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).     

Activation of ATP-sensitive K+ channels by ADP and K+ channel openers: homology model of sulfonylurea receptor carboxyl-termini

The multispecific organic anion transporters have been indicated to be involved in the transmembrane transport of various anionic substances. The kidney and liver possess the distinct organic anion transport pathways for the elimination of potentially toxic anionic drugs and metabolites. In the kidney, proximal tubular cells actively excrete organic anions of both endogenous and exogenous origin. We have isolated the renal multispecific organic anion transporter, OAT1 (organic anion transporter 1), from the rat kidney. OAT1 is a 551-amino acid residue protein with 12 putative membrane spanning domains. OAT1 mediates sodium-independent, anion exchange for a variety of organic anions including p-aminohippurate, cyclic nucleotides, prostanoides, dicarboxylates, and anionic drugs including beta-lactams, non-steroidal antiinflammatory drugs, diuretics and antiviral drugs. So far, three other isoforms have been identified. OATs comprise a new family of multispecific organic anion transporter, i.e., the OAT family. OATs show weak structural similarity to organic cation transporters (OCTs) and OCTN/carnitine transporters. All of the members of the OAT family are commonly expressed in the kidney, suggesting its significance in the renal organic anion excretion. In addition, OAT members appear to be responsible for the distribution/elimination of water soluble anionic drugs into/from the liver, brain and fetus.     

Renal Organic Anion Transporters (SLC22 Family): Expression, Regulation, Roles in Toxicity, and Impact on Injury and Disease

Organic solute flux across the basolateral and apical membranes of renal proximal tubule cells is a key process for maintaining systemic homeostasis. It represents an important route for the elimination of metabolic waste products and xenobiotics, as well as for the reclamation of essential compounds. Members of the organic anion transporter (OAT, SLC22) family expressed in proximal tubules comprise one pathway mediating the active renal secretion and reabsorption of organic anions. Many drugs, pesticides, hormones, heavy metal conjugates, components of phytomedicines, and toxins are OAT substrates. Thus, through transporter activity, the kidney can be a target organ for their beneficial or detrimental effects. Detailed knowledge of the OATs expressed in the kidney, their membrane targeting, substrate specificity, and mechanisms of action is essential to understanding organ function and dysfunction. The intracellular processes controlling OAT expression and function, and that can thus modulate kidney transport capacity, are also critical to this understanding. Such knowledge is also providing insight to new areas such as renal transplant research. This review will provide an overview of the OATs for which transport activity has been demonstrated and expression/function in the kidney observed. Examples establishing a role for renal OATs in drug clearance, food/drug–drug interactions, and renal injury and pathology are presented. An update of the current information regarding the regulation of OAT expression is also provided.     

Membrane transport function in primary cultures of human proximal tubular cells

To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions and cations, peptides, and neutral amino acids. p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug-drug interactions in human kidney.     

Mechanisms of renal anionic drug transport

By utilizing filtration, active secretion and reabsorption processes, the kidney can conserve essential nutrients, and eliminate drugs and potentially toxic compounds. Active uptake of organic anions and cations across the basolateral membrane, and their extrusion into the urine across the brush border membrane mainly takes place in the renal proximal tubule cells, and is facilitated via a range of substrate-specific tubular transporters. Many drugs and their phase II conjugates are anionic compounds, and therefore renal organic anion transporters are important determinants of their distribution and elimination. Competition for renal excretory transporters may cause drugs to accumulate in the body leading to toxicity, which is a potential hazard of concomitant drug administration. Here, we present a brief update on the most prominent human proximal tubule organic anion transporters, which either belong to the ATP-binding cassette (ABC) or the solute carrier transporter (SLC) families. We focus on the participation of the individual transporters in renal anionic drug elimination, in an attempt to understand their overall biological and pharmacological significance, hoping to inspire further studies in the renal transporters field.     

The role of organic ion transporters in drug disposition: an update

Transporters for organic anions and organic cations in kidney, liver, intestine, brain, and placenta play essential roles in drug disposition. The cloning and characterization of these transporters have significantly advanced our understanding of the molecular and cellular mechanisms of the drug disposition process. This review aims at updating the recent knowledge of general properties, structure-function relationships, and regulation mechanisms of the organic anion transporters (OATs) and the organic cation transporters (OCTs). Such information will be essential for the design and development of new drugs to maximize therapeutic efficacy and minimize drug-induced toxicity as well as unwanted drug-drug interactions.     

Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1)

An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 μM and 4.4 μM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates. This study was supported in part by the Deutsche Krebshilfe (Bonn, Germany).     

