The multispecific organic anion transporter family - Cell Press

Review

TRENDS in Pharmacological Sciences

Vol.25 No.12 December 2004

The multispecific organic anion transporter family: properties and pharmacological significance Hiroki Miyazaki1,2, Takashi Sekine3 and Hitoshi Endou1 1 Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan 2 Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Kumamoto-shi, Kumamoto 860-8556, Japan 3 Department of Pediatrics, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan

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. Organic anions are chemically heterogeneous substances that contain carbon backbones and a net negative charge at physiological pH. Numerous compounds, such as drugs, environmental substances, plant and animal toxins, and metabolites of both foreign and endogenous origins are classified as organic anions (Table 1). Because many of these substances are toxic to the body, their elimination is essential for homeostasis. The kidneys and liver are the major routes for organic anion elimination. In the kidneys, the transepithelial transport of organic anions occurs predominantly in proximal tubular cells [1,2]. The most prominent feature of the renal organic anion transport system is its ‘multispecificity’ in substrate recognition [1–3]. This feature became recognized widely in the field of transporters following the discovery of P-glycoprotein (multidrug resistance 1) encoded by ABCB1. Historically, however, the renal organic anion transport system was the first characterized ‘multispecific’ transport system and, because of its significance in renal physiology and pharmacology, was investigated extensively using tissue slices, isolated and perfused tubules, cultured cells, membrane vesicles and peritubular capillary perfusion techniques [1–3]. For these studies of the Corresponding author: Hitoshi Endou ([email protected]).

renal organic anion system, p-aminohippurate (PAH) was used as a prototypical substrate and therefore the organic anion transport system was alternatively referred to as the PAH transport system. Because xenobiotics possess diverse chemical structures, the multispecificity of the PAH transporter appears rational for the elimination pathway. In addition to the kidneys, similar organic anion transport systems have been identified in several tissues of epithelial origin, such as those of the liver and the choroid plexus [1,2]. In 1997, the PAH transporter was isolated and designated OAT1 (organic anion transporter 1) [4–6]. Subsequently, several structurally related proteins have been cloned and the identification of OAT family members has increased our knowledge of organic anion transporter systems. Members of the OAT family OAT1: classical renal organic anion transporter Several research groups cloned the first member of the OAT family OAT1 [4–6]. OAT1 is identical to the previously isolated clone novel kidney-specific transporter (NKT), which had unknown function [7]. OAT1 mRNA is expressed predominantly in the kidneys and weakly in the brain. In the kidneys, OAT1 protein is localized at the basolateral membrane of proximal tubular cells. OAT1mediated uptake of PAH is stimulated by an outwardly directed concentration gradient of dicarboxylates such as a-ketoglutarate, which is consistent with the previous notion that OAT1 is an organic anion–dicarboxylate exchanger [8]. The substrate selectivity of OAT1 is markedly broad, and substrates include endogenous substances, such as dicarboxylates, cyclic nucleotides and prostaglandins, and exogenous substances, such as various anionic drugs and environmental compounds (Table 1) [9]. The affinities of OAT1 for these compounds are similar to reported values for the basolateral PAH transporter [3] and the functional properties and localization of OAT1 are identical to those of the renal PAH transport system. The alternative splice variants of OAT1 have been identified [10,11]: OAT1-1 and OAT1-2 appear to be almost identical functionally, whereas no functions have been detected for OAT1-3 and OAT1-4 [12].

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Table 1. Typical substances that are transported by or interact with OAT1a,b Type of compound Cyclic nucleotides Dicarboxylates Eicosanoids Others Drugs Antibiotics Antiviral drugs NSAIDs Diuretics Antihypertensive agents Anti-neoplasmics Anti-epileptics Uricosuric drugs Conjugates Sulfate conjugates Cysteine conjugates Glucuronide conjugates Glycine conjugates Mycotoxins

