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In vitro and in vivo studies have established the organic anion transporters OAT1 (SLC22A6, NKT) and OAT3 (SLC22A8) among the main multi-specific "drug" transporters. They also transport numerous endogenous metabolites, raising the possibility of drug-metabolite interactions (DMI). To help understand the role of these drug transporters on metabolism across scales ranging from organ systems to organelles, a formal multi-scale analysis was performed. Metabolic network reconstructions of the omics-alterations resulting from Oat1 and Oat3 gene knockouts revealed links between the microbiome and human metabolism including reactions involving small organic molecules such as dihydroxyacetone, alanine, xanthine, and p-cresol-key metabolites in independent pathways. Interestingly, pairwise organ-organ interactions were also disrupted in the two Oat knockouts, with altered liver, intestine, microbiome, and skin-related metabolism. Compared to older models focused on the "one transporter-one organ" concept, these more sophisticated reconstructions, combined with integration of a multi-microbial model and more comprehensive metabolomics data for the two transporters, provide a considerably more complex picture of how renal "drug" transporters regulate metabolism across the organelle (e.g. endoplasmic reticulum, Golgi, peroxisome), cellular, organ, inter-organ, and inter-organismal scales. The results suggest that drugs interacting with OAT1 and OAT3 can have far reaching consequences on metabolism in organs (e.g. skin) beyond the kidney. Consistent with the Remote Sensing and Signaling Theory (RSST), the analysis demonstrates how transporter-dependent metabolic signals mediate organ crosstalk (e.g., gut-liver-kidney) and inter-organismal communication (e.g., gut microbiome-host).

How organs sense circulating metabolites is a key question. Here, we show that the multispecific organic anion transporters of drugs, OAT1 (SLC22A6 or NKT) and OAT3 (SLC22A8), play a role in organ sensing. Metabolomics analyses of the serum of Oat1 and Oat3 knockout mice revealed changes in tryptophan derivatives involved in metabolism and signaling. Several of these metabolites are derived from the gut microbiome and are implicated as uremic toxins in chronic kidney disease. Direct interaction with the transporters was supported with cell-based transport assays. To assess the impact of the loss of OAT1 or OAT3 function on the kidney, an organ where these uptake transporters are highly expressed, knockout transcriptomic data were mapped onto a "metabolic task"-based computational model that evaluates over 150 cellular functions. Despite the changes of tryptophan metabolites in both knockouts, only in the Oat1 knockout were multiple tryptophan-related cellular functions increased. Thus, deprived of the ability to take up kynurenine, kynurenate, anthranilate, and N-formylanthranilate through OAT1, the kidney responds by activating its own tryptophan-related biosynthetic pathways. The results support the Remote Sensing and Signaling Theory, which describes how "drug" transporters help optimize levels of metabolites and signaling molecules by facilitating organ cross talk. Since OAT1 and OAT3 are inhibited by many drugs, the data implies potential for drug-metabolite interactions. Indeed, treatment of humans with probenecid, an OAT-inhibitor used to treat gout, elevated circulating tryptophan metabolites. Furthermore, given that regulatory agencies have recommended drugs be tested for OAT1 and OAT3 binding or transport, it follows that these metabolites can be used as endogenous biomarkers to determine if drug candidates interact with OAT1 and/or OAT3.

How organs sense circulating metabolites is a key question. Here, we show that the multispecific organic anion transporters of drugs, OAT1 (SLC22A6 or NKT) and OAT3 (SLC22A8), play a role in organ sensing. Metabolomics analyses of the serum of Oat1 and Oat3 knockout mice revealed changes in tryptophan derivatives involved in metabolism and signaling. Several of these metabolites are derived from the gut microbiome and are implicated as uremic toxins in chronic kidney disease. Direct interaction with the transporters was supported with cell-based transport assays. To assess the impact of the loss of OAT1 or OAT3 function on the kidney, an organ where these uptake transporters are highly expressed, knockout transcriptomic data were mapped onto a "metabolic task"-based computational model that evaluates over 150 cellular functions. Despite the changes of tryptophan metabolites in both knockouts, only in the Oat1 knockout were multiple tryptophan-related cellular functions increased. Thus, deprived of the ability to take up kynurenine, kynurenate, anthranilate, and N-formylanthranilate through OAT1, the kidney responds by activating its own tryptophan-related biosynthetic pathways. The results support the Remote Sensing and Signaling Theory, which describes how "drug" transporters help optimize levels of metabolites and signaling molecules by facilitating organ cross talk. Since OAT1 and OAT3 are inhibited by many drugs, the data implies potential for drug-metabolite interactions. Indeed, treatment of humans with probenecid, an OAT-inhibitor used to treat gout, elevated circulating tryptophan metabolites. Furthermore, given that regulatory agencies have recommended drugs be tested for OAT1 and OAT3 binding or transport, it follows that these metabolites can be used as endogenous biomarkers to determine if drug candidates interact with OAT1 and/or OAT3.