Organic Cation Transporter OCTs (SLC22) and MATEs (SLC47) in the Human Kidney

In the kidney, human organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are the major transporters for the secretion of cationic drugs into the urine. In the human kidney, OCT2 mediates the uptake of drugs from the blood at the basolateral membrane of tubular epithelial cells, and MATE1 and MATE2-K secrete drugs from cells into the lumen of proximal tubules. However, the expression of these transporters depends on the species of the animal. In the rodent kidney, OCT1 and OCT2 are expressed at the basolateral membrane, and MATE1 localizes at the brush-border membrane. Together, these transporters recognize various compounds and have overlapping, but somewhat different, substrate specificities. OCTs and MATEs can transport important drugs, such as metformin and cisplatin. Therefore, functional variation in OCTs and MATEs, including genetic polymorphisms or inter-individual variation, may seriously affect the pharmacokinetics and/or pharmacodynamics of cationic drugs. In this review, we summarize the recent findings and clinical importance of these transporters.     

Carrier-mediated transport in the hepatic distribution and elimination of drugs,with special reference to the category of organic cations

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.     

Carrier-mediated transport in the hepatic distribution and elimination of drugs, with special reference to the category of organic cations

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.     

Polyspecific Organic Cation Transporters: Structure,Function, Physiological Roles,and Biopharmaceutical Implications

The body is equipped with broad-specificity transporters for the excretion and distribution of endogeneous organic cations and for the uptake, elimination and distribution of cationic drugs, toxins and environmental waste products. This group of transporters consists of the electrogenic cation transporters OCT1-3 (SLC22A1-3), the cation and carnitine transporters OCTN1 (SLC22A4), OCTN2 (SLC22A5) and OCT6 (SLC22A16), and the proton/cation antiporters MATE1, MATE2-K and MATE2-B. The transporters show broadly overlapping sites of expression in many tissues such as small intestine, liver, kidney, heart, skeletal muscle, placenta, lung, brain, cells of the immune system, and tumors. In epithelial cells they may be located in the basolateral or luminal membranes. Transcellular cation movement in small intestine, kidney and liver is mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux transporters that operate as cation/proton antiporters. Recent data showed that OCT-type transporters participate in the regulation of extracellular concentrations of neurotransmitters in brain, mediate the release of acetylcholine in non-neuronal cholinergic reactions, and are critically involved in the regulation of histamine release from basophils. The recent identification of polymorphisms in human OCTs and OCTNs allows the identification of patients with an increased risk for adverse drug reactions. Transport studies with expressed OCTs will help to optimize pharmacokinetics during development of new drugs.     

Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations

OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.28 mM at intravesicular and extravesicular pH values of 6.0 and 7.4, respectively. Tetraethylammonium uptake was pH-dependent, and overshoot uptake was observed in the presence of an outwardly directed proton gradient. A protonophore and membrane potential affected the overshoot uptake. Furthermore, preloading tetraethylammonium in the vesicles significantly increased the rate of uptake of [14C]tetraethylammonium. In mouse kidney, OCTN1 was expressed predominantly at the apical membrane of cortical proximal tubular epithelial cells. It was concluded that OCTN1 is involved in renal excretion of organic cations across the apical membrane in a pH-dependent, membrane potential-sensitive manner and is affected significantly by the organic cations on the trans side, showing counter transport activity.     

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.     

Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells

Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.     

A multispecific uptake system for taurocholate, cardiac glycosides and cationic drugs in the liver.

To test the hypothesis of multiplicity in carrier-mediated uptake mechanisms for organic cations in the liver and to study the possible relation with bile acid and cardiac glycoside uptake mechanisms, mutual interaction during uptake of various radiolabeled quaternary amines has been studied in isolated rat hepatocytes. Inhibition patterns at low concentrations (1 microM) of the presumed type I monovalent organic cation tri-n-butylmethylammonium were markedly different from those at relatively high concentrations (25 microM). Both the cardiac glycoside K-strophantoside and the bile acid taurocholate clearly reduced the uptake rate of tri-n-butylmethylammonium at 25 microM whereas these agents completely failed to reduce the uptake at low concentrations of the cation. Subsequently, inhibition of uptake of some multivalent amphipathic organic cations (muscle relaxants) for the type II uptake system was investigated. It was found that the uptake of these muscle relaxants both at tracer concentrations (< 1 microM) and at relatively high concentrations (25 microM) was decreased in the presence of low concentrations of the cardiac glycoside K-strophantoside, while taurocholate only inhibited the uptake at the concentration range > 25 microM of the muscle relaxants. Procainamide ethobromide, a typical type I organic cation, did not affect the uptake either at the low or high concentration range of the muscle relaxants. It is concluded that for each of the type I-like compounds and type II-like compounds tested at least two systems are involved in uptake into hepatocytes: tri-n-butylmethylammonium in a concentration range < or = 1 microM is mainly taken up by the type I uptake system and at concentrations > or = 25 microM also by system(s) that can be inhibited by taurocholate and K-strophantoside. Bulky amphipathic organic (type II) cations at concentrations < 1 microM are also transported by an uptake system that is inhibitable by cardiac glycosides but not by bile salts. At concentrations > 25 microM these compounds are predominantly accommodated by an uptake system that possibly mediates uptake of both cardiac glycosides and bile acids. This concept was supported by the observation that both type II organic cations and bile salts can inhibit ouabain uptake, while type II organic cations as well as the cardiac glycosides reduce taurocholate uptake rate. The present data support the idea that the liver seems to be equipped with a "multispecific" uptake system that transports hydrophobic compounds irrespective of charge, including some type I and type II organic cations at relatively high substrate concentrations.     