Examples cAMP, cGMP a–Ketoglutarate, succinate, glutarate PGE1, PGE2, PGF2a, PGD2, PGI2, TXB2, 6-keto-PGF1a Urate, folate, octatanoate, neurotransmitter metabolites Penicillin antibiotics, cephem antibiotics, carbapenem antibiotics Aziothymidine, acyclovir, amantadine, adefovir, cidofovir Salicylate, acetylsalicylate, indomethacin, diclofenac, ibuprofen, piroxicam Loop diuretics, thiazide diuretics ACE inhibitors, angiotensin AT2 receptor antagonists Anti-metabolites (methotrexate, azathioprine, Ara-C, 5-FU); alkylating agents (cyclophosphamide, chlorambucil); antibiotics (doxorubicin, aclarubicin) Valproate Probenecid, benzbromarone Estrone sulfate, dehydroepiandrosterone sulfate, p-nitrophenyl–S S-benzyl–Cys, CTFC, DCVC, N-acetyl-S-farnesyl–Cys b-Estradiol–17-G, p-nitrophenyl–G, 4-methyumbelliferyl–G, a-naphtyl–G p-Aminohippurate, o-hydroxyhippurate Ochratoxin A, ochratoxin B, aflatoxin G1, patulin, citrinin

a

OAT1 interacts with O100 substances of both endogenous and exogenous origins. Abbreviations: ACE, angiotensin-converting enzyme; Ara-C, cytosine arabinoside; CTFC, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; 5-FU, 5-fluorouracil; –G, glucuronide conjugate; NSAIDs, non-steroidal anti-inflammatory drugs; OAT1, organic anion transporter 1; PG, prostaglandin; –S, sulfate conjugate; TX, thromboxane.

b

Other OAT members To date, five structurally related OATs, namely, OAT2 [13], OAT3 [14], OAT4 [15], URAT1 [16] and OAT5 [17] have been identified (Table 2). In addition to these transporters, several uncharacterized transporter proteins that are structurally related to the OAT family members exist (Figure 1). OAT2 was isolated originally from the rat liver as a novel liver-specific transport protein with unknown function [18]. Because of its structural similarities to OAT1, OAT2 was functionally characterized [13]. OAT2 is expressed predominantly in the liver and weakly in the kidneys. The typical substrates of OAT2 are salicylate, acetylsalicylate, prostaglandin E2 (PGE2), dicarboxylates and PAH. OAT3 was isolated from the rat [14], and is identical to Roct, which had been identified as a transporter-like

protein that exhibits reduced expression in osteosclerosis mice [19]. OAT3 mRNA is expressed in the kidneys, liver, brain and eye [14]. In the kidneys, OAT3 is localized at the basolateral membrane of proximal tubular cells [20]. In the brain, OAT3 is localized at the brush border membrane of choroid plexus cells [21,22] and in capillary endothelial cells [23]. Like OAT1, OAT3 recognizes a broad spectrum of substrates, and mediates the high-affinity transport of PAH, estrone sulfate, ochratoxin A and various drugs, including the cationic drug cimetidine in exchange for dicarboxylates inside cells [24,25]. OAT4 was cloned from human kidneys [15]. OAT4 mRNA is expressed in the kidneys and is localized at the apical membrane of proximal tubular cells. In the placenta, OAT4 is expressed on the foetal side of the syncytiotrophoblast cells [26]. When expressed in Xenopus

Table 2. Properties of each OAT isoforma Gene product (gene symbol)

Identified homologues

OAT1 (SLC22A6)

OAT4 (SLC22A11)

Human [10,11,36], rat [4,5], mouse [7], flounder [6], C. elegans [81], rabbit [82], pig [83] Human [28], rat [13], mouse [45] Human [20], rat [14], mouse [22] Human [15]

URAT1 (SLC22A12) OAT5 (SLC22A19)

Human [16], mouse [47,80] Mouse [17]

OAT2 (SLC22A7)

OAT3 (SLC22A8)

a

Chromosome localization in human 11q12.3

Tissue distribution

Transport mechanism

Brain, kidney, placenta, smooth muscle [10]; brain, kidney [4]

OA–DC exchanger [4,5]

6q21.1–2

Kidney, liver [13,45]

11q12.3

Brain, kidney, smooth muscle [20]; brain, eye, kidney, liver [14] Kidney, placenta [15]

11q13.1 11q13.1 –

Kidney [16]; brain, kidney [80] Kidney [17]

Renal PT localization in human Basolateral

PDZ-motif at C-terminalb No (NGL*)

Unknown

Basolateral

No (EDV*)

OA–DC exchanger [24,25]

Basolateral

No (GSS*)

OA–DC exchanger [27] Urate–anion exchanger [16] Unknown

Apical

Yes (TSL*)

Apical

Yes (TQF*)

Unknown

Yes (TPL*)

