Molecular Insights into the Structure–Function Relationship of Organic Anion Transporters OATs

Abstract The organic anion transporter (OAT) family encoded by SLC22A mediates the absorption, distribution, and excretion of a diverse array of environmental toxins, and clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories, and therefore is critical for the survival of mammalian species. Several OATs have been identified: OAT1 (SLC22A6), OAT2 (SLC22A7), OAT3 (SLC22A8), OAT4 (SLC22A11), OAT5 (SLC22A19) OAT6 (SLC22A20) and URAT1 (SLC22A12). The expressions of these OATs have been detected in key organs such as kidney, liver, brain and placenta. OAT dysfunction in these organs may contribute to the renal, hepatic, neurological and fetal toxicity and diseases. In this review, we summarize, according to the work done by our laboratory as well as by others, the most updated molecular studies on these OAT members, especially on the aspect of their structure–function relationships. The functional roles of N-glycosylation, transmembrane domains and individual amino acids, cell surface assembly, as well as associating proteins will be discussed. In addition, we will show the recent analyses of coding region polymorphisms of OATs, which give us information on the genetic variants of OATs and their potential effects on OAT functions.     

Impact of genetic polymorphisms in transmembrane carrier-systems on drug and xenobiotic distribution

Active transport across biological membranes has become a noticeable factor in the absorption, distribution, and excretion of an increasing number of drugs. Different transmembrane transport systems including organic anion transporters (OATP, solute carrier family SLC21A), organic cation transporters (OCT, SLC22A), dipeptide transporters (PEPT, SLC15A), nucleoside transporters (CNT, SLC28A), monocarboxylate carriers (MCT, SLC2A), and members of the large ATP-binding cassette family (ABC, SLC3A) are involved in drug disposition. Genetic polymorphisms in transport proteins frequently occur and contribute to interindividual differences in the efficacy and safety of pharmatherapy. Currently, the most advanced research has been done on P-glycoprotein (ABCB1, SLC3A1.201.1). Knowledge of this transporter indicates that haplotype analysis rather than association with single nucleotide polymorphisms (SNPs) provides the most appropriate interpretation of pharmacogenetic data from drug transporters. This review gives an overview and update on the pharmacological impact of genetic variants in transmembrane transporters.An erratum to this article can be found at     

Functional maturation of drug transporters in the developing, neonatal, and postnatal kidney

Because renal function in newborns is immature, the pharmacokinetics of drugs administered to neonates vary significantly from adult patients. The establishment of drug transport systems is a key process in the functional maturation of the nephron. However, a thorough examination of the expression of the main drug transporters in the kidney throughout all stages of development (embryonic, postnatal, and mature) has yet to be carried out, and the functional (physiological) impact is not well understood. Using time-series microarray data, we analyzed the temporal behavior of mRNA levels for a wide range of SLC and ABC transporters in the rodent kidney throughout a developmental time series. We find dynamic increases between the postnatal and mature stages of development for a number of transporters, including the proximal tubule-specific drug and organic anion transporters (OATs) OAT1 (SLC22a6) and OAT3 (SLC22a8). The OATs are the major multispecific basolateral drug, toxin, and metabolite transporters in the proximal tubule responsible for handling of many drugs, as well as the prototypical OAT substrate para-aminohippurate (PAH). We therefore performed specific in vivo pharmacokinetic analysis of the transport of PAH in postnatal and maturing rodent kidney. We show that there is a 4-fold increase in PAH clearance during this period. Clearance studies in Oat1 and Oat3 knockouts confirm that, as in the adult, Oat1 is the principle transporter of PAH in the postnatal kidney. The substantial differences observed supports the need for better understanding of pharmacokinetics in the newborn and juvenile kidney compared with the adult kidney at the basic and clinical level.     

