Multi-level Analysis of Organic Anion Transporters 1, 3, and 6 Reveals Major Differences in Structural Determinants of Antiviral Discrimination
2008; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês
10.1074/jbc.m708615200
ISSN1083-351X
AutoresDavid M. Truong, Gregory Kaler, Akash Khandelwal, Peter W. Swaan, Sanjay K. Nigám,
Tópico(s)Viral Infections and Immunology Research
ResumoLong-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues. Organic anion transporters (OATs) are believed to mediate the cellular uptake, and hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various OAT isoforms has been lacking. To characterize substrate interactions with mOat1, mOat3, and mOat6, a fluorescence-based competition assay in Xenopus oocytes as well as wild-type and knock-out whole embryonic kidney (WEK) organ culture systems was developed using 6-carboxyfluorescein, 5-carboxyfluorescein, and fluorescein. Of nine common antiviral drugs assessed in oocytes, many manifested higher affinity for SLC22a6 (mOat1), originally identified as NKT (e.g. adefovir and cidofovir), two (ddC and ddI) manifested significantly higher affinity for mOat3, while mOat6 had comparatively low but measurable affinity for certain antivirals. A live organ staining approach combined with fluorescent uptake in WEK cultures allowed the visualization of OAT-mediated uptake ex vivo into developing proximal tubule-like structures, as well as quantification of substrate interactions of individual OAT isoforms. In general, antiviral specificity of SLC22a6 (Oat1) (in Oat3-/- WEK culture) and SLC22a8 (Oat3) (in Oat1-/- WEK culture) was consistent with the Xenopus oocyte data. The combined observations suggest SLC22a8 (Oat3) is the major transporter interacting with ddC and ddI. Finally, quantitative structure-activity relationship analysis of the nine antivirals' physicochemical descriptors with their OAT affinity indicates that antiviral preferences of mOat1 are explained by high polar surface areas (e.g. phosphate groups), whereas mOat3 prefers hydrogen bond acceptors (e.g. amines, ketones) and low rotatable bond numbers. In contrast, hydrogen bond donors (e.g. amides, alcohols) diminish binding to mOat6. This suggests that, despite sharing close overall sequence homology, Oat1, Oat3, and Oat6 have signficantly different binding pockets. Taken together, the data provide a basis for understanding potential drug interactions in combination antiviral therapy, as well as suggesting structural mdifications for drug design, especially in the context of targeting toward or away from specific tissues. Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues. Organic anion transporters (OATs) are believed to mediate the cellular uptake, and hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various OAT isoforms has been lacking. To characterize substrate interactions with mOat1, mOat3, and mOat6, a fluorescence-based competition assay in Xenopus oocytes as well as wild-type and knock-out whole embryonic kidney (WEK) organ culture systems was developed using 6-carboxyfluorescein, 5-carboxyfluorescein, and fluorescein. Of nine common antiviral drugs assessed in oocytes, many manifested higher affinity for SLC22a6 (mOat1), originally identified as NKT (e.g. adefovir and cidofovir), two (ddC and ddI) manifested significantly higher affinity for mOat3, while mOat6 had comparatively low but measurable affinity for certain antivirals. A live organ staining approach combined with fluorescent uptake in WEK cultures allowed the visualization of OAT-mediated uptake ex vivo into developing proximal tubule-like structures, as well as quantification of substrate interactions of individual OAT isoforms. In general, antiviral specificity of SLC22a6 (Oat1) (in Oat3-/- WEK culture) and SLC22a8 (Oat3) (in Oat1-/- WEK culture) was consistent with the Xenopus oocyte data. The combined observations suggest SLC22a8 (Oat3) is the major transporter interacting with ddC and ddI. Finally, quantitative structure-activity relationship analysis of the nine antivirals' physicochemical descriptors with their OAT affinity indicates that antiviral preferences of mOat1 are explained