A current model for the transepithelial flux of organic anions in the proximal tubule is shown in Figure 5–13. Two primary transporters on the basolateral membrane mediate the flux of organic anions from interstitial fluid to tubule cell: OAT1 (SLC22A6) and OAT3 (SLC22A8). Energetically, hydrophilic organic anions are transported across the basolateral membrane against an electrochemical gradient in exchange with intracellular α-ketoglutarate, which moves down its concentration gradient from cytosol to blood. The outwardly directed gradient of α-ketoglutarate is maintained at least in part by a basolateral Na+-dicarboxylate transporter (NaDC3). The Na+ gradient that drives NaDC3 is maintained by Na+,K+-ATPase. Transport of small-molecular-weight organic anions by the cloned transporters OAT1 and OAT3 can be driven by α-ketoglutarate; coupled transport of α-ketoglutarate and small-molecular-weight organic anions (e.g., p-aminohippurate) occurs in isolated basolateral membrane vesicles. The molecular pharmacology and molecular biology of OATs have recently been reviewed (El-Sheikh et al., 2008; Srimaroeng et al., 2008).
The mechanism responsible for the apical membrane transport of organic anions from tubule cell cytosol to tubular lumen remains controversial. Some studies suggest that OAT4 may serve as the luminal membrane transporter for organic anions. However, recent studies show that the movement of substrates via this transporter can be driven by exchange with α-ketoglutarate, suggesting that OAT4 may function in the reabsorptive, rather than secretory, flux of organic anions. Other studies have suggested that in the pig kidney, OATV1 serves as an electrogenic facilitated transporter on the apical membrane (Jutabha et al., 2003). The human ortholog of OATV1 is NPT1, or NaPi-1, originally cloned as a phosphate transporter. NPT1 can support the low-affinity transport of hydrophilic organic anions such as PAH. Other transporters that may play a role in transport across the apical membrane include MRP2 and MRP4, multidrug-resistance transporters in the ATP binding cassette family C (ABCC). Both transporters interact with some organic anions and may actively pump their substrates from tubule cell cytosol to tubular lumen.
OAT1 (SLC22A6). OAT1 was cloned from rat kidney (Sekine et al., 1997; Sweet et al., 1997). This transporter is > 30% identical to OCTs in the SLC22 family. Mouse, human, pig, and rabbit orthologs have been cloned and are ∼80% identical to human OAT1. Mammalian isoforms of OAT1 vary in length from 545-551 amino acids. The gene for the human OAT1 is mapped to chromosome 11 and is found in an SLC22 cluster that includes OAT3 and OAT4. There are four splice variants in human tissues, termed OAT1-1, OAT1-2, OAT1-3, and OAT1-4. OAT1-2, which includes a 13-amino-acid deletion, transports PAH at a rate comparable with OAT1-1. These two splice variants use the alternative 5′-splice sites in exon 9. OAT1-3 and OAT1-4, which result from a 132-bp (44-amino-acid) deletion near the carboxyl terminus of OAT1, do not transport PAH. In humans, rat, and mouse, OAT1 is expressed primarily in the kidney, with some expression in brain and skeletal muscle.
Immunohistochemical studies suggest that OAT1 is expressed on the basolateral membrane of the proximal tubule in human and rat, with highest expression in the middle segment, S2. Based on quantitative PCR, OAT1 is expressed at a third of the level of OAT3. OAT1 exhibits saturable transport of organic anions such as PAH. This transport is trans-stimulated by other organic anions, including α-ketoglutarate. Thus, the inside negative-potential difference drives the efflux of the dicarboxylate α-ketoglutarate, which, in turn, supports the influx of monocarboxylates such as PAH. Regulation of expression levels of OAT1 in the kidney appears to be controlled by sex steroids.
OAT1 generally transports small-molecular-weight organic anions that may be endogenous (e.g., PGE2 and urate) or ingested drugs and toxins. Some neutral compounds are also transported by OAT1 at a lower affinity (e.g., cimetidine). Key residues that contribute to transport by OAT1 include the conserved K394 and R478, which are involved in the PAH–glutarate exchange mechanism.
OAT2 (SLC22A7) OAT2 was cloned first from rat liver (and named NLT at the time) (Sekine et al., 1998; Simonson et al., 1994). OAT2 is present in both kidney and liver. In the kidney, the transporter is localized to the basolateral membrane of the proximal tubule, and appears to function as a transporter for nucleotides, particularly guanine nucleotides such as cyclic GMP (Cropp et al., 2008). Cellular studies suggest that OAT2 functions in both the influx and efflux of guanine nucleotides. Organic anions such as PAH and methotrexate are also transported with low affinity by OAT2, which transports PGE2 with a high affinity.
