JANUARY 7, 2011 VOLUME 286 NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 243

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1 THE JURAL F BILGICAL CHEMISTRY VL. 286,. 1, pp , January 7, by Te American Society for Biocemistry and Molecular Biology, Inc. Printed in te U.S.A. Analysis of Tree-dimensional Systems for Developing and Mature Kidneys Clarifies te Role of AT1 and AT3 in Antiviral Handling * Received for publication, April 30, 2010, and in revised form, ctober 1, 2010 Publised, JBC Papers in Press, ctober 4, 2010, DI /jbc.M Mega A. agle, David M. Truong, Ankur V. Dnyanmote, Sun-Young An, Satis A. Eraly, Wei Wu, and Sanjay K. igam ** 1 From te Departments of Pediatrics, Cellular and Molecular Medicine, and **Bioengineering and te Division of eprology and Hypertension, Department of Medicine, University of California San Diego, La Jolla, California and te Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas Te organic anion transporters AT1 (SLC22A6, originally identified by us as KT) and AT3 (SLC22A8) are critical for andling many toxins, metabolites, and drugs, including antivirals (Truong, D. M., Kaler, G., Kandelwal, A., Swaan, P. W., and igam, S. K. (2008) J. Biol. Cem. 283, ). Altoug microinjected Xenopus oocytes and/or transfected cells indicate overlapping specificities, te individual contributions of tese transporters in te tree-dimensional context of te tissues in wic tey normally function remain unclear. Here, andling of HIV antivirals (stavudine, tenofovir, lamivudine, acyclovir, and zidovudine) was analyzed wit tree-dimensional ex vivo functional assays using knock-out tissue. To investigate te contribution of AT1 and AT3 in various nepron segments, te AT-selective fluorescent tracer substrates 5-carboxyfluorescein and 6-carboxyfluorescein were used. Altoug AT1 function (uptake in oat3 / tissue) was confined to portions of te cortex, consistent wit a proximal tubular localization, AT3 function (uptake in oat1 / tissue) was apparent trougout te cortex, indicating localization in te distal as well as proximal nepron. Tis functional localization indicates a complex tree-dimensional context, wic needs to be considered for metabolites, toxins, and drugs (e.g. antivirals) andled by bot transporters. Tese results also raise te possibility of functional differences in te relative importance of AT1 and AT3 in antiviral andling in developing and mature tissue. Because te HIV antivirals are used in pregnant women, te results may also elp in understanding ow tese drugs are andled by developing organs. * Tis work was supported, in wole or in part, by ational Institutes of Healt Grants AI057695, DK079784, and GM88824 (to S. K..) and HL (to S. A. E.). Tis work was also supported by a postdoctoral fellowsip from te Society of Toxicology and Colgate Palmolive (to A. V. D.). 1 To wom correspondence sould be addressed: Dept. of Medicine, University of California San Diego, 9500 Gilman Dr., La Jolla, CA snigam@ucsd.edu. 2 Te abbreviations used are: AT, organic anion transporter; ABC, ATPbinding cassette; 5CF, 5-carboxyfluorescein; 6CF, 6-carboxyfluorescein; TRITC, tetrametylrodamine isotiocyanate; K, knock-out. rganic anion transporters (ATs) 2 are widely distributed across epitelia of multiple organs and are responsible for te andling and excretion of a diverse array of xenobiotics as well as endogenous metabolites. Among te former are commonly used drugs suc as penicillins, nonsteroidal anti-inflammatory drugs, diuretics, and various antivirals (1 3). oat1, initially identified by us as nkt (novel kidney transporter) (1 4), and oat3, initially identified as roct (5, 6), are two major transporters belonging to te SLC22 family of solute carriers, and along wit oter SLC transporter families (e.g. organic