Linkage of organic anion transporter-1 to metabolic pathways through integrated "omics"-driven network and functional analysis

Abstract

The main kidney transporter of many commonly prescribed drugs (e.g. penicillins, diuretics, antivirals, methotrexate, and non-steroidal anti-inflammatory drugs) is organic anion transporter-1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478). Targeted metabolomics in knockouts have shown that OAT1 mediates the secretion or reabsorption of many important metabolites, including intermediates in carbohydrate, fatty acid, and amino acid metabolism. This observation raises the possibility that OAT1 helps regulate broader metabolic activities. We therefore examined the potential roles of OAT1 in metabolic pathways using Recon 1, a functionally tested genome-scale reconstruction of human metabolism. A computational approach was used to analyze in vivo metabolomic as well as transcriptomic data from wild-type and OAT1 knock-out animals, resulting in the implication of several metabolic pathways, including the citric acid cycle, polyamine, and fatty acid metabolism. Validation by in vitro and ex vivo analysis using Xenopus oocyte, cell culture, and kidney tissue assays demonstrated interactions between OAT1 and key intermediates in these metabolic pathways, including previously unknown substrates, such as polyamines (e.g. spermine and spermidine). A genome-scale metabolic network reconstruction generated some experimentally supported predictions for metabolic pathways linked to OAT1-related transport. The data support the possibility that the SLC22 and other families of transporters, known to be expressed in many tissues and primarily known for drug and toxin clearance, are integral to a number of endogenous pathways and may be involved in a larger remote sensing and signaling system (Ahn, S. Y., and Nigam, S. K. (2009) Mol. Pharmacol. 76, 481-490, and Wu, W., Dnyanmote, A. V., and Nigam, S. K. (2011) Mol. Pharmacol. 79, 795-805). Drugs may alter metabolism by competing for OAT1 binding of metabolites.

FIGURE 1.

Overall schema for data analysis and experimental testing of predictions. The gene expression profiles and urinary metabolomic data were analyzed separately and compared with one another, and based on the results, in vitro assays in Xenopus oocytes and cell cultures, together with ex vivo uptake assays in kidney slices, were carried out.

FIGURE 2.

Global metabolic analysis using urinary metabolomic profiles. The analysis of the urinary metabolomic data was carried out in two phases; the first (unshaded boxes and black arrows) involved tailoring Recon 1 into global OAT1 WT and OAT1 KO models, and the second (shaded box and arrow) involved comparative analyses between the two models. Twenty-four-hour urine metabolomic excretion profiles were used to define transport flux constraints on the networks. The difference in network capabilities between the two models, using flux span calculations, was then compared, keeping only the top 5% of the identified changes.

FIGURE 3.

Number of increased and decreased intracellular reaction activities (p < 0.05) in OAT1-associated metabolic pathways based on kidney gene expression data. Consistency scores for each metabolic reaction determined based on the GIMME algorithm were compared between OAT1 knock-out versus wild-type mice. Intracellular pathways mediating nucleotide, fatty acid, arginine, tyrosine, and pyruvate metabolism were implicated to be broadly perturbed in the kidney when the OAT1 function was absent. Only metabolic pathway subsystems containing more than one perturbed reaction are shown in this figure.

FIGURE 4.

Inhibition of OAT1-mediated tracer uptake by endogenous metabolites. Concentration-dependent inhibition of fluorescent tracer (6CF) uptake by mOAT1 was noted for β-hydroxybutyrate, spermidine, spermine, arginine, pyruvate, and α-ketoglutarate. β-Hydroxybutyrate, spermidine, spermine, and α-ketoglutarate were tested in Xenopus oocyte transport assays, whereas arginine and pyruvate were tested in CHO cell culture assays.

FIGURE 5.

Inhibition of 6CF uptake by polyamines in adult kidney slices. A–D, fluorescent photomicrographs of 6CF (1.0 μm) uptake (white) in adult kidney slices in the presence of either vehicular control (A), 2.0 mm probenecid (B), 2.0 mm spermidine (C), or 2.8 mm arginine (D). The bar graph (E) shows quantitative analysis of the fluorescent signal from 6CF absorption in adult kidney slices (*, p < 0.05). Images are representative of quadruplet slices from the same experiment. Scale bar, 0.2 mm. Error bars, S.E.

FIGURE 6.

Inhibition of [³H]methotrexate uptake by spermidine in mOAT1-expressing CHO cells. Uptake of [³H]methotrexate was measured in the absence of inhibitor (control) and the presence of 1000 μm of spermidine. Each bar represents mean ± S.E. (error bars) of triplicate samples, percentage of control.