by high polar surface areas (e.g. phosphate groups), whereas mOat3 prefers hydrogen bond acceptors (e.g. amines, ketones) and low rotatable bond numbers. In contrast, hydrogen bond donors (e.g. amides, alcohols) diminish binding to mOat6. This suggests that, despite sharing close overall sequence homology, Oat1, Oat3, and Oat6 have signficantly different binding pockets. Taken together, the data provide a basis for understanding potential drug interactions in combination antiviral therapy, as well as suggesting structural mdifications for drug design, especially in the context of targeting toward or away from specific tissues. The organic anion transporters (OATs) 2The abbreviations used are:OATorganic anion transporterPAHp-aminohippurateESestrone-3-sulfateWEKwhole embryonic kidney culture5-CF5-carboxyfluorescein6-CF6-carboxyfluorescein4-CFDM4-carboxyfluorescein-dimethylesterCFcarboxyfluoresceinNRTInucleotide reverse transcriptase inhibitorQSARquantitative structure activity relationshipHIVhuman immunodeficiency virusAZTazidothymidinePSApolar surface areaRBrotatable bondsWTwild typeTRITCtetramethyl-rhodamine isothiocyanate. 2The abbreviations used are:OATorganic anion transporterPAHp-aminohippurateESestrone-3-sulfateWEKwhole embryonic kidney culture5-CF5-carboxyfluorescein6-CF6-carboxyfluorescein4-CFDM4-carboxyfluorescein-dimethylesterCFcarboxyfluoresceinNRTInucleotide reverse transcriptase inhibitorQSARquantitative structure activity relationshipHIVhuman immunodeficiency virusAZTazidothymidinePSApolar surface areaRBrotatable bondsWTwild typeTRITCtetramethyl-rhodamine isothiocyanate. mediate the uptake of structurally diverse substrates: endogenous compounds (steroids, odorants, cyclic nucleotides, neurotransmitters), drugs (non-steroidal anti-inflammatory drugs, diuretics, and antibiotics), environmental toxins (ochratoxin A and mercuric chlorides), and other organic wastes (1Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (53) Google Scholar, 2Eraly S.A. Bush K.T. Sampogna R.V. Bhatnagar V. Nigam S.K. Mol. Pharmacol. 2004; 65: 479-487Crossref PubMed Scopus (66) Google Scholar). It has recently been hypothesized that OATs and other SLC22 family members participate in a remote sensing mechanism for small molecules between tissues and also between organisms (1Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (53) Google Scholar, 23Kaler G. Truong D.M. Khandelwal A. Nagle M. Eraly S.A. Swaan P.W. Nigam S.K. J. Biol. Chem. 2007; 282: 23841-23853Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The prototype, now called Oat1, was initially identified as NKT (3Lopez-Nieto C.E. You G. Barros E.J.G. Beier D.R. Nigam S.K. J. Am. Soc. Nephrol. 1996; 7: 1301Google Scholar, 4Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5Sweet D.H. Wolff N.A. Pritchard J.B. J. Biol. Chem. 1997; 272: 30088-30095Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Since then, at least six OATs have been identified, all of which are members of the larger SLC22 family of transporters. Most closely related to SLC22a6 (Oat1) are SLC22a8 (Oat3), originally identified as Roct (6Brady K.P. Dushkin H. Fornzler D. Koike T. Magner F. Her H. Gullans S. Segre G.V. Green R.M. Beier D.R. Genomics. 1999; 56: 254-261Crossref PubMed Scopus (74) Google Scholar), and the recently identified SLC22a20 (Oat6), expressed in olfactory mucosa (1Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (53) Google Scholar, 7Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (91) Google Scholar). Importantly, the OATs have been postulated to constitute the initial step in the uptake of antiviral nucleoside and nucleobase analog antiretroviral, uptake (8Burckhardt B.C. Burckhardt G. Rev. Physiol. Biochem. Pharmacol. 2003; 146: 95-158Crossref PubMed Scopus (256) Google Scholar, 9Sweet D.H. Toxicol. Appl. Pharmacol. 