anion-transporting polypeptides and SLC21) as well as members of te ATP-binding cassette (ABC) transporter family, tey are implicated in te renal secretion of drugs. However, it is AT1 and AT3, wic are bot located on te basolateral membrane of te proximal renal tubule, tat are primarily responsible for renal uptake and are terefore likely to play rate-limiting roles in regulating te distribution of drugs, toxins, and metabolites between blood and urine. However, most studies ave been performed using simple in vitro systems, and many unanswered questions remain about te function at te level of te wole organ. Moreover, it as been suggested tat remote sensing and signaling occur between oat omologs in multiple tissues/organs and fluid compartments (e.g. blood and urine) via organic anion metabolites and/or signaling molecules (1, 7, 8). AT-mediated tubular accumulation of some drugs, including certain antivirals, can result in neprotoxicity (9). Detailed functional information concerning kidney transporters present at te blood-urine barrier is crucial for understanding te neprotoxicity of tese antivirals and would elp advance te development of new terapeutics. Data on compartmentalization of antivirals between blood and urine could also elp provide a systemic view of antiviral distribution, elimination, and toxicity. Tis type of analysis as not been attempted due to a paucity of tecniques to address te role of specific genes in tree-dimensional contexts. Previous studies on antivirals ave concluded tat AT1 and, to a lesser extent, AT3 play a major role in transport of antiviral drugs suc as adefovir, cidofovir, and tenofovir. For example, AT1 as been sown to be te major transporter for adefovir (10, 11), wereas an equal contribution to te transport of tenofovir by AT1 and AT3 was reported by anoter group (12). Te interaction of AT1 and AT3 wit a larger set of antivirals as also been investigated in vitro (3, 13). Analyses of specific ATs in te tree-dimensional context of te adult and developing kidneys are critical for under- JAUARY 7, 2011 VLUME 286 UMBER 1 JURAL F BILGICAL CHEMISTRY 243

2 Role of AT1 and AT3 in Antiviral Handling standing te distribution of antivirals between blood and urine. We terefore studied te interaction of te ATs wit stavudine, tenofovir, lamivudine, acyclovir, and zidovudine in tree-dimensional systems for adult and embryonic kidneys from oat1 and oat3 knock-outs (13 15). Tis enabled us to isolate te contribution of eac transporter. Moreover, a novel visualization approac for fluorescent organic anion uptake in adult renal slices provided information on te functional localization of te ATs tat functionally establises te likely contributions of particular nepron segments to drug and toxin transport. Te results also raise te possibility of different ATs being important in antiviral andling at different developmental stages. EXPERIMETAL PRCEDURES Materials Water-soluble probenecid was purcased from Molecular Probes. Te fluorescent tracers 5-carboxyfluorescein (5CF) and 6-carboxyfluorescein (6CF) and tetrametylrodamine isotiocyanate (TRITC)-conjugated lectins (Dolicos biflorus lectin and Lotus lectin) were obtained from Sigma. Antivirals (stavudine, lamivudine, tenofovir, zidovudine, and acyclovir) were purcased from eiter Moravek Biocemicals (Brea, CA) or Sigma. Adult Renal Slice Uptake Assay and Imaging Kidneys from adult wild type and oat1 and oat3 knock-outs were dissected and maintained in 4 C L-15 medium until coronal 0.2-mm slices were cut wit a Stadie-Riggs microtome (14). Slices were maintained in PBS at 4 C, and symmetrical pieces were ten selected and placed in 24-well plates containing minimal saline wit eiter 1 M 5CF or 1 M 6CF along wit eiter TRITC-conjugated D. biflorus or Lotus