Abbreviations: C. elegans, Caenorhabditis elegans; DC, dicarboxylate; OA, organic anion; OAT, organic anion transporter; PDZ, PSD-95/DglA/ZO-1 homology; PT, proximal tubules. b The last three amino acids at the C-terminal of each transporter are shown in parentheses; asterisks denote stop codons. www.sciencedirect.com

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hFLIPT1 hOAT2 hOAT1 hOAT3 hOAT4 hURAT1 mOAT5* hUST3 hOAT5* hORCTL3 hORCTL4 hOCT3 hOCT1 hOCT2

CeOAT1 hBOCT

CeOCT1 hCT2 hOCTN1 hOCTN2 0.1 TRENDS in Pharmacological Sciences

Figure 1. Phylogenetic tree analysis of transporters of the SLC22A family. The alignment program CLUSTAL W (http://www.ebi.ac.uk/clustalw/) and the phylogenetic tree display program TreeViewPPC (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) were used to generate the tree. Human, Caenorhabditis elegans and mouse transporters are denoted by ‘h’, ‘Ce’ and ‘m’, respectively. Mainly human clones are described to simplify and facilitate the understanding of the figure. In the case of OAT5, a human homologue has not been identified; however, mouse OAT5 is described. mOAT5* and hOAT5*, which was identified by Sun et al. [28], are not homologues of each other. Because of their developmental significance, CeOAT1 and CeOCT1 are also described. The sequence identity between the Caenorhabditis elegans transporters (CeOAT1 and CeOCT1) and the human transporters (the organic anion transporter family and the organic cation transporter family) is !29%. Organic anion transporters (OATs) are shown in red, the organic cation/carnitine transporter (OCTN/CT) family is shown in blue, and organic cation transporters (OCTs) are shown in green. The transporter proteins with unknown function, such as hFLIPT1, hUST3, hOAT5, hORCTL3, hORCTL4 and hBOCT, are shown in black. hUST3 is identical to hOAT4, which was isolated by Sun et al. [28]. The length of bars indicates the number of substitutions per residue, with 0.1 corresponding to a distance of 10 substitutions per 100 residues. Abbreviations: BOCT, brain-type organic cation transporter; CT, carnitine transporter; FLIPT, Fly-like putative transporter; ORCTL, organic-cation transporter like; UST, unknown solute transporter.

oocytes, OAT4 mediates the NaC-independent, highaffinity transport of estrone sulfate, dehydroepiandrosterone sulfate, ochratoxin A, and PGE2 and PGF2a. A recent study demonstrated that OAT4 functions as an organic anion–dicarboxylate exchanger [27]. URAT1 is expressed exclusively in the kidneys where it is located in the apical membrane of proximal tubular cells [16]. URAT1 exhibits NaC-independent uptake of urate and regulates the serum urate level; genetic defects in URAT1 are the predominant causes of idiopathic renal hypouricemia (discussed later). OAT5 was recently identified from mouse [17], and is expressed exclusively in the kidneys and mediates the transport of ochratoxin A. Other than these six clones, Sun et al. has reported two OAT-related clones and designated them as hOAT4 and hOAT5, respectively [28]. However, Sun et al. did not demonstrate any transport function, and human OAT4, identified by Cha and colleagues [15], and mouse OAT5, identified by Youngblood and Sweet [17], are not identical to these two clones. www.sciencedirect.com

Molecular structure of OATs and crucial residues for substrate recognition The members of the OAT family show homology to members of the organic cation transporter (OCT) family [29], and the organic cation/carnitine transporter (OCTN/CT) family. Thus, OATs, OCTs and OCTN/CTs together with uncharacterized orphan transporters comprise a transporter superfamily, namely, the organic ion transporter family encoded by SLC22A (Figure 1). There is no structural similarity between the OAT family and other multispecific organic anion transporter families, such as multidrug resistance-associated proteins (MRPs) and organic anion transporting polypeptides (OATPs). OAT members consist of w500–550 amino acid residues and exhibit common structural features (Figure 2). The significance of the glycosylation and phosphorylation sites of OATs is discussed in the following section. Multispecific substrate recognition is the most significant feature of OATs, and the structural similarities between OATs and OCTs provide clues regarding this unique property. In addition to the requirement of charge