2005; 204: 198-215Crossref PubMed Scopus (129) Google Scholar). To combat chronic disease because of HIV, herpes, smallpox, and other viruses, antiviral drugs are often administered, with HIV also requiring prolonged combination therapies as either triple or quadruple drug "cocktails" (10Young B. AIDS Patient Care STDS. 2005; 19: 286-297Crossref PubMed Scopus (31) Google Scholar). Unfortunately, long-term exposure to antivirals is often associated with nephrotoxicity, lactic acidosis, hepatic failure, and skeletal myopathy (11Lewis W. Prog. Cardiovasc. Dis. 2003; 45: 305-318Crossref PubMed Scopus (34) Google Scholar, 12Anderson P.L. Kakuda T.N. Lichtenstein K.A. Clin. Infect. Dis. 2004; 38: 743-753Crossref PubMed Scopus (121) Google Scholar), problems that can be further exacerbated by additional, as yet undefined, drug interactions. organic anion transporter p-aminohippurate estrone-3-sulfate whole embryonic kidney culture 5-carboxyfluorescein 6-carboxyfluorescein 4-carboxyfluorescein-dimethylester carboxyfluorescein nucleotide reverse transcriptase inhibitor quantitative structure activity relationship human immunodeficiency virus azidothymidine polar surface area rotatable bonds wild type tetramethyl-rhodamine isothiocyanate. organic anion transporter p-aminohippurate estrone-3-sulfate whole embryonic kidney culture 5-carboxyfluorescein 6-carboxyfluorescein 4-carboxyfluorescein-dimethylester carboxyfluorescein nucleotide reverse transcriptase inhibitor quantitative structure activity relationship human immunodeficiency virus azidothymidine polar surface area rotatable bonds wild type tetramethyl-rhodamine isothiocyanate. Despite their plausible role via OAT transport in cytotoxicity, data on the relative affinity of antiviral drugs for OATs (Km or IC50) is limited mostly to Oat1 (13Wada S. Tsuda M. Sekine T. Cha S.H. Kimura M. Kanai Y. Endou H. J. Pharmacol. Exp. Ther. 2000; 294: 844-849PubMed Google Scholar, 14Cihlar T. Lin D.C. Pritchard J.B. Fuller M.D. Mendel D.B. Sweet D.H. Mol. Pharmacol. 1999; 56: 570-580Crossref PubMed Scopus (305) Google Scholar), with some data indicating the involvement of Oat3 (15Takeda M. Khamdang S. Narikawa S. Kimura H. Kobayashi Y. Yamamoto T. Cha S.H. Sekine T. Endou H. J. Pharmacol. Exp. Ther. 2002; 300: 918-924Crossref PubMed Scopus (228) Google Scholar, 16Cha S.H. Sekine T. Fukushima J.I. Kanai Y. Kobayashi Y. Goya T. Endou H. Mol. Pharmacol. 2001; 59: 1277-1286Crossref PubMed Scopus (444) Google Scholar, 17Uwai Y. Ida H. Tsuji Y. Katsura T. Inui K. Pharm. Res. (N. Y.). 2007; 24: 811-815Crossref PubMed Scopus (146) Google Scholar). However, the relative contribution of each OAT to the uptake of antivirals in epithelial and other tissues is unknown. The analysis is further confounded by the presence of multiple, simultaneously expressed transporters; varying degrees of substrate specificity at the (individual) transporter level; as well as a complex pattern of tissue expression and cellular localization. These issues have yet to be addressed in a single system or in a single set of experiments; unfortunately, widely used heterologous systems for addressing OAT affinity do not take these issues into account. In the original description of NKT/Oat1, it was shown that the gene is expressed during kidney development (4Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Whole organ culture could conceivably provide a system for studying substrate interactions in a complete environment, providing native cellular machinery recapitulating the whole tissue response ex vivo. Rat whole embryonic kidney (WEK) cultures have previously been shown to accumulate fluorescein in a probenecid-sensitive fashion (18Sweet D.H. Eraly S.A. Vaughn D.A. Bush K.T. Nigam S.K. Kidney Int. 2006; 69: 837-845Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Exploiting OATs expressed in WEKs, OAT isoform-specific fluorescent anion "trackers" capable of visualizing OAT-mediated uptake in organ systems could conceivably dissect the role of individual Oats in intact tissue. In fact, it has been shown that 6-carboxyfluorescein is an effective fluorescent tracer for studying hOat1-mediated translocation in transfected Chinese hamster ovary cells (19Cihlar T. Ho E.S. Anal. Biochem. 2000; 283: 49-55Crossref PubMed Scopus (63) Google Scholar). In general, carboxyfluoresceins (CF) have better kinetic properties for studying cellular OAT-mediated uptake than fluorescein, because the extra negative charge on the molecule allows for better cell retention (20De Clerck L.S. Bridts C.H. Mertens A.M. Moens M.M. Stevens W.J. J. Immunol. Methods. 