lectin and in te presence of eiter PBS or an inibitor (probenecid (2 mm), stavudine (1 mm), acyclovir (200 M), tenofovir (100 M), zidovudine (100 M), or lamivudine (200 M)). Slices were incubated at 25 C for 1 and ten wased tree times wit PBS before being placed on a slide wit Fluoromount and imaged wit a ikon D-Eclipse C1 confocal microscope (A. G. Heinze, Lake Forest, CA). rgan Culture Uptake Assay and Imaging Embryonic kidneys from embryonic day 13.5 oat1 and oat3 knock-outs were cultured for 4 days at 37 C in DMEM/F-12 wit 10% FBS and 1% penicillin/streptomycin as described previously (3).n day 4 of culture, te cultures (n 3) were wased once wit PBS containing CaCl 2 (0.1 mm) and MgCl 2 (1 mm) at room temperature. 1 M 6CF was added to eac well, and cultures were incubated (in te dark) wit or witout probenecid (2 mm), stavudine (1 mm), acyclovir (200 M), tenofovir (100 M), zidovudine (100 M), and lamivudine (200 M). Cultured kidneys were wased four to five times wit PBS (containing CaCl 2 and MgCl 2 ) and imaged. Images were obtained by confocal microscopy. Knock-outs and wild types were generated as described previously (14, 15). Knock-outs were backcrossed to C57BL/6J for seven generations for oat1 (Deltagen, Inc., Redwood City, CA) and for four generations for oat3. Embryonic day 13.5 (oat1 and oat3 knock-out (K)) tissue was used for embryonic kidney culture, followed by te assays described below (13). Experiments on adult renal slices were performed on tissue from age-matced adult non-pregnant female oat1 and oat3 knock-outs between 12 and 20 weeks of age. Quantitative Image Analysis Images were taken at 4 magnification. Te kidney was outlined, and pixel intensities were measured wit IH Image J software as described previously (3). Microarray Data: Expression of Transporters in oat3 Knock-outs As described previously (16), gene expression profiling in oat3 K kidney was carried out using Affymetrix mouse GeneCip 230 according to te manufacturer s specification. RESULTS To begin to understand te relative contribution of AT1 and AT3 to antiviral andling in different tissues regulating te blood-urine interface, we employed tree-dimensional assays utilizing wild-type and knock-out kidneys. Two kinds of tree-dimensional ex vivo kidney assays were used: adult kidney slices and cultured wole embryonic kidney (wic is allowed to differentiate in vitro). Te latter as te considerable advantage of being a live assay in tat te tissue continues to grow for 2 3 weeks. Functional Localization of AT1 (in oat3 Ks) and AT3 (in oat1 Ks) in Adult Kidney Using Fluorescent Tracers A classic assay for analyzing transport in te adult kidney is te use of renal tissue slices, but tis assay is not well validated for te determination of AT1- and AT3-specific transport. In fact, current data are contradictory. Altoug some studies suggest tat tis process occurs primarily in te proximal tubule, oter immunolocalization data suggest tat oat expression beyond tis part of te nepron includes even te collecting ducts, a notion tat does not ave muc pysiological support (17). Te functional consequence of te localization of AT1 and AT3 transport as not been previously establised. Tis is obviously important for designing combination terapies, like tose employing multiple antivirals as in HIV terapy, were competition at te transport step may be an important consideration. In addition, te increasing problem of drug-induced neprotoxicity furter illustrates te importance of understanding te molecular interaction between te transporters in te kidney and antivirals. To functionally define te relative contribution of AT1 (in oat3 Ks) and AT3 (in oat1 Ks) transporters to antiviral andling, as well as wic nepron segments are involved, we determined te uptake