Torsemide renal clearance and genetic variation in luminal and basolateral organic anion transporters

AIMS: To investigate the association between torsemide renal clearance and genetic variation in the basolaterally expressed renal organic anion transporters OAT1 and OAT3 and in the luminally situated OAT4. METHODS: We analysed 22 polymorphisms in the OAT coding genes SLC22A6, SLC22A8 and SLC22A11 and their haplotypes and measured torsemide renal clearance in 95 healthy men. In addition, the effect of torsemide on the OAT-mediated transport was studied in vitro. RESULTS: In stably transfected HEK293 cells torsemide (100 microm) inhibited the uptake by human OAT1, OAT3 and OAT4 by 63.1, 58.1 and 68.0%, respectively. Torsemide renal clearance ranged from 6.5 to 43.1 ml min(-1) with a log-normal distribution and a geometric mean of 15.6 ml min(-1) (15.0-16.1 +/- SEM). No clear outlier group was observed. AA carriers of the polymorphism rs11231809 in SLC22A11 had a torsemide renal clearance of 13.3 ml min(-1) (12.7-13.9) compared with 15.1 ml min(-1) (14.5-15.8) in AT and 18.0 ml min(-1) (16.7-19.5) in TT carriers (P = 0.002). The two most frequent haplotypes at SLC22A11 showed an association with torsemide renal clearance. Homozygous carriage of these two haplotypes resulted in renal clearances of 21.2 ml min(-1) (19.0-23.7) and 11.8 ml min(-1) (10.5-13.5), respectively. No association between reanl clearance and genetic variation in SLC22A6 or SLC22A8 was observed. CONCLUSIONS: Genetic variation in the gene encoding the luminally expressed OAT4 rather than in the basolaterally expressed OATs may affect the renal clearance of torsemide.     

Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy

Organic cation transporters (OCTs) of the solute carrier family (SLC) 22 and multidrug and toxin extrusion (MATE) transporters of the SLC47 family have been identified as uptake and efflux transporters, respectively, for xenobiotics including several clinically used drugs such as the antidiabetic agent metformin, the antiviral agent lamivudine, and the anticancer drug oxaliplatin. Expression of human OCT1 (SLC22A1) and OCT2 (SLC22A2) is highly restricted to the liver and kidney, respectively. By contrast, OCT3 (SLC22A3) is more widely distributed. MATEs (SLC47A1, SLC47A2) are predominantly expressed in human kidney. Data on in vitro studies reporting a large number of substrates and inhibitors of OCTs and MATEs are systematically summarized. Several genetic variants of human OCTs and in part of MATE1 have been reported, and some of them result in reduced in vitro transport activity corroborating data from studies with knockout mice. A comprehensive overview is given on currently known genotype-phenotype correlations for variants in OCTs and MATE1 related to protein expression, pharmacokinetics/-dynamics of transporter substrates, treatment outcome, and disease susceptibility.     

Pathway-based pharmacogenomics of gemcitabine pharmacokinetics in patients with solid tumors

Aim: The aim of this study was to evaluate the association of gemcitabine pathway SNPs with detailed pharmacokinetic measures obtained from solid tumor patients receiving gemcitabine-based therapy. Materials & methods: SNPs within nine gemcitabine pathway genes, namely CDA, CMPK, DCK, DCTD, NT5C2, NT5C3, SLC28A1, SLC28A3 and SLC29A1 were analyzed for association with gemcitabine pharmacokinetics. Results: Significant association of gemcitabine clearance with SNPs in NT5C2 was identified. Clearance of 2′,2′-difluorodeoxyuridine, a gemcitabine metabolite was significantly predicted by CDA, SLC29A1 and NT5C2 SNPs. This study reports an association of formation clearance of 2′,2′-difluoro-2′-deoxycytidine triphosphate, an active form of gemcitabine with SNPs within uptake transporters SLC28A1, SLC28A3 and SLC29A1. Conclusion: Genetic variation in gemcitabine pathway genes is associated with its pharmacokinetics and hence could influence gemcitabine response. Our study identified pharmacogenetic markers that could be further tested in larger patient cohorts and could open up opportunities to individualize therapy in solid tumor patients. Original submitted 10 February 2012; Revision submitted 27 April 2012.     

Inhibition of oat3-mediated renal uptake as a mechanism for drug-drug interaction between fexofenadine and probenecid.