1994; 172: 115-124Crossref PubMed Scopus (217) Google Scholar). In light of this, we developed and characterized a fluorescence-based assay for Xenopus oocytes and WEKs for determining OAT isoform substrate interactions using differently substituted carboxyfluoresceins. Additionally, we have utilized the uptake of fluorescent OAT substrates in the in vitro and the whole organ system to determine the affinity values for a subset of nucleotide reverse transcriptase inhibitors (NRTI) and acyclic nucleotide/nucleoside antivirals interacting with mOat1, mOat3, and mOat6: adefovir, cidofovir, tenofovir, acyclovir, ddC, ddI, 3TC, d4T, and AZT (21De Clercq E. J. Clin. Virol. 2004; 30: 115-133Crossref PubMed Scopus (832) Google Scholar). We demonstrate that mouse WEKs can be used for quantitative analyses and, using a novel live organ lectin staining procedure, localize distinct proximal tubule-like uptake patterns in WEK. By using mOat1 and mOat3 WEK knock-out strains, we confirm that adefovir and cidofovir interact predominantly with mOat1, although ddC and ddI seem to primarily interact with mOat3. The ex vivo data are generally consistent with our in vitro oocyte transport results obtained in this study. Finally, quantitative structure-activity relationship (QSAR) analysis was used to correlate the physicochemical properties of the nine antiviral molecules with their relative binding interactions to mOat1, mOat3, and mOat6. These characteristics include polar surface area, presence of hydrogen bond donors and acceptors, and the number of rotatable bonds. Thus, we have quantitatively and systematically analyzed the interaction of commonly used antiviral drugs with OATs on multiple levels. These results should aid in the design of antivirals directed toward or away from OAT expressing tissues with the goal of enhancing or diminishing transport (and presumably toxicity) into tissue compartments, and help in predicting drug-drug interactions and cytotoxicity during combination therapy. The quantitative multilevel analysis should also help provide a "systems" basis for understanding antiviral handling via OATs. The radiolabeled tracers [3H]p-aminohippurate (PAH) (specific activity 4.2 Ci/mmol) and [3H]estrone-3-sulfate (ES) (57 Ci/mmol) were purchased from PerkinElmer Life Sciences. [3H]ddC (30 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). Fluorescent anions, TRITC-conjugated lectins (Dolichos biflorus lectin and Lotus lectin (Sigma)), and prototypical OAT compounds (ES, PAH), and fluorescent dyes (4-carboxyfluorescein dimethylester (4-CFDM), 5-carboxyfluorescein (5-CF), 6-carboxyfluorescein (6-CF), fluorescein, eosin Y, and resorufin) were obtained from Sigma. Antivirals (adefovir, cidofovir, tenofovir, acyclovir, zidovudine (AZT), lamivudine (3TC), stavudine (d4T), zalcitabane (ddC), and didanosine (ddI)) were obtained from Moravek Biochemicals or Sigma. Xenopus oocyte assays were performed as described previously (1Kaler G. Truong D.M. Sweeney D.E. Logan D.W. Nagle M. Wu W. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2006; 351: 872-876Crossref PubMed Scopus (53) Google Scholar, 22Eraly S.A. Vallon V. Vaughn D.A. Gangoiti J.A. Richter K. Nagle M. Monte J.C. Rieg T. Truong D.M. Long J.M. Barshop B.A. Kaler G. Nigam S.K. J. Biol. Chem. 2006; 281: 5072-5083Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Capped RNA was synthesized from linearized plasmid DNA for SLC22a6 (mOat1) (Image clone 4163278), SLC22a8 (mOat3) (Image clone 4239544), and SLC22a20 (mOat6) (Image clone 6309674;) using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX). Briefly, oocytes were isolated and maintained in Barths buffer growth medium, injected with cRNA solution (0.5 μg/μl, 23 nl/oocyte), and allowed to mature for 3-4 days post-injection prior to transport assays. Experimental groups of 16-20 oocytes were placed in a 24-well plate with 1 ml of Barths buffer containing 1 μCi of a 3H-labeled organic anion tracer or a fluorescent anion tracer: 20 μm 6-CF (for mOat1-injected oocytes), 100 μm 5-CF (mOat3), or 30 μm