patterns of te fluorescent AT substrates 6CF and 5CF (3) in adult kidney slices from te wild type and oat1 and oat3 Ks. Te use of tese fluorescent substrates to study AT1 and AT3 uptake is well establised (3, 8). Tis metod allows for te direct visualization of transport and te anatomical localization of AT function. Initially, we assessed CF uptake in fres renal slices (coronal sections) from WT adults. Tese assays revealed discrete fluorescence trougout te cortex of te kidney (Fig. 1, A and B), wit only background fluorescence in te outer medullary stripe for bot 5CF and 6CF. Co-staining wit D. biflorus lectin, a lectin tat binds to collecting ducts (18, 19), demonstrated, importantly, a lack of CF uptake in collecting duct rays of te inner medulla as well tose of te cortical collect- 244 JURAL F BILGICAL CHEMISTRY VLUME 286 UMBER 1 JAUARY 7, 2011

3 Role of AT1 and AT3 in Antiviral Handling FIGURE 1. Fluorescent organic anion tracer uptake in adult WT kidney slices. A D, potomicrograps of coronal sections of WT adult kidney stained wit D. biflorus lectin (red). Sown are te results from te examination of te AT-mediated uptake of te fluorescent tracers (green) 5CF (1 M)(A and C) and 6CF (1 M)(B and D) in te absence (A and B) and presence (C and D) of 2 mm probenecid. ccd, cortical collecting duct;, nepron. Scale bar 200 m. ing ducts. Inibition of CF uptake by 2 mm probenecid confirmed AT-mediated transport (Fig. 1, C and D). Interestingly, te cortical fluorescence patterns of 5CF and 6CF uptake were distinct. 5CF sowed uptake trougout te cortical tubule, wereas 6CF manifested discrete areas of uptake in te juxtamedullary portion of te cortex, consistent wit proximal tubule localization (Fig. 1, A and B). Given te differences in te kinetics of tese tracers wit respect to AT1 and AT3 (3), te different fluorescence patterns observed are likely to reflect true differences in te functional transport by AT1 and AT3. To directly determine te intrarenal location of AT3 function, we used renal slices from oat1 knock-outs. As we sow below, tese knock-outs do not exibit significant gene expression canges in ABC and SLC transporters oter tan te oat knock-outs studied ere. Because we are studying probenecid-inibitable uptake, it is unlikely tat te effects were AT-independent. Two types of slices were generated: first, a coronal cut to delineate te uptake differences between te FIGURE 2. Localization of fluorescent tracer 5CF in oat1 knock-out and fluorescent tracer 6CF in oat3 knock-out as a function of AT3 and AT1, respectively. Potomicrograps sow coronal sections of adult kidney from oat1 Ks (A, C, and E) and AT3 Ks (B, D, and F). A, C, and E, examination of 5CF uptake (green) in adult oat1 Ks co-stained wit eiter D. biflorus lectin (red)(a) or Lotus lectin (red)(c and E). B, D, and F, examination of 6CF uptake (green) in adult oat3 Ks co-stained wit eiter D. biflorus lectin (red)(b) or Lotus lectin (red)(d and F). ccd, cortical collecting duct; G, glomerulus; M, outer medulla; IM, inner medulla. Te asterisks indicate occlusions in te pattern of tracer uptake. mat, mouse AT. Scale bars 200 mina D and 60 mine and F. cortex, outer medulla, inner medulla, and pelvis; and second, a sallow coronal cut troug just te cortex to determine te intracortical location of CF accumulation. As expected, loss of AT1 eliminated te differences between 5CF and 6CF uptake (data not sown), indicating tat eiter could be used to assess AT3-mediated transport. Te results of 5CF uptake in oat1 Ks (function of AT3) are summarized in Fig. 2 (A, C, and E). o probenecid-sensitive uptake was seen in eiter te inner or outer medulla or renal pelvis; furtermore, D. biflorus lectin co-staining (Fig. 2A) sowed tat cortical collecting ducts also did not accumulate CF. However, as revealed by co-staining wit Lotus lectin (Fig. 2, C and E), wic preferentially binds to te mesencymally derived nepron, proximal and distal tubules trougout te cortex accumulated significant cellular levels of CF, wic was competed by probenecid. Circular gaps in te uptake pattern in te cortex resembled glomeruli in sape and location. Tese results suggest tat AT3 function is present in distal as well as proximal tubules (but not in glomeruli or collecting ducts). JAUARY 7, 2011 VLUME 286 UMBER 1 JURAL F BILGICAL CHEMISTRY 245