Organic anion transporter 1 (OAT1/SLC22A6) is a drug transporter with numerous xenobiotic and endogenous substrates. The Remote Sensing and Signaling Theory suggests that drug transporters with compatible ligand preferences can play a role in "organ crosstalk," mediating overall organismal communication. Other drug transporters are well known to transport lipids, but surprisingly little is known about the role of OAT1 in lipid metabolism. To explore this subject, we constructed a genome-scale metabolic model using omics data from the Oat1 knockout mouse. The model implicated OAT1 in the regulation of many classes of lipids, including fatty acids, bile acids, and prostaglandins. Accordingly, serum metabolomics of Oat1 knockout mice revealed increased polyunsaturated fatty acids, diacylglycerols, and long-chain fatty acids and decreased ceramides and bile acids when compared with wildtype controls. Some aged knockout mice also displayed increased lipid droplets in the liver when compared with wildtype mice. Chemoinformatics and machine learning analyses of these altered lipids defined molecular properties that form the structural basis for lipid-transporter interactions, including the number of rings, positive charge/volume, and complexity of the lipids. Finally, we obtained targeted serum metabolomics data after short-term treatment of rodents with the OAT-inhibiting drug probenecid to identify potential drug-metabolite interactions. The treatment resulted in alterations in eicosanoids and fatty acids, further supporting our metabolic reconstruction predictions. Consistent with the Remote Sensing and Signaling Theory, the data support a role of OAT1 in systemic lipid metabolism.

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.

Species differences in renal drug transporters continue to plague drug development with animal models failing to adequately predict renal drug toxicity. For example, adefovir, a renally excreted antiviral drug, failed clinical studies for human immunodeficiency virus due to pronounced nephrotoxicity in humans. In this study, we demonstrated that there are large species differences in the kinetics of interactions of a key class of antiviral drugs, acyclic nucleoside phosphonates (ANPs), with organic anion transporter 1 [(OAT1) SLC22A6] and identified a key amino acid residue responsible for these differences. In OAT1 stably transfected human embryonic kidney 293 cells, the Km value of tenofovir for human OAT1 (hOAT1) was significantly lower than for OAT1 orthologs from common preclinical animals, including cynomolgus monkey, mouse, rat, and dog. Chimeric and site-directed mutagenesis studies along with comparative structure modeling identified serine at position 203 (S203) in hOAT1 as a determinant of its lower Km value. Furthermore, S203 is conserved in apes, and in contrast alanine at the equivalent position is conserved in preclinical animals and Old World monkeys, the most related primates to apes. Intriguingly, transport efficiencies are significantly higher for OAT1 orthologs from apes with high serum uric acid (SUA) levels than for the orthologs from species with low serum uric acid levels. In conclusion, our data provide a molecular mechanism underlying species differences in renal accumulation of nephrotoxic ANPs and a novel insight into OAT1 transport function in primate evolution.