Fexofenadine, a nonsedating antihistamine drug, is effective for the treatment of seasonal allergic rhinitis and chronic urticaria. Simultaneous administration of probenecid increases the plasma concentration of fexofenadine due to an inhibition of its renal elimination in healthy volunteers (Clin Pharmacol Ther 77:17-23, 2005). The purpose of the present study is to investigate the possibility that the drug-drug interaction between fexofenadine and probenecid involves the renal basolateral uptake process. The uptake of fexofenadine was determined in HEK293 cells expressing human organic anion transporter 1 (OAT1/SLC22A6), OAT2 (SLC22A7), OAT3 (SLC22A8), and organic cation transporter 2 (OCT2/SLC22A2). Only hOAT3-HEK showed a significantly greater accumulation of fexofenadine than that in vector-HEK, which was saturable with K(m) and V(max) values of 70.2 microM and 120 pmol/min/mg protein, respectively. Inhibition potency of probenecid for the uptake of fexofenadine was compared between hOAT3 and organic anion-transporting peptide 1B3 (hOATP1B3), a transporter responsible for the hepatic uptake of fexofenadine (Drug Metab Dispos 33:1477-1481, 2005). The K(i) values were determined to be 1.30 and 130 microM for hOAT3 and hOATP1B3, respectively, with Hill coefficients of 0.76 and 0.64, respectively. The K(i) value of probenecid for hOAT3, but not for hOATP1B3, was significantly lower than the maximum unbound plasma concentration of probenecid at clinical dosages. These results suggest that the renal drug-drug interaction between fexofenadine and probenecid is probably explained by an inhibition of the renal uptake of fexofenadine via hOAT3, at least in part.     

In vitro and in vivo evidence of the importance of organic anion transporters (OATs) in drug therapy

Organic anion transporters 1-10 (OAT1-10) and the urate transporter 1 (URAT1) belong to the SLC22A gene family and accept a huge variety of chemically unrelated endogenous and exogenous organic anions including many frequently described drugs. OAT1 and OAT3 are located in the basolateral membrane of renal proximal tubule cells and are responsible for drug uptake from the blood into the cells. OAT4 in the apical membrane of human proximal tubule cells is related to drug exit into the lumen and to uptake of estrone sulfate and urate from the lumen into the cell. URAT1 is the major urate-absorbing transporter in the apical membrane and is a target for uricosuric drugs. OAT10, also located in the luminal membrane, transports nicotinate with high affinity and interacts with drugs. Major extrarenal locations of OATs include the blood-brain barrier for OAT3, the placenta for OAT4, the nasal epithelium for OAT6, and the liver for OAT2 and OAT7. For all transporters we provide information on cloning, tissue distribution, factors influencing OAT abundance, interaction with endogenous compounds and different drug classes, drug/drug interactions and, if known, single nucleotide polymorphisms.     

Impact of Genetic Polymorphisms on the Efficacy of HMG-CoA Reductase Inhibitors

Although pharmacologic treatment for cholesterol reduction represents an advance in cardiovascular and atherosclerosis treatment, the benefits of such therapy are still limited because of interindividual variability in the response to these drugs. Disease severity, treatment adherence, physiologic conditions, biologic conditions, and the patient's genetic profile could be cited as important factors in the evaluation of interindividual variability. In regard to the latter consideration, three large groups of genes could be investigated: (i) genes that code for proteins involved in metabolism and/or drug transport, thereby influencing the pharmacokinetics of these compounds; (ii) genes that code for proteins involved in the mechanism of action and/or in the metabolic pathway of drug action, and which therefore influence pharmacodynamics; and (iii) genes that code for proteins involved in direct development of the disease or in intermediate phenotypes. In this review we discuss pharmacogenetic studies of the HMG-CoA reductase inhibitors (statins) and the implications of pharmacogenetic considerations for predicting treatment efficacy and reducing the adverse effects of these drugs. Once new studies have been performed and most of the genetic variability associated with drug action has been revealed, the great challenge will be to apply this knowledge in clinical medicine.     

Proteomic analysis for the impact of hypercholesterolemia on expressions of hepatic drug transporters and metabolizing enzymes

1. Our objective is to investigate the alterations of hepatic drug transporters and metabolizing enzymes in hypercholesterolemia. Male Sprague–Dawley rats were fed high-cholesterol chows for 8 weeks to induce hypercholesterolemia. Protein levels of hepatic drug transporters and metabolizing enzymes were analyzed by iTRAQ labeling coupled with LC TRIPLE-TOF.