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Outside Membrane

1

2

3

4

5

Inside NH

6 P

7

8

9

10 11 12

P

P P

COOH

: N-glycosylation site P

: PKC phosphorylation site TRENDS in Pharmacological Sciences

Figure 2. Predicted membrane topology of organic anion transporters (OATs). The OATs have common structures such as: (i) 12 putative transmembrane domains (TMDs), with the intracellular localization of both N- and C-termini; (ii) a large extracellular hydrophobic loop containing several glycosylation sites between the first and second TMDs; and (iii) a large intracellular hydrophobic loop containing multiple phosphorylation sites between the sixth and seventh TMDs. Thr36 in human OAT1 [31] and His34 in flounder OAT1 [33] are crucial for substrate recognition. Both Lys394 and Arg478 in flounder OAT1 have an important role in the organic anion–dicarboxylate exchange mechanism [33]. The importance of Lys370 and Arg454 was also confirmed in rat OAT3 [34].

in substrate recognition by OATs and OCTs, the general mechanism that underlies such substrate recognition should be common between OATs and OCTs. Hydrophobicity and strength of the anionic charge are important for substrates of ‘the classical organic anion transport system’, as demonstrated by Ullrich [3]. For example, the hydrophobic part of a substrate must be of a certain ˚ ) to bind to OATs and the strength of the size (4–10 A anionic charge contributes to the interaction of substrates with OATs [3]. Recent studies using site-directed mutagenesis have provided important information regarding substrate recognition and membrane targeting of OATs [30] and shown that the following residues are important: Thr36 in human OAT1 (hOAT1) [31], His34, Lys394 and Arg478 in flounder OAT1 (fOAT1) [33], and Lys370 and Arg454 in rat OAT3 (rOAT3) [34]. Regulation of OATs Glycosylation Glycosylation sites in the first extracellular loop between transmembrane domains (TMDs) 1 and 2 are conserved in OATs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited PAH transport activity in mOAT1transfected COS7 cells [32]. Immunofluorescence revealed that the mOAT1 protein remained mainly in the intracellular compartment after tunicamycin treatment [32]. This study indicates that the glycosylation of the mOAT1 protein is necessary for the proper trafficking of the protein to the plasma membrane. Other experiments have demonstrated that disrupting Asp39 (one of the glycosylated sites) in mice resulted in a complete loss of transport activity of OAT1 without affecting its surface expression [35]. Thus, glycosylation could also be responsible for substrate recognition. Phosphorylation All OAT isoforms have several sites for phosphorylation by protein kinase C (PKC) in the large intracellular loop www.sciencedirect.com


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between the sixth and seventh TMDs. Several studies have revealed that the activation of PKC decreases the transport activity of OATs [36–39]. This inhibitory effect is also associated with altered substrate selectivity. A reduced OAT-mediated transport activity is rescued by PKC inhibitors [36,37]. Furthermore, recent studies have demonstrated that OAT1 activity is stimulated by epidermal growth factor (EGF) via mitogen-activated protein kinases (MAPKs) [40]. In addition to the sites for phosphorylation by PKC, OAT isoforms also have putative sites for phosphorylation by PKA, casein kinase II or tyrosine kinase. It is not clear whether these protein kinases are involved in the regulation of transporter functions. Protein–protein interaction Several renal apical transporters possess the PDZ motif at their C-terminus [41]. The PDZ motif is one of the protein– protein interaction modules, and is composed of three amino acid residues: S/T-X-f (where X is any amino acid and f is a hydrophobic residue). The renal apical organic anion transporters OAT4 and URAT1 possess the PDZ motif at their C-terminus. Yeast two-hybrid experiments revealed that both OAT4 and URAT1 interact with the multivalent PDZ domain-containing protein PDZK1 via their C-terminal PDZ motifs [42]. The coexpression of URAT1 and PDZK1 in HEK293 cells increases URAT1 transport activity. This synergic effect is abolished when the C-terminal PDZ motif deletion mutant of URAT1 is coexpressed with PDZK1. These results indicate that PDZK1 regulates transport activities via interaction with the PDZ motif. Gender and developmental differences Gender differences in mRNA and/or protein expression have been reported for OAT1 [43,44], OAT2 [44–46], OAT3 [43,44,46] and URAT1 [47], and suggest that some OAT members are regulated by sex hormones. Developmental changes of expression have been reported in OATs [44,48,49]: the mRNA expression levels of OAT1, OAT2 and OAT3 increased during postnatal development. Pharmacological aspects of the OAT members Pharmacokinetics As mentioned earlier, OAT family members have crucial roles in the elimination of various drugs from the body. OAT1 interacts with a wide range of organic anion drugs such as b-lactam antibiotics, antiviral drugs, diuretics, anti-tumor drugs, angiotensin-converting enzyme (ACE) inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs) [9]. OAT3 also interacts with various drugs and endogenous substances such as NSAIDs, anti-tumor drugs, histamine H2 receptor antagonists, diuretics, ACE inhibitors, b-lactam antibiotics and estrone sulfate. In the kidneys, OAT1 and OAT3 take up drugs and drug metabolites from the blood into proximal tubular cells (Figure 3a). In the liver, OAT2 and OAT3 are localized at the basolateral membrane of hepatocytes. The difference in the distribution of OAT family members in the kidneys and liver should be one of the determinants of the