In vitro data indicates that the kidney proximal tubule (PT) transporters of uremic toxins and solutes (e.g., indoxyl sulfate, p-cresol sulfate, kynurenine, creatinine, urate) include two "drug" transporters of the organic anion transporter (OAT) family: OAT1 (SLC22A6, originally NKT) and OAT3 (SLC22A8). Here, we have examined new and prior metabolomics data from the Oat1KO and Oat3KO, as well as newly obtained metabolomics data from a "chemical double" knockout (Oat3KO plus probenecid). This gives a picture of the in vivo roles of OAT1 and OAT3 in the regulation of the uremic solutes and supports the centrality of these "drug" transporters in independently and synergistically regulating uremic metabolism. We demonstrate a key in vivo role for OAT1 and/or OAT3 in the handling of over 35 uremic toxins and solutes, including those derived from the gut microbiome (e.g., CMPF, phenylsulfate, indole-3-acetic acid). Although it is not clear whether trimethylamine-N-oxide (TMAO) is directly transported, the Oat3KO had elevated plasma levels of TMAO, which is associated with cardiovascular morbidity in chronic kidney disease (CKD). As described in the Remote Sensing and Signaling (RSS) Hypothesis, many of these molecules are involved in interorgan and interorganismal communication, suggesting that uremia is, at least in part, a disorder of RSS.

In vitro data indicates that the kidney proximal tubule (PT) transporters of uremic toxins and solutes (e.g., indoxyl sulfate, p-cresol sulfate, kynurenine, creatinine, urate) include two "drug" transporters of the organic anion transporter (OAT) family: OAT1 (SLC22A6, originally NKT) and OAT3 (SLC22A8). Here, we have examined new and prior metabolomics data from the Oat1KO and Oat3KO, as well as newly obtained metabolomics data from a "chemical double" knockout (Oat3KO plus probenecid). This gives a picture of the in vivo roles of OAT1 and OAT3 in the regulation of the uremic solutes and supports the centrality of these "drug" transporters in independently and synergistically regulating uremic metabolism. We demonstrate a key in vivo role for OAT1 and/or OAT3 in the handling of over 35 uremic toxins and solutes, including those derived from the gut microbiome (e.g., CMPF, phenylsulfate, indole-3-acetic acid). Although it is not clear whether trimethylamine-N-oxide (TMAO) is directly transported, the Oat3KO had elevated plasma levels of TMAO, which is associated with cardiovascular morbidity in chronic kidney disease (CKD). As described in the Remote Sensing and Signaling (RSS) Hypothesis, many of these molecules are involved in interorgan and interorganismal communication, suggesting that uremia is, at least in part, a disorder of RSS.

The organic anion transporters OAT1 (SLC22A6) and OAT3 (SLC22A8) have similar substrate specificity for drugs, but it is far from clear whether this holds for endogenous substrates. By analysis of more than 600 metabolites in the Oat3KO (Oat3 knockout) by LC/MS, we demonstrate OAT3 involvement in the movement of gut microbiome products, key metabolites, and signaling molecules, including those flowing through the gut-liver-kidney axis. Major pathways affected included those involved in metabolism of bile acids, flavonoids, nutrients, amino acids (including tryptophan-derivatives that are uremic toxins), and lipids. OAT3 is also critical in elimination of liver-derived phase II metabolites, particularly those undergoing glucuronidation. Analysis of physicochemical features revealed nine distinct metabolite groups; at least one member of most clusters has been previously validated in transport assays. In contrast to drugs interacting with the OATs, endogenous metabolites accumulating in the Oat1KO (Oat1 knockout) versus Oat3KO have distinct differences in their physicochemical properties; they are very different in size, number of rings, hydrophobicity, and molecular complexity. Consistent with the Remote Sensing and Signaling Hypothesis, the data support the importance of the OAT transporters in inter-organ and inter-organismal remote communication via transporter-mediated movement of key metabolites and signaling molecules (e.g. gut microbiome-to-intestine-to-blood-to-liver-to-kidney-to-urine). We discuss the possibility of an intimate connection between OATs and metabolite sensing and signaling pathways (e.g. bile acids). Furthermore, the metabolomics and pathway analysis support the view that OAT1 plays a greater role in kidney proximal tubule metabolism and OAT3 appears relatively more important in systemic metabolism, modulating levels of metabolites flowing through intestine, liver, and kidney.

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10-8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established.

One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.

One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10-8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established.