2. Total 239 differentially expressed proteins were identified using proteomic analysis. Among those, protein levels of hepatic drug transporters (MRP2, ABCD3, OAT2, SLC25A12, SCL38A3, SLC2A2 and SLC25A5) and metabolizing enzymes (CYP2B3, CYP2C7, CYP2C11, CYP2C13, CYP4A2 and UGT2B) were markedly reduced, but the levels of CYP2C6 and CYP2E1 were increased in hypercholesterolemia group compared to control. Decreased expressions of drug transporters MRP2 and OAT2 were further confirmed by real time quantitative PCR (RT-qPCR) and western blot.

3. Ingenuity pathway analysis revealed that these differentially expressed proteins were regulated by various signaling pathways including nuclear receptors and inflammatory cytokines. One of the nuclear receptor candidates, liver X receptor alpha (LXRα), was further validated by RT-qPCR and western blot. Additionally, LXRα agonist T0901317 rescued the reduced expressions of MRP2 and OAT2 in HepG2 cells in hypercholesterolemic serum treatment.

4. Our present results indicated that hypercholesterolemia affected the expressions of various drug transporters and metabolizing enzymes in liver via nuclear receptors pathway. Especially, decreased function of LXRα contributes to the reduced expressions of MRP2 and OAT2.     

The multispecific organic anion transporter family: properties and pharmacological significance

Physiological and pharmacological studies indicate that the renal and hepatic organic anion transport systems are responsible for the elimination of numerous compounds, such as drugs, environmental substances and metabolites of both endogenous and exogenous origins. Recently, the molecular identity of the organic anion transport system, the OAT family, was revealed. To date, six OAT members have been identified and shown to have important roles not only in detoxification in the kidneys, liver and brain, but also in the reabsorption of essential compounds such as urate. The OAT family members are closely associated with the pharmacokinetics, drug-drug interactions and toxicity of anionic substances such as nephrotoxic drugs and uremic toxins. The molecular characterization of the OAT family encoded by SLC22A will be discussed.     

Site and mechanism of the action of diuretics

Abstract: The mechanism of action of diuretics can be established by studying the molecular mechanism of action, the site of action within the nephron, and the relationship between the pharmacokinetics of the diuretic and its effect. The molecular mechanism of action is known for diuretic agents such as acetazolamide (carbonic anhydrase), theophylline (phosphodiesterase), digitalis glucosides (Na-K-ATPase), spironolactone (aldosterone antagonism) and dopamine (specific receptors?). The “receptor” for the clinically most important diuretics, i.e. loop diuretics, thiazides, and other potassium-sparing diuretics is, however, unknown. It appears from recent studies of the ion transport in the dilating segment that there probably is a sodium-chloride co-transport in this segment and that loop diuretics specificly inhibit the active chloride transport. The main site of diuretic action is well established for the different groups of diuretics: carbonic anhydrase inhibitors act on the proximal tubulus, loop diuretics on the diluting segment, thiazides on the cortical diluting segment/distal tubulus, and potassium-sparing agents on distal tubulus/collecting ducts. Moreover, some diuretics have additional tubular sites of action. It is also important to realize that other effects of diuretics, e.g. inhibition of the tubuloglomerular feedback mechanism or renal and extrarenal hemodynamic effects, can modify the tubular diuretic effect. Finally, the renal handling of diuretics is of importance to the diuretic effect by determining the concentration of the drug at the “receptor” sit (s). It is emphasized that knowledge of the different aspects of the mechanisms of action of diuretics is a prerequisite for rational use of diuretics, clinically as well as experimentally.     