fluorescein (mOat6). Samples contained different concentrations of an unlabeled organic anion. After a 1-h incubation at 25 °C, oocytes were washed 3 times with 4 °C Barths buffer and each experimental group was divided into four samples of 4-5 oocytes. Radioactivity was measured by scintillation counting (Rackbeta, Beckman-Coulter) or by fluorescence (PolarStar plate reader, BMG Labtechnologies, Durham, NC). To calculate the OAT-mediated component, the background tracer uptake was measured in uninjected oocytes and was subtracted from the uptake of OAT-injected oocytes in all experimental samples. Substrate activity was calculated for fluorescent tracers as the fluorescence clearance (CL) from the incubation medium (CL = Vtransport/S), by dividing the fluorescence (relative fluorescence units) absorbed per oocyte per unit time (Vtransport, relative fluorescence units/oocyte/h) by the tracer concentration in the incubation medium (S, relative fluorescence units/ml). [3H] transport rates utilize counts/min instead of the relative fluorescence unit term. Affinity of an inhibitor organic anion (IC50, Ki, or Km) was determined by measuring tracer uptake in the presence of 3-4 inhibitor concentrations in 10-fold increments. Each data point is the average of 2-3 experiments. Inhibition data were curve fitted using nonlinear regression in Prism software 5.0 (GraphPad Inc., San Diego, CA) to calculate the IC50 ± S.E., the Michaelis-Menten equation for Km, and the Ki value was determined as shown previously (23Kaler G. Truong D.M. Khandelwal A. Nagle M. Eraly S.A. Swaan P.W. Nigam S.K. J. Biol. Chem. 2007; 282: 23841-23853Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Maximum uptake (Vmax) was calculated as Vmax = CL · ([S]+ Ki), where CL values were measured in a single oocyte experiment with all fluorescent compounds tested at the concentration, S = 100 μm, and Ki values were from Table 1.TABLE 1OAT-binding affinities and transport characteristics of fluorescent substratesOATFluorescent substrateKiaCalculated from data of Fig. 1, the mean ± S.E.CLbFor each of the transporters, OAT-mediated clearance values were determined for all fluorescent tracers in a singie preparation of OAT-injected oocytes. For each transporter, data of a typical single experiment is presented (data obtained with different oocyte preparations are not comparable to each other).VmaxcCalculated as Vmax = CL · (S + Kd), with all fluorescent compounds tested at a concentration, S = 100 μm.μmnanoliter/oocyte/hpmol/oocyte/hmOat1 (SLC22a6)Fluorescein3.0 ± 0.112.7 ± 1.51.3 ± 0.25-Carboxyfluorescein523.4 ± 803.6 ± 1.02.2 ± 1.46-Carboxyfluorescein56.6 ± 326.7 ± 5.54.2 ± 1.14-Carboxyfluorescein-DM34.8 ± 48.7 ± 2.61.2 ± 0.4Eosin-Y0.1 ± 0.031.5 ± 1.70.2 ± 0.2Resorufin4.7 ± 0.2NDdND, uptake of resorufin was difficult to measure because it was rapidly decolorized (apparently metabolized) after being taken up by oocytes.NDmOat3 (SLC22a8)Fluorescein35.0 ± 281.6 ± 32.711.0 ± 4.85-Carboxyfluorescein373.3 ± 2895.5 ± 21.545.2 ± 16.96-Carboxyfluorescein10.7 ± 0.133.5 ± 9.23.7 ± 1.04-Carboxyfluorescein-DM17.6 ± 0.930.3 ± 8.83.6 ± 1.0Eosin-Y0.3 ± 0.040.6 ± 0.40.06 ± 0.04Resorufin8.3 ± 0.1NDNDmOat6 (SLC22a20)Fluorescein93.3 ± 6.226.4 ± 12.35.1 ± 2.65-Carboxyfluorescein3581.0 ± 5821.4 ± 1.8emOat6-mediated transport rate was not measurable for 5-CF and 6-CF because their uptake in OAT-injected oocytes was not significantly different from uninjected oocytes.NDemOat6-mediated transport rate was not measurable for 5-CF and 6-CF because their uptake in OAT-injected oocytes was not significantly different from uninjected oocytes.6-Carboxyfluorescein3162.3 ± 2411.4 ± 2.1emOat6-mediated transport rate was not measurable for 5-CF and 6-CF because their uptake in OAT-injected oocytes was not significantly different from uninjected oocytes.NDemOat6-mediated transport rate was not measurable for 5-CF and 6-CF because their uptake in OAT-injected oocytes was not significantly different from uninjected oocytes.4-Carboxyfluorescein-DM4456 ± 1173.8 ± 0.52.1 ± 0.9Eosin-Y4.8 ± 0.737.4 ± 26.63.9 ± 2.9Resorufin>3000NDNDa Calculated from data of Fig. 1, the mean ± S.E.b For each of the