Inhibition of rat renal C-S lyase: assessment using kidney slice methodology

Renal C-S lyase enzymes are implicated in the biotransformation of xenobiotics into potentially toxic metabolites by a deviation from the normal pathways of glutathione conjugate processing. C-S lyase enzymes occur in gastro-intestinal bacteria, and in liver as well as in mammalian and avian kidney. The renal enzyme cleaves the carbon-sulphur bond in cysteine conjugates derived from halogenated olefins (e.g. tetrafluoroethene, trichloroethene, and hexachlorobutadiene). Substituted S-nitrophenyl conjugates, which are analogues of a substrate for the hepatic C-S lyase enzyme (S-2,4-dinitrophenyl-L-cysteine), are demonstrated to display significant inhibition of rat renal C-S lyase using kidney slice methodology. They are also shown to disrupt the tubular uptake of organic cations and anions.     

Transporter-mediated renal handling of nafamostat mesilate

Nafamostat mesilate (NM) is a serine-protease inhibitor that is rapidly eliminated from the circulation and accumulated in the kidney. This study was conducted to characterize the mechanism of NM transport in the kidney because a serious side effect of NM-induced hyperkalemia may be related to accumulation of NM in the kidney. Measurements of uptake of NM in vivo by the kidney uptake index (KUI) method and of transport in an in vitro-cultured LLC-PK1 cell system suggested the involvement of an organic cation transporter (OCT). To clarify the involvement of OCTs located in the basolateral membrane of proximal tubules, we evaluated NM transport by OCTs expressed in Xenopus laevis oocytes. The IC(50) values of NM on [(14)C]TEA ([(14)C]tetraethylammonium) uptake by rOCT1, rOCT2, and hOCT2 were 50, 0.5, and 20 microM, respectively, and NM was concluded to be a substrate of OCTs. To investigate the transport of NM across the brush-border membrane, we examined the uptake of NM into brush-border membrane vesicles (BBMVs) isolated from rat renal cortex. NM was taken up into the BBMVs, and the uptake was decreased by unlabeled NM and temperature, implying that a transporter(s) is also involved in NM transport across the apical membrane. NM was not a substrate of hOCTN1, hOCTN2, or P-gp, implying the involvement of some unknown transporter(s). Thus, renal accumulation of NM can be explained by the involvement of the basolateral OCTs, though the influence of the apical membrane transporter remains to be clarified.     

Inhibitory effects of indoxyl sulfate and creatinine on the renal transport of meropenem and biapenem in rats

Carbapenem antibiotics are excreted preferentially in the urine after intravenous administration, with organic anion transporters (OATs) known to be involved in the renal tubular secretion of carbapenem antibiotics. Various uremic toxins (UTs) accumulate in the blood of patients with end-stage renal failure, and some UTs such as indoxyl sulfate (IS) and creatinine (Cr) are excreted in the urine via OATs. However, information about the possible interactions between these UTs and carbapenems in the renal secretion remains limited. In this study, we investigated the effects of IS and Cr on the renal transport of anionic meropenem and zwitterionic biapenem by using rat renal cortical slices. The uptake of meropenem and biapenem in the renal cortical slices was significantly decreased in the presence of 0.1 mM IS or 1 mM Cr. When biapenem and Cr were co-administered to rats intravenously, biapenem clearance from the plasma was clearly retarded, reflecting the current in vitro results. However, IS and Cr exerted no inhibitory effect on the uptake of metformin, a substrate of renal organic cation transporter (OCT) 2, in the renal cortical slices. Thus, our findings indicate that IS and Cr interfere with the renal secretion of carbapenem antibiotics by preferentially inhibiting OATs.