing CDDP secretion. Hepatic CDDP accumulation increased 250% in NASH rats relative to healthy rats. Hepatic organic cation transporter-1 induction and multidrug and toxin extrusion protein-1 and multidrug resistance-associated protein-4 reduction may contribute to hepatic CDDP sequestration in NASH rats, although no drug-related toxicity was observed. These data provide a link between NASH-induced hepatic and renal transporter expression changes and CDDP renal clearance, which may alter nephrotoxicity.     

Animal species differences in the pyridoxine transport function of SLC19A3: Absence of Slc19a3-mediated pyridoxine uptake in the rat small intestine

The thiamine transporters, SLC19A2 and SLC19A3, have recently been shown to transport pyridoxine in addition to thiamine, the originally identified substrate, in our study on human orthologs. Based on these results, we characterized the rat and mouse orthologs for pyridoxine transport function. Through the assessment of pyridoxine uptake in human embryonic kidney 293 cells transiently expressing the SLC19A2/3 orthologs, we found that both rat and mouse Slc19a2 can transport pyridoxine, but rat or mouse Slc19a3 cannot. However, all SLC19A2/3 orthologs were capable of thiamine transport. We subsequently demonstrated in the rat small intestine that a carrier-mediated mechanism exists for thiamine uptake, but not for pyridoxine uptake. This is supported by the finding that rat Slc19a3, for which the human ortholog operates for the intestinal uptake of both pyridoxine and thiamine, lacks the pyridoxine transport function. Thus, SLC19A3s from different animal species exhibit differences in pyridoxine transport. Rats and mice, in which Slc19a3 lacks this function, are not suitable model animals for studies involving pyridoxine disposition and related issues.     

Systematic identification and characterization of cynomolgus macaque solute carrier transporters

Cynomolgus macaques are used in preclinical studies in part because of their evolutionary closeness to humans. However, drug transporters [including solute carrier (SLC) transporters] essential for the absorption and excretion of drugs have not been fully investigated at the molecular level in cynomolgus macaques. We identified and characterized cynomolgus macaque SLC15A1, SLC15A2, SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLC47A1, and SLC47A2, along with SLCO (formerly SLC21A) transporters SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1. These cynomolgus SLC transporters had high amino acid sequence identities (92–97%) with their human orthologs and contained sequence motifs characteristic of SLC transporters. Phylogenetic analysis showed that these cynomolgus SLC transporters were more closely clustered with their human orthologs than with those of dogs, rats, or mice. Gene structure and genomic organization were similar in macaques and humans. Cynomolgus SLC transporter mRNAs showed distinct tissue expression patterns, being most abundantly expressed in jejunum (SLC15A1), liver (SLC22A1, SLCO1B1, and SLCO2B1), and kidney (SLC15A2, SLC22A2, SLC22A6, SLC22A8, SLC47A1, SLC47A2, and SLCO1A2). In contrast, cynomolgus SLCO2B1 mRNA was more ubiquitously expressed. Among these SLC mRNAs, the most abundant in liver was SLCO1B1, in jejunum SLC15A1, and in kidney SLC22A2. These results suggest similar characteristics of SLC transporters in cynomolgus macaques and humans.     

Zileuton ameliorates depressive-like behaviors,hippocampal neuroinflammation,apoptosis and synapse dysfunction in mice exposed to chronic mild stress

Our previous study has found that zileuton, a selective 5-lipoxygenase (5LO) inhibitor, abrogated lipopolysaccharide-induced depressive-like behaviors and hippocampal neuroinflammation. Herein, we further extended our curiosity to investigate effects of zileuton on stress-induced depressive-like behaviors. Our data indicated that zileuton significantly ameliorated depressive-like behaviors in mice subjected to chronic mild stress (CMS), as shown in the tail suspension test, forced swimming test and novelty-suppressed feeding test. The further studies indicated that zileuton suppressed hippocampal neuroinflammation, evidenced by lower levels of TNF-α, IL-1β and nuclear NF-κB p65 as well as decreased number of Iba1-positive cells. It also significantly ameliorated hippocampal apoptosis, indicated by deceased number of TUNEL-positive cells, deceased ratio of cleaved caspase-3/procaspase-3 and increased ratio of Bcl-2/Bax. More importantly, zileuton increased the level of synaptic proteins PSD-95 and SYN and the number of NeuN⁺/BrdU⁺ cells in the hippocampus. Over all, zileuton alleviated CMS-induced depressive-like behaviors, neuroinflammatory and apoptotic responses, abnormalities of synapse and neurogenesis in the hippocampus, suggesting that it might has beneficial effects on depression.     

Involvement of sex hormonal regulation of K+ channels in electrophysiological and contractile functions of muscle tissues

Sex hormones, such as testosterone, progesterone, and 17β-estradiol, control various physiological functions. This review focuses on the sex hormonal regulation of K⁺ channels and the effects of such regulation on electrophysiological and contractile functions of muscles. In the cardiac tissue, testosterone and progesterone shorten action potential, and estrogen lengthens QT interval, a marker of increased risk of ventricular tachyarrhythmias. We have shown that testosterone and progesterone in physiological concentration activate KCNQ1 channels via membrane-delimited sex hormone receptor/eNOS pathways to shorten the action potential duration. Mitochondrial K⁺ channels are also involved in the protection of cardiac muscle. Testosterone and 17β-estradiol directly activate mitochondrial inner membrane K⁺ channels (Ca²⁺ activated K⁺ channel (K_Ca channel) and ATP-sensitive K⁺ channel (K_ATP channel)) that are involved in ischemic preconditioning and cardiac protection. During pregnancy, uterine blood flow increases to support fetal growth and development. It has been reported that 17β-estradiol directly activates large-conductance Ca²⁺-activated K⁺ channel (BK_Ca channel) attenuating arterial contraction. Furthermore, 17β-estradiol increases expression of BK_Ca channel β1 subunit which enhances BK_Ca channel activity by DNA demethylation. These findings are useful for understanding the mechanisms of sex or generation-dependent differences in the physiological and pathological functions of muscles, and the mechanisms of drug actions.     

Effects of Probenecid on Hepatic and Renal Disposition of Hexadecanedioate,an Endogenous Substrate of Organic Anion Transporting Polypeptide 1B in Rats

The aim of the present study was to investigate changes in plasma concentrations and tissue distribution of endogenous substrates of organic anion transporting polypeptide (OATP) 1B, hexadecanedioate (HDA), octadecanedioate (ODA), tetradecanedioate (TDA), and coproporphyrin-III, induced by its weak inhibitor, probenecid (PBD), in rats. PBD increased the plasma concentrations of these four compounds regardless of bile duct cannulation, whereas liver-to-plasma (K_p,liver) and kidney-to-plasma concentration ratios of HDA and TDA were reduced. Similar effects of PBD on plasma concentrations and K_p,liver of HDA, ODA, and TDA were observed in kidney-ligated rats, suggesting a minor contribution of renal disposition to the overall distribution of these three compounds. Tissue uptake clearance of deuterium-labeled HDA (d-HDA) in liver was 16-fold higher than that in kidney, and was reduced by 80% by PBD. This was compatible with inhibition by PBD of d-HDA uptake in isolated rat hepatocytes. Such inhibitory effects of PBD were also observed in the human OATP1B1-mediated uptake of d-HDA. Overall, the disposition of HDA is mainly determined by hepatic OATP-mediated uptake, which is inhibited by PBD. HDA might, thus, be a biomarker for OATPs minimally affected by urinary and biliary elimination in rats.