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(a) Renal proximal tubular cells Apical

Basolateral

ATP MRP2,4

OA DC ADP

+

OATv1 (NPT1?)

OAT1 OA

OA –

OA

Urate URAT1

DC

OA OA

OAT3 OAT4

OA DC

(b) Choroid plexus epithelial cells

(c) Brain capillary endothelial cells OA

OA

OAT3

MRP4

OA Brush border membrane (CSF side)

OAT3 DC

ATP ADP MRP1 Basolateral membrane (blood side)

DC

RST?

ATP ADP

Abluminal membrane (brain side)

ATP ADP MRP1

RST?

MRP4,5

Blood side OA

OA

OA

OA

OA TRENDS in Pharmacological Sciences

Figure 3. The role of organic anion transporter (OAT) members in (a) renal proximal tubular cells, (b) choroid plexus epithelial cells and (c) brain capillary endothelial cells. (a) Uptake of organic anions (OAs) across the basolateral membrane is mediated by two OA–dicarboxylate (DC) exchangers, OAT1 and OAT3. The multidrug resistanceassociated proteins MRP2 and MRP4 mediate primary active transport for OA excretion. OATv1 (and its possible human homologue NPT1) is also involved in OA efflux by electro-gradient. OAT4 might be involved in the reabsorption of OAs via exchange mechanisms. URAT1 mediates urate reabsorption. The outwardly directed gradient of DCs is maintained by NaC–dicarboxylate exchangers in addition to the tricarboxylic acid cycle. (b,c) SLC22A members involved in organic anion transport across the blood– cerebrospinal fluid (CSF) barrier (BCSFB) and blood–brain barrier (BBB) are shown. Recent studies have indicated the significant roles of organic anion transporters in the BBB and BCSFB. However, more-detailed molecular information on OATs in the brain is essential for understanding the pharmacokinetics in the brain. In the BCSFB and BBB, many other organic anion transporters, such as MRPs and organic anion transporting polypeptides (OATPs), are expressed and function. P-glycoprotein and peptide transporters, which also mediate the transport of neutral substances, are also expressed (not shown). Abbreviation: RST, renal-specific transporter.

elimination route of drugs. In the brain, OAT3 is localized on the brush border membrane of choroid plexus cells, and takes up drugs and drug metabolites from the cerebrospinal fluid (CSF), thus functioning as one of the key molecules of the blood–CSF barrier (Figure 3b,c) [21,22]. Another important aspect is drug–drug interactions. Drugs present in plasma could affect the transport of these drugs individually, and mutually influence the pharmacokinetics of the drugs. A notable example is the concomitant use of probenecid and penicillin G; the halflife of penicillin G is significantly prolonged when combined with probenecid compared with when it is administered alone. It has also been reported that the administration of methotrexate (MTX) with acidic drugs, such as NSAIDs, and b-lactam antibiotics, causes a severe suppression of bone marrow. NSAIDs and b-lactam antibiotics inhibit the tubular secretion of MTX, thereby reducing its renal clearance. As a consequence, unwanted side-effects, such as bone marrow suppression, could occur as a result of the increase in plasma MTX levels [50,51]. www.sciencedirect.com

These phenomena can be explained at the level of OAT1 and OAT3. Similar OAT-mediated drug–drug interactions have been reported for diuretics [52], nucleoside analogues [53] and antiviral drugs [54].