Organic Anion Transporters of the SLC22 Family: Biopharmaceutical,Physiological, and Pathological Roles

The human organic anion transporters OAT1, OAT2, OAT3, OAT4 and URAT1 belong to a family of poly-specific transporters mainly located in kidneys. Selected OATs occur also in liver, placenta, and brain. OATs interact with endogenous metabolic end products such as urate and acidic neutrotransmitter metabolites, as well as with a multitude of widely used drugs, including antibiotics, antihypertensives, antivirals, anti-inflammatory drugs, diuretics and uricosurics. Thereby, OATs play an important role in renal drug elimination and have an impact on pharmacokinetics. In this review we focus on the interaction of human OATs with drugs. We report the affinities of human OATs for drug classes and compare the putative importance of individual OATs for renal drug excretion. The role of OATs as sites of drug-drug interaction and mediators cell toxicity, their gender-dependent regulation in health and diseased states, and the possible impact of single nucleotide polymorphisms are also dealt with.     

Pharmacogenetics of antihypertensive treatment

Hypertension is a common disorder associated with increased cardiovascular morbidity and mortality. Unfortunately, in the US only about one-third of those who are aware of their hypertensive status have their blood pressure adequately controlled. One reason for this is the variable and unpredictable response individuals have to pharmacologic treatment. Clinicians often resort to "trial-and-error" to match patients with effective drug treatment. Hypertension pharmacogenetics seeks to find genetic predictors of drug response. To date, more than forty studies have investigated associations between genetic polymorphisms and response to antihypertensive drugs. Angiotensin-converting enzyme inhibitors and beta blockers have been most frequently studied, followed by angiotensin II blockers, diuretics, adrenergic alpha-agonists, and calcium channel blockers. Renin-angiotensin-aldosterone system genes have been the most widely studied, with the angiotensin-converting enzyme I/D variant being typed in about one-half of all hypertension pharmacogenetic studies. In total, 160 possible gene polymorphism-drug interactions have been explored, with about one-quarter of these showing that genes predict drug response. However, disparate and conflicting findings have been the rule rather than the exception, and the discovery of clinically relevant antihypertensive drug-response genes remains elusive. While there is a growing enthusiasm that pharmacogenetics of hypertension is important, the translation of pharmacogenetic findings to clinical practice in the future will depend on additional studies to enhance our pharmacogenetics knowledge base, the availability of pharmacogenetic screening tests that are affordable and easy to implement in clinical practice, a cohort of clinicians who are trained to interpret genetic test results, and health care systems that pay for them. Caution regarding the future of hypertension pharmacogenetics is warranted.     

Pharmacogenetics of irinotecan,doxorubicin and docetaxel transporters in Asian and Caucasian cancer patients: a comparative review

Drug efflux and influx transporters play critical roles in regulating the cellular drug disposition and modulating the pharmacokinetics and pharmacodynamics of anti-cancer agents, which may potentially alter treatment outcomes. The efficiency of drug transport is often dependent on the expression and activity of these membrane-bound proteins, factors which have been shown to be regulated by genes that are known to be highly polymorphic in different ethnic populations. The role of drug transporters becomes even more critical for anti-cancer agents due to the narrow therapeutic windows that separate treatment response and toxicities for these agents. Moreover, high inter-individual variability in the disposition of anti-cancer agents often results in variable treatment outcomes among patients receiving standard doses of the same drug. Such variability has been attributed at least in part to polymorphisms in genes encoding drug-metabolizing enzymes and transporter. To date, numerous pharmacogenetic studies have investigated the associations between variants in the ABC and SLC transporters genes with drug disposition, treatment outcomes and drug-induced toxicities. However, the strengths of these associations and their clinical relevance in different ethnic populations have not been critically examined. This review aims to summarize and evaluate the implications of pharmacogenetic variants in the ABC and SLC transporters genes on the pharmacokinetics and clinical outcomes of three anti-cancer agents: irinotecan, docetaxel and doxorubicin in Caucasian and Asian patients.     

Molecular cloning and functional characterization of a novel gene encoding human prostaglandin carrier, hPrC

