Abstract
The organic anion transporters OAT1 (SLC22A6, originally identified by us as NKT) and OAT3 (SLC22A8) are critical for handling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Khandelwal, A., Swaan, P. W., and Nigam, S. K. (2008) J. Biol. Chem. 283, 8654-8663). Although microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, the individual contributions of these transporters in the three-dimensional context of the tissues in which they normally function remain unclear. Here, handling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed with three-dimensional ex vivo functional assays using knock-out tissue. To investigate the contribution of OAT1 and OAT3 in various nephron segments, the OAT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Although OAT1 function (uptake in oat3(-/-) tissue) was confined to portions of the cortex, consistent with a proximal tubular localization, OAT3 function (uptake in oat1(-/-) tissue) was apparent throughout the cortex, indicating localization in the distal as well as proximal nephron. This functional localization indicates a complex three-dimensional context, which needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) handled by both transporters. These results also raise the possibility of functional differences in the relative importance of OAT1 and OAT3 in antiviral handling in developing and mature tissue. Because the HIV antivirals are used in pregnant women, the results may also help in understanding how these drugs are handled by developing organs.
Highlights
2 The abbreviations used are: Organic anion transporters (OATs), organic anion transporter; ABC, ATPbinding cassette; 5CF, 5-carboxyfluorescein; 6CF, 6-carboxyfluorescein; TRITC, tetramethylrhodamine isothiocyanate; KO, knock-out. Monly used drugs such as penicillins, nonsteroidal anti-inflammatory drugs, diuretics, and various antivirals [1,2,3]. oat1, initially identified by us as nkt [1,2,3,4], and oat3, initially identified as roct [5, 6], are two major transporters belonging to the SLC22 family of solute carriers, and along with other SLC transporter families as well as members of the ATP-binding cassette (ABC) transporter family, they are implicated in the renal secretion of drugs
Functional Localization of OAT1 and OAT3 in Adult Kidney Using Fluorescent Tracers—A “classic” assay for analyzing transport in the adult kidney is the use of renal tissue slices, but this assay is not well validated for the determination of OAT1- and OAT3-specific transport
OAT1 was initially proposed to be either an organic anion transporter or an organic cation transporter [4], and it is clear that OAT1 and OAT3 can be both, organic anions remain the preferred substrates
Summary
Materials—Water-soluble probenecid was purchased from Molecular Probes. The fluorescent tracers 5-carboxyfluorescein (5CF) and 6-carboxyfluorescein (6CF) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins (Dolichos biflorus lectin and Lotus lectin) were obtained from Sigma. Slices were maintained in PBS at 4 °C, and symmetrical pieces were selected and placed in 24-well plates containing minimal saline with either 1 M 5CF or 1 M 6CF along with either TRITC-conjugated D. biflorus or Lotus lectin and in the presence of either PBS or an inhibitor (probenecid (2 mM), stavudine (1 mM), acyclovir (200 M), tenofovir (100 M), zidovudine (100 M), or lamivudine (200 M)). Organ Culture Uptake Assay and Imaging—Embryonic kidneys from embryonic day 13.5 oat and oat knock-outs were cultured for 4 days at 37 °C in DMEM/F-12 with 10% FBS and 1% penicillin/streptomycin as described previously [3].On day 4 of culture, the cultures (n ϭ 3) were washed once with PBS containing CaCl2 (0.1 mM) and MgCl2 (1 mM) at room temperature. Microarray Data: Expression of Transporters in oat Knock-outs—As described previously [16], gene expression profiling in oat KO kidney was carried out using Affymetrix mouse GeneChip 230 according to the manufacturer’s specification
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