transporters, OAT-mediated clearance values were determined for all fluorescent tracers in a singie preparation of OAT-injected oocytes. For each transporter, data of a typical single experiment is presented (data obtained with different oocyte preparations are not comparable to each other).c Calculated as Vmax = CL · (S + Kd), with all fluorescent compounds tested at a concentration, S = 100 μm.d ND, uptake of resorufin was difficult to measure because it was rapidly decolorized (apparently metabolized) after being taken up by oocytes.e mOat6-mediated transport rate was not measurable for 5-CF and 6-CF because their uptake in OAT-injected oocytes was not significantly different from uninjected oocytes. Open table in a new tab WEK Culture/Tissue Preparation—Knockouts of mOat1 and mOat3 were bred as described previously (22Eraly S.A. Vallon V. Vaughn D.A. Gangoiti J.A. Richter K. Nagle M. Monte J.C. Rieg T. Truong D.M. Long J.M. Barshop B.A. Kaler G. Nigam S.K. J. Biol. Chem. 2006; 281: 5072-5083Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 24Sweet D.H. Miller D.S. Pritchard J.B. Fujiwara Y. Beier D.R. Nigam S.K. J. Biol. Chem. 2002; 277: 26934-26943Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). As described previously (18Sweet D.H. Eraly S.A. Vaughn D.A. Bush K.T. Nigam S.K. Kidney Int. 2006; 69: 837-845Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whole kidneys were microdissected from embryonic day 13.5 mice (E13.5) and grown on polycarbonate Transwell filters with Dulbecco's modified Eagle's medium/F-12 (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Mediatech) and 1% penicillin/streptomycin (Invitrogen) at 37 °C for 4 days. All animal protocols conformed to NIH guidelines for acceptable animal use. Whole Embryonic Kidney Uptake Assay and Confocal Microscopy—After 4 days of culture, E13.5 kidneys were washed once in phosphate-buffered saline, and WEKs (on the filter) were incubated for 1 h at 25 °C in a minimal saline solution (phosphate-buffered saline, 0.1 mm CaCl2, and 1 mm MgCl2) with 1 μm fluorescent tracker (5-CF, 6-CF, or fluorescein) and with or without tracker uptake inhibitor (1-2 mm probenecid: PAH, ES, ddC, ddI, adefovir, or cidofovir). WEKs were also incubated with 20 μg/ml D. biflorus lectin, a collecting duct marker, or Lotus lectin, a proximal tubule marker (25Laitinen L. Virtanen I. Saxen L. J. Histochem. Cytochem. 1987; 35: 55-65Crossref PubMed Scopus (153) Google Scholar). Following the 1-h uptake, WEKs were washed 3 times in 4 °C phosphate-buffered saline, cut from the Transwell filter, and mounted on a glass slide with fluormount (Southern Biotech, Birmingham, AL). To prevent fluorescent tracker diffusion out of the cell, samples were kept on ice until imaged. Confocal images were taken with the Nikon D-eclipse C1 confocal microscope (A.G. Heinze, Lake Forest, CA). In each individual experiment images were taken using the same settings (e.g. gain, saturation) for intensity comparisons. Quantitative Image Analysis—Image analysis was carried out using the ImageJ software (NIH, Bethesda, MD) using pixel intensity measurements. Images were manually outlined twice to measure intensity, and then the average value obtained was taken as the total intensity of a kidney. Four representative background areas were traced in interstitial spaces. Average background measurements were then subtracted from the total intensity. Significance values were determined using the t test and all values represent triplicate kidney samples. QSAR Analysis—The antiviral affinity data for mOat1, mOat3, and mOat6 were correlated with physicochemical descriptors using multiple linear regression. IC50 values were converted to Ki before conversion to the -log value (pKi). The descriptors, molecular weight, number of rotatable bonds (RB), LogP, and the calculated molar refractivity were calculated using Sybyl software (Tripos Discovery Informatics, St. Louis, MO) where as number of hydrogen bond donor, number of hydrogen bond acceptor, and polar surface area (PSA) were calculated using the Chemaxon package. The affinity data (pKi) and various descriptors were used as dependent and independent