In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the human prostaglandin carrier (hPrC). The isolated cDNA consisted of 1431 base pairs that encoded a 477-amino acid protein, and we found that isolated hPrC does not belong to any drug transporter families. RT-PCR analysis revealed that the hPrC mRNA is expressed in various human tissues ubiquitously. When expressed in Xenopus laevis oocytes, hPrC mediated the transport of [(3)H]prostaglandin E(2) (PGE(2)) in a sodium-independent manner. The uptake of [(3)H] PGE(2) was not trans-stimulated by PG analogous. Although there are several PG transporters such as multidrug resistance-associated protein 4 (MRP4), organic cation transporter 1 (OCT1) [solute carrier (SLC) 22A1], organic anion transporter 1-3 (OAT1-3) [SLC22A6-8], OAT4 [SLC11], OATP-1 (LST-1) [SLCO1B1], OATP2B1 [SLCO2B1], OATP2A1 (PGT) [SLCO2A1], OATP4A1 (OATP-E) [SLCO4A1] have been isolated and well characterized, our findings suggest that hPrC functions as a novel transport peptide responsible for PG uptake. Our results should provide insight into the novel mechanism of the PG transport in the human body.     

Pharmacogenetically driven treatments for alcoholism: are we there yet?

Pharmacogenetic analyses of treatments for alcohol dependence attempt to predict treatment response and side-effect risk for specific medications. We review the literature on pharmacogenetics relevant to alcohol dependence treatment, and describe state-of-the-art methods of pharmacogenetic research in this area. Two main pharmacogenetic study designs predominate: challenge studies and treatment-trial analyses. Medications studied include US FDA-approved naltrexone and acamprosate, both indicated for treating alcohol dependence, as well as several investigational (and off-label) treatments such as sertraline, olanzapine and ondansetron. The best-studied functional genetic variant relevant to alcoholism treatment is rs1799971, a single-nucleotide polymorphism in exon 1 of the OPRM1 gene that encodes the μ-opioid receptor. Evidence from clinical trials suggests that the presence of the variant G allele of rs1799971 may predict better treatment response to opioid receptor antagonists such as naltrexone. Evidence from clinical trials also suggests that several medications interact pharmacogenetically with variation in genes that encode proteins involved in dopaminergic and serotonergic neurotransmission. Variation in the DRD4 gene, which encodes the dopamine D(4) receptor, may predict better response to naltrexone and olanzapine. A polymorphism in the serotonin transporter gene SLC6A4 promoter region appears related to differential treatment response to sertraline depending on the subject's age of onset of alcoholism. Genetic variation in SLC6A4 may also be associated with better treatment response to ondansetron. Initial pharmacogenetic efforts in alcohol research have identified functional variants with potential clinical utility, but more research is needed to further elucidate the mechanism of these pharmacogenetic interactions and their moderators in order to translate them into clinical practice.     

Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney

The role of carrier-mediated transport in determining the pharmacokinetics of drugs has become increasingly evident with the discovery of genetic variants that affect expression and/or function of a given drug transporter. Drug transporters are expressed at numerous epithelial barriers, such as intestinal epithelial cells, hepatocytes, renal tubular cells and at the blood-brain barrier. Several recent studies have associated alterations in substrate uptake with the presence of SNPs. Here, we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion-transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT1; SLC15 family).     

Exploring Variation in Known Pharmacogenetic Variants and its Association with Drug Response in Different Mexican Populations

Purpose

Information on genetic variants that affect the pharmacokinetics and pharmacodynamics (PK/PD) of drugs in different populations from Mexico is still an ongoing endeavor. Here, we investigate allele frequencies on pharmacogenetic targets in Mexican Mestizos and Natives from three different States and its association with drug efficacy in individuals receiving either anticoagulants or antipsychotic drugs.

Methods

Natives from three different states and Mestizo patients receiving acenocoumarol or antipsychotics were genotyped using the DMET microarray (Affymetrix).

Results

We provide a collection of genetic variants that indicate that there are 3-times more variation than similarities between populations from Mexico and major continental groups. These differences were observed in several relevant targets including ABCB1, SLCO1A1, NAT2, UGTs, TYMS, VKORC1, and NR1I3. Moreover, Mexican Mestizos also showed allele frequency differences when compared to Natives for variants on DPYD, ADH1A, CYP3A4, SLC28A3, and SLC28A1. Significant allele differences also arose among the three Native groups here studied, mostly for transporters of the ABC-binding cassette and the solute carrier gene family. Finally, we explored genotype-drug response associations and pinpointed variants on FMOs (coumarins), and GSTM1 (haloperidol).

Conclusions

These findings confirm previous results and further delve into the pharmacogenetics of Mexican populations including different Native groups.