4 Role of AT1 and AT3 in Antiviral Handling Similarly, te location of AT1 function was determined using renal slices from te oat3 knock-outs. As in oat1 Ks, bot 5CF and 6CF exibited similar patterns. Bot coronal and sallow cortex sections were exposed to 6CF (te preferred AT1 substrate (3)) (Fig. 2, B, D, and F). o probenecid-sensitive accumulation was seen in te medulla, renal pelvis, cortical collecting ducts, or presumptive glomeruli. otably, probenecid-inibitable uptake was seen in distinct patces in te cortex, similar to te pattern seen in wild-type slices incubated wit 6CF (Fig. 2D). In te sallow coronal slice, uptake was seen in a grid-like pattern (Fig. 2D), wit rectangular regions ( occlusions ) lacking uptake. Higer magnification revealed distinct tubules wit or witout AT function (Fig. 2F). Tese results suggest tat AT1 is functionally sequestered into a distinct tubule type, consistent wit proximal tubule localization. Importantly, tis implies tat AT1- and AT3-mediated transport is anatomically and functionally distinct. Tese results appear to be te first demonstration of te distribution of transport witin te context of te wole organ and ave broad implications for understanding system-wide drug, toxin, and metabolite andling. Tese considerations are particularly important for te design of combination antiviral terapies because in vitro oocyte transport data sow tat antivirals can be transported by AT1, AT3, or bot (3). Interaction of Antivirals wit AT3 (in oat1 Ks) and AT1 (in oat3 Ks) in Adult Kidney Slices Having establised 5CF and 6CF uptake in renal slices as a novel ex vivo metod of examining AT-mediated transport, we developed te metod furter by utilizing a competitive assay between antivirals and 6CF. Te reductions in signal were normalized against CF uptake alone and probenecid inibition. As described above, tissues from te oat K animals were used to distinguis AT1- and AT3-mediated transport. AT3- specific (in oat1 Ks) and AT1-specific (in oat3 Ks) uptake of 6CF (1 M) was tested in adult renal slices (Figs. 3 and 4). Binding was demonstrated in oat1 knock-out kidneys (function of te AT3 transporter) in te presence of zidovudine (100 M) (Fig. 3, C and H), acyclovir (200 M) (Fig. 3, D and H), tenofovir (100 M) (Fig. 3, E and H), lamivudine (200 M) (Fig. 3, F and H), and stavudine (1 mm) (Fig. 3, G and H). Similarly, blocking of fluorescence uptake in oat3 knock-out slices (function of te AT1 transporter) (Fig. 4, C H) was tested and compared in te presence of te antivirals at te same concentrations as for oat1 knock-out slices (function of te AT3 transporter). Te antivirals were tested in adult and embryonic kidneys at concentrations similar to IC 50 values gatered from te Xenopus oocyte data (Table 1 (3, 20 26) and Table 2 (3, 16, 21 25)). Inibition of 6CF tracer uptake by te antivirals was similar in oat1 and oat3 K renal slices (Figs. 3 and 4). Except for tenofovir (Fig. 3E), wic inibited uptake by 90% in oat1 K slices (function of te AT3 transporter) (Fig. 3), all of te antivirals in oat1 (function of AT3) and oat3 (function of AT1) knock-out slices (Figs. 3 and 4, respectively) manifested moderate inibition of 6CF tracer uptake in te 30 70% range. FIGURE 