variables, respectively. The final model for each OAT contains statistically significant descriptors (p < 0.05). Fluorescent Tracers for OAT-mediated Uptake—To identify fluorescent compounds with the potential to serve as tracers of OAT-mediated transport, a panel of six fluorescent anions was tested for suitable characteristics. Key criteria included a high Vmax (translocation rate) and a suitable Ki (affinity constant) to minimize the chance of tracer competition with the test compound. The affinity values for each fluorescent anion for mOat1, mOat3, and mOat6 were determined by inhibiting the uptake of a radiolabeled tracer, either PAH or ES (Fig. 1 and Table 1). The fluorescent compounds (fluorescein, 4-CFDM, 5-CF, 6-CF, eosin Y, and resorufin) were all found to have significant interaction with all OATs tested. Because this does not necessarily indicate they are substrates, the uptake rates (Vmax) of the fluorescent compounds were also determined for each transporter (Fig. 1 and Table 1). The uptake of 5 of the compounds (simultaneously tested at 100 μm and used to calculate the Vmax) were obtained in a single typical experiment. All compounds tested proved to be translocated by (i.e. to be substrates for) at least one of the three transporters. The data from Table 1 was then used to determine the best "tracers" (i.e. probes for cellular accumulation) and trackers (i.e. visualization of transport in organ culture systems) for the different transporters. Based on the Vmax values determined, 6-CF, 5-CF, and fluorescein had the best characteristics for being used as probes of the transport activities of, respectively, mOat1, mOat3, and mOat6. 6-CF had the best characteristics for mOat1 transport compared with other compounds tested exhibiting a Ki of 57.3 μm and a Vmax of 4.2 pmol/oocyte/h (Table 1). Although 6-CF binding affinity for mOat1 proved to be lower than that of fluorescein, 4-CFDM, eosin Y, and resorufin, the translocation rate, Vmax, was the highest (Table 1). Similarly, whereas 5-CF showed the poorest affinity for mOat3 (Ki, of 373.3 μm, compared with the second poorest, fluorescein, which was 35.1 μm), it had the highest Vmax value, 45.2 pmol/oocyte/h. Therefore, 5-CF has the best characteristics for use as a fluorescent mOat3 substrate. For mOat6, fluorescein had the second strongest binding affinity (Ki, 93.2 μm, compared with eosin Y, 5 μm) as well as the highest Vmax (5.1 pmol/oocyte/h), qualities that suggest its use as an effective tracer for mOat6. OAT Antiviral Affinities—Whereas mOat1 and mOat3 are coexpressed in many tissues, the relative proportion, as well as functionality, can be variable. For example, mOat1 is expressed in mouse olfactory mucosa (along with mOat6), whereas in retina, mOat3 expression appears higher than that of mOat1 (7Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (91) Google Scholar). Thus, expression patterns are likely to be important determinants of tissue penetration for various antivirals. Using the identified fluorescent probes (5-CF, 6-CF, and fluorescein), binding affinities for a diverse group of antiviral drugs were determined for mOat1, mOat3, and mOat6 in Xenopus oocytes. The uptake of many of these compounds has been determined for hOat1 (8Burckhardt B.C. Burckhardt G. Rev. Physiol. Biochem. Pharmacol. 2003; 146: 95-158Crossref PubMed Scopus (256) Google Scholar, 9Sweet D.H. Toxicol. Appl. Pharmacol. 2005; 204: 198-215Crossref PubMed Scopus (129) Google Scholar, 12Anderson P.L. Kakuda T.N. Lichtenstein K.A. Clin. Infect. Dis. 2004; 38: 743-753Crossref PubMed Scopus (121) Google Scholar), although not side by side in the same study. Due to structural homology, Oat3 and Oat6 might translocate them as well, although this remains to be determined. Because of this, and specifically for these compounds, IC50 (half-maximal inhibition of transport) should be a good approximation of transport affinity (Km), because translocation by a SLC22a family member has been established. Adefovir, tenofovir, cidofovir, and acyclovir all demonstrated significantly greater affinity for mOat1 than for its h
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