Statistical analysis was performed on physicochemical descriptors of ∼250 drugs known to interact with one or more SLC22 "drug" transporters (i.e., SLC22A6 or OAT1, SLC22A8 or OAT3, SLC22A1 or OCT1, and SLC22A2 or OCT2), followed by application of machine-learning methods and wet laboratory testing of novel predictions. In addition to molecular charge, organic anion transporters (OATs) were found to prefer interacting with planar structures, whereas organic cation transporters (OCTs) interact with more three-dimensional structures (i.e., greater SP3 character). Moreover, compared with OAT1 ligands, OAT3 ligands possess more acyclic tetravalent bonds and have a more zwitterionic/cationic character. In contrast, OCT1 and OCT2 ligands were not clearly distinquishable form one another by the methods employed. Multiple pharmacophore models were generated on the basis of the drugs and, consistent with the machine-learning analyses, one unique pharmacophore created from ligands of OAT3 possessed cationic properties similar to OCT ligands; this was confirmed by quantitative atomic property field analysis. Virtual screening with this pharmacophore, followed by transport assays, identified several cationic drugs that selectively interact with OAT3 but not OAT1. Although the present analysis may be somewhat limited by the need to rely largely on inhibition data for modeling, wet laboratory/in vitro transport studies, as well as analysis of drug/metabolite handling in Oat and Oct knockout animals, support the general validity of the approach-which can also be applied to other SLC and ATP binding cassette drug transporters. This may make it possible to predict the molecular properties of a drug or metabolite necessary for interaction with the transporter(s), thereby enabling better prediction of drug-drug interactions and drug-metabolite interactions. Furthermore, understanding the overlapping specificities of OATs and OCTs in the context of dynamic transporter tissue expression

There has been a recent interest in the broader physiological importance of multispecific "drug" transporters of the SLC and ABC transporter families. Here, a novel multi-tiered systems biology approach was used to predict metabolites and signaling molecules potentially affected by the in vivo deletion of organic anion transporter 1 (Oat1, Slc22a6, originally NKT), a major kidney-expressed drug transporter. Validation of some predictions in wet-lab assays, together with re-evaluation of existing transport and knock-out metabolomics data, generated an experimentally validated, confidence ranked set of OAT1-interacting endogenous compounds enabling construction of an "OAT1-centered metabolic interaction network." Pathway and enrichment analysis indicated an important role for OAT1 in metabolism involving: the TCA cycle, tryptophan and other amino acids, fatty acids, prostaglandins, cyclic nucleotides, odorants, polyamines, and vitamins. The partly validated reconstructed network is also consistent with a major role for OAT1 in modulating metabolic and signaling pathways involving uric acid, gut microbiome products, and so-called uremic toxins accumulating in chronic kidney disease. Together, the findings are compatible with the hypothesized role of drug transporters in remote inter-organ and inter-organismal communication: The Remote Sensing and Signaling Hypothesis (Nigam, S. K. (2015) Nat. Rev. Drug Disc. 14, 29). The fact that OAT1 can affect many systemic biological pathways suggests that drug-metabolite interactions need to be considered beyond simple competition for the drug transporter itself and may explain aspects of drug-induced metabolic syndrome. Our approach should provide novel mechanistic insights into the role of OAT1 and other drug transporters implicated in metabolic diseases like gout, diabetes, and chronic kidney disease.

The multispecific organic anion drug transporters OAT6 (SLC22A20) and OAT1 (SLC22A6) are expressed in nasal epithelial cells and both can bind odorants. Sequence analysis of OAT6 revealed an evolutionarily conserved 79-amino acid (AA) fragment present not only in OAT6 but also in other SLC22 transporters, such as the organic anion transporter (OAT), organic carnitine transporter (OCTN), and organic cation transporter (OCT) subfamilies. A similar fragment is also conserved in some odorant receptors (ORs) in both humans and rodents. This fragment is located in regions believed to be important for ligand/substrate preference and recognition in both classes of proteins, raising the possibility that it may be part of a potential common ligand/substrate recognition site in certain ORs and SLC22 transporters. In silico screening of an odorant database containing known OR ligands with a pharmacophore hypothesis (generated from a set of odorants known to bind OAT6 and/or OAT1), followed by in vitro uptake assays in transfected cells, identified OR ligands capable of inhibiting OAT6- and/or OAT1-mediated transport, albeit with different affinities. The conservation of the AA fragments between these two different classes of proteins, together with their coexpression in olfactory as well as other tissues, suggests the possibility that ORs and SLC22 transporters function in concert, and raises the question as to whether these transporters function in remote sensing and signaling and/or as transceptors.