Pharmacodynamics Several drugs alter urate excretion. In addition to probenecid and benzbromarone, which are uricosuric drugs, sulfinpyrazone and losartan enhance uric acid secretion. By contrast, pyrazinoic acid, the metabolite of the antituberculous agent pyrazinamide, inhibits urate excretion by an as yet unknown mechanism, and is thus called an anti-uricosuric drug. In a study using URAT1 cRNA-injected Xenopus oocytes, the cis-inhibitory effect of uricosuric drugs and the trans-stimulatory effect of anti-uricosuric drugs on the URAT1mediated transport of urate were demonstrated [16]. These results indicate that URAT1 is the target molecule that modulates the serum level of urate.

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Toxicological aspects Nephrotoxic drugs OATs are involved in the development of organ-specific toxicity of drugs and their metabolites. For example, the nephrotoxic effects of b-lactam antibiotics (e.g. cephaloridine) and carbapenem antibiotics are closely associated with OATs [55], and OATs are also responsible for the nephrotoxicity of antiviral drugs, such as adefovir and cidofovir [56]. Indeed, b-lactam antibiotics and antiviral drugs exhibit a significantly high cytotoxicity in OAT1transfected cell cultures [57,58]. The nephrotoxicity of all these compounds could be reduced by the coadministration of other substrates of OATs or inhibitors of OATs. Indeed, recently, a new application of probenecid as a nephroprotectant in therapy with the antiviral drug cidofovir was determined [59]. When probenecid is coadministered with cidofovir, probenecid inhibits the tubular accumulation of cidofovir, thus reducing its potential risk. Environmental substances Ochratoxin A is a mycotoxin that contaminates cereals and is thought to be responsible for Balkan nephropathy [84], an endemic nephropathy that exhibits characteristic chronic tubulointerstitial changes. In OAT1-expressing oocytes and OAT1-transfected cell cultures, the addition of ochratoxin A to the culture media decreases cell viability [60]. This decreased cell viability is rescued by the nontoxic substrates of OAT1, such as PAH. Uremic toxin The uremic toxins and their metabolites, produced during the catabolism process within the body, seem to be associated with the exacerbation of renal function in renal failure. Indoxyl sulfate, a uremic toxin derived from dietary proteins, is a substrate of OATs [61–63]. Immunohistochemical analyses revealed that 5/6-nephrectomized rats, an animal model of chronic renal failure, showed higher intensities of OAT1 and OAT3 proteins than shamoperated rats [63]. These data suggest that OATs are also involved in the progression of chronic renal failure. In the body, uremic substances that cannot be eliminated by glomerular filtration during renal failure should be removed via tubular secretion mediated by OATs. In these cases, OAT protein expression levels increased, resulting in the accumulation of toxic substances in the tubules. OAT1 and OAT3 are also involved in the uptake of other uremic toxins such as 3-carboxy-4-methyl-5-propyl2-furanpropionate, indoleacetate and hippurate [64]. Pathophysiological significance OAT3 OAT3 knockout mice were generated recently [22]. These mice show a substantial loss of organic anion transport in the kidneys and the brain choroid plexus. For example, these mice exhibit markedly lower uptake of taurocholate, estrone sulfate and PAH in renal slices than wild-type mice, and, in the brain, the accumulation of fluorescein is reduced by w75% in the choroid plexus cells of OAT3 knockout mice compared with wild-type mice. By contrast, the capillary accumulation of fluorescein–methotrexate is www.sciencedirect.com