OAT3 (SLC22A8). OAT3 (SLC22A8) was cloned originally from rat kidney (Kusuhara et al., 1999). Human OAT3 consists of two variants, one of which transports a wide variety of organic anions, including model compounds, PAH and estrone sulfate, as well as many drug products (e.g., pravastatin, cimetidine, 6-mercaptopurine, and methotrexate) (Srimaroeng et al., 2008). The longer OAT3 in humans, a 568-amino-acid protein, does not support transport. It is likely that the two OAT3 variants are splice variants. Human OAT3 is confined to the basolateral membrane of the proximal tubule.
OAT3 clearly has overlapping specificities with OAT1, although kinetic parameters differ. For example, estrone sulfate is transported by both OAT1 and OAT3, but OAT3 has a much higher affinity in comparison with OAT1. The weak base cimetidine (an H2 receptor antagonist) is transported with high affinity by OAT1, whereas the cation TEA is not transported.
OAT4 (SLC22A9). OAT4 (SLC22A9) was cloned from a human kidney cDNA library (Cha et al., 2000). OAT4 is expressed in human kidney and placenta; in human kidney, OAT4 is present on the luminal membrane of the proximal tubule. At first, OAT4 was thought to be involved in the second step of secretion of organic anions, i.e., transport across the apical membrane from cell to tubular lumen. However, studies demonstrate that organic anion transport by OAT4 can be stimulated by transgradients of α-ketoglutarate (Ekaratanawong et al., 2004), suggesting that OAT4 may be involved in the reabsorption of organic anions from tubular lumen into cell. The specificity of OAT4 includes the model compounds estrone sulfate and PAH, as well as zidovudine, tetracycline, and methotrexate (El-Sheikh et al., 2008; Srimaroeng et al., 2008). Interestingly, the affinity for PAH is low (>1 mM). Collectively, emerging studies suggest that OAT4 may be involved not in secretory flux of organic anions but in reabsorption instead.
Other Anion Transporters. URAT1 (SLC22A12), first cloned from human kidney, is a kidney-specific transporter confined to the apical membrane of the proximal tubule (Enomoto et al., 2002). Data suggest that URAT1 is primarily responsible for urate reabsorption, mediating electroneutral urate transport that can be trans-stimulated by Cl− gradients. The mouse ortholog of URAT1 is involved in the renal secretory flux of organic anions including benzylpenicillin and urate.
NPT1 (SLC17A1), cloned originally as a phosphate transporter in humans, is expressed in abundance on the luminal membrane of the proximal tubule as well as in the brain (Werner et al., 1991). NPT1 transports PAH, probenecid, and penicillin G. It appears to be part of the system involved in organic anion efflux from tubule cell to lumen.
MRP2 (ABCC2), an ABC transporter, initially called the GS-X pump (El-Sheikh et al., 2008; Ishikawa et al., 1990; Toyoda et al., 2008), has been considered to be the primary transporter involved in efflux of many drug conjugates such as glutathione conjugates across the canalicular membrane of the hepatocyte. However, MRP2 is also found on the apical membrane of the proximal tubule, where it is thought to play a role in the efflux of organic anions into the tubular lumen. Its role in the kidney may be to secrete glutathione conjugates of drugs, but it also may support the translocation (with glutathione) of various non-conjugated substrates. In general, MRP2 transports larger, bulkier compounds than do most of the organic anion transporters in the SLC22 family.
MRP4 (ABCC4) is found on the apical membrane of the proximal tubule and transports a wide array of conjugated anions, including glucuronide and glutathione conjugates (El-Sheikh et al., 2008; Toyoda et al., 2008). However, unlike MRP2, MRP4 appears to interact with various drugs, including methotrexate, cyclic nucleotide analogs, and antiviral nucleoside analogs. Recent studies in Mrp4 knockout mice suggest that the transporter is involved in the renal elimination of the antiviral drugs adefovir and tenofovir (Imaoka et al., 2006). Other MRP efflux transporters also have been identified in human kidney, including MRP3 and MRP6, both on the basolateral membrane. Their roles in the kidney are not yet known.
Polymorphisms of OATs Polymorphisms in OAT1 and OAT3 have been identified in ethnically diverse human populations (Leabman et al., 2003; Srimaroeng et al., 2008). Two amino acid polymorphisms (allele frequencies >1%) in OAT1 have been identified in African-American populations. Three amino acid polymorphisms and seven rare amino acid variants in OAT3 have been identified in ethnically diverse U.S. populations (see www.pharmgkb.org).