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unchanged, indicating that the loss of OAT3 function is restricted to the entry step into choroid plexus cells, in which OAT3 is localized at the brush border membrane. To date, only OAT3 knockout mice have been generated, and knockout mice for other OAT family members have not yet been produced. URAT1 URAT1 mediates urate reabsorption and regulates the serum level of urate [16]. The pivotal role of URAT1 was confirmed by the observation that genetic defects of URAT1 cause idiopathic renal hypouricemia [16]. Patients with idiopathic renal hypouricemia exhibit increased renal excretion of urate and a decreased serum level of urate. In Japanese patients with this disorder, the W258X nonsense mutation in the gene encoding URAT1 (SLC22A12) was identified in up to 70–80% of these patients [65,66]. URAT1 is a urate–anion exchanger and therefore releases intracellular anions, including lactates, in exchange for urate. This efflux of anions via URAT1 might have other physiological effects. Other topics in the SLC22A family Carnitine transporters (OCTN/CTs) Carnitine is an essential compound for the b-oxidation of fatty acids. Carnitine is a zwitterion, possessing both the quaternary ammonium group and a carboxylic group. To date, three carnitine transporters, namely, OCTN1 [67], OCTN2/CT1 [68–70] and CT2 [71] belonging to the SLC22A family, have been identified. OCTN1 is expressed in the kidneys, skeletal muscle, placenta, prostate and heart. OCTN1 transports monovalent organic cations such as tetraethylammonium (TEA), quinidine, pyrilamine, verapamil and carnitine. OCTN2/CT1 is expressed ubiquitously in most tissues, and is localized at the apical membrane in the kidneys and intestine. OCTN2/CT1 mediates high-affinity carnitine transport, which is partly dependent on NaC. The physiological importance of OCTN2/CT1 is evident in the inherited human systemic carnitine deficiency in which carnitine cannot be absorbed from the intestine nor reabsorbed from the glomerular filtrate in the kidneys. Mutations in the gene encoding OCTN2/CT1 have been demonstrated in many patients with systemic carnitine deficiency [72]. CT2 is another high-affinity carnitine transporter [71], and is identical to OCT6 isolated from haematopoietic cells (with the exception of its N-terminal amino acids) [73]. CT2 is expressed mainly in the testes where it is localized in Sertoli cells and epithelial cells of the epididymal ducts. Carnitine is considered to be essential for the motility of spermatozoa, and CT2 might have some roles in the male fertility system. Recently, two studies using positional cloning technique have revealed that OCTN1 is associated with rheumatoid arthritis [74] and Crohn disease [75]. Because of its importance in energy production, defects in the carnitine transport system might be responsible for the development several inflammatory diseases. Perspectives Identification of the OAT family, together with two other organic anion transporter families (OATP and MRP) has

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brought us a step further in the elucidation of the molecular mechanisms for drug elimination and distribution in the kidneys, liver and brain. Nonetheless, many questions remain to be addressed. First, several uncharacterized (orphan) transporters in the SLC22A superfamily remain (Figure 1) and thus further investigation is required to determine their functions. Second, the physiological significance of OAT2 and OAT4 need to be clarified. In vivo studies such as those using transgenic and knockout mice should provide some clues. Third, knowledge of the regulation mechanisms of OAT is required. Gender differences in the expression level of OAT members and the PDZ interaction on apical OATs are of significant interest. Fourth, the clarification of the three-dimensional (3D) structure of OATs is necessary to understand the broad substrate recognition of OATs. Because of difficulties in the purification and crystallization of membrane transporter proteins, the 3D structures of transporters are yet to be clarified. The recent successful identification of Escherichia coli major facilitator superfamily proteins (glycerol-3-phosphate transporter:GlpT and lactose permease:LacY) [76,77] might accelerate this issue. Fifth, the apical exit pathway for organic anions in the kidneys is an important issue. Recently, a voltage-driven apical organic anion efflux transporter, OATv1, identified from the pig kidney [78], and the renal specific transporter (RST), previously cloned from mouse kidney with unknown function [79], were characterized [80]. These transporters show broad substrate selectivity similar to OAT family members, and are candidates for the apical exit pathway for organic anions. The amino acid sequence of OATv1 exhibits the highest identity (60–65%) to that of NaC-dependent inorganic phosphate transporter type I (NPT1), which belongs to the SLC17A family. On the basis of the high amino acid sequence identity (74%), RST is considered to be the mouse homologue of hURAT1 [47]. Finally, the pathological significance of OATs is of great interest. Originally, studies of OATs were focused on the molecular mechanisms that underlie the elimination of xenobiotics. However, the identification of URAT1 has provided a novel aspect that OATs are involved in both the reabsorption and the excretion of organic anions. Several organic anions, such as urate, carnitine, sulfate conjugates of steroid hormone, prostaglandins and cyclic nucleotides, are essential for the maintenance of homeostasis. Thus, genetic defects of OATs might give rise to pathophysiological states.

Acknowledgements We gratefully thank Taku Hirata, Naohiko Anzai and Hiroyuki Kusuhara for kindly providing Figure 1, Figure 3a and Figure 3b,c, respectively. This work was supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Uehara Memorial Foundation, Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Sciences (JSPS), Grants-in-Aid for Scientific Research and Bioventure project from the Science Research Promotion Fund of the Japan Private School Promotion Foundation, Grants-in-Aid from the Tokyo Biochemical Research Foundation, and Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation. www.sciencedirect.com


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