3. Inibition of 6CF uptake by antivirals in oat1 knock-out adult kidney slices (function of te AT3 transporter). Fluorescent potomicrograps sow 6CF (1 M) uptake (green) in adult oat1 K kidney slices in te presence of a veicle control (A),2mM probenecid (B), 100 M zidovudine (C), 200 M acyclovir (D), 100 M tenofovir (E), 200 M lamivudine (F), and 1 mm stavudine (G). Images are representative of triplicate slices from te same experiment. Te bar grap (H) sows relative fluorescent signal strengt derived from quantitative image analysis of 6CF in adult oat1 K kidney slices. Scale bar 0.2 mm. Interaction of Antivirals wit AT1 and AT3 in rgan Culture We previously establised organ culture of wole embryonic kidney as a novel model for te analysis of organic anion transporter function (3, 13). Tese cultures, wic alter 246 JURAL F BILGICAL CHEMISTRY VLUME 286 UMBER 1 JAUARY 7, 2011

5 Role of AT1 and AT3 in Antiviral Handling TABLE 1 Kinetics ( M) of antiviral drugs for AT1 (literature-based compilation of data) Kinetic parameters (K m, K i, and IC 50 ) for antiviral drugs in mouse (m), rat (r), and uman () AT1 were analyzed in different in vitro assay systems. Te individual structures of antiviral drugs are also sown. I, inibition; S, substrate; S2, cell line from te second segment of te proximal tubule; LLC-PK1, pig renal epitelial cell; CH, Cinese amster ovary cells; HEK293, uman embryonic kidney cell line; X. laevis, Xenopus laevis oocytes. See references below for details. Table 1 a Kinetics (µm) of antivirals for at1 Assay Drugs at1 Binding Km Ki IC50 Structure System Acyclovir Lamivudine AT1 S/I S b H 2 mat1 I X. laevis 209 c I LLC-PK1 981 d H rat1 S/I X. laevis 242 e mat1 I X. laevis 104 c S rat1 S X. laevis e H H H 2 Stavudine mat1 I X. laevis 628 c rat1 S/I X. laevis e H H S CH 33.8 f H AT1 2 g Tenofovir S HEK293 mat1 I X. laevis 81 c I S b AT1 S HEK293 H Zidovudine mat1 I X. laevis 78 c - S LLC-PK d + rat1 S/I X. laevis 68 e a adapted from ref. 19; b ref. 23; c ref. 3; d ref. 21; e ref. 25; f ref. 20; g ref. 24; ref. 22. P H H TABLE 2 Kinetics ( M) of antiviral drugs for AT3 (literature-based compilation of data) Kinetic parameters (K m, K i, and IC 50 ) for antiviral drugs in mouse (m), rat (r), and uman () AT3 were analyzed in different in vitro assay systems. Te individual structures of antiviral drugs are also sown. I, inibition; S, substrate; S2, cell line from te second segment of te proximal tubule; LLC-PK1, pig renal epitelial cell; CH, Cinese amster ovary cells; HEK293, uman embryonic kidney cell line; X. laevis, X. laevis oocytes. See references below for details. Table: 2 a Kinetics (µm) of antivirals for at3 Drugs at3 Binding Assay System Km Ki IC50 Structure b I S2 g AT3 I X. laevis f S HEK293 Acyclovir S I Knockout mat3 H I X. laevis 729 c rat3 I LLC-PK d I Knockout Lamivudine mat3 S I X. laevis 140 c H I Knockout Stavudine mat3 H I X. laevis 2113 c e AT3 S HEK293 H 2 Tenofovir I Knockout P mat3 H I X. laevis 384 c AT3 S/I S b I Knockout H H Zidovudine mat3 - I X. laevis 38 c + rat3 I LLC-PK1 143 d a adapted from ref. 19; b ref. 23; c ref. 3; d ref. 21; e ref. 24; f ref. 22; g ref. 26; ref. 27. H H 2 H 2 H H FIGURE 4. Inibition of 6CF uptake by antivirals in oat3 knock-out adult kidney slices (function of te AT1 transporter). Fluorescent potomicrograps sow 6CF (1 M) uptake (green) in adult oat3 K kidney slices in te presence of a veicle control (A),2mM probenecid (B), 100 M zidovudine (C), 200 M acyclovir (D), 100 M tenofovir (E), 200 M lamivudine (F), and 1 mm stavudine (G). Te bar grap (H) sows relative fluorescent signal strengt derived from quantitative image analysis of 6CF absorption in adult oat3 K kidney slices. Scale bar 0.2 mm. *, p wit period of differentiation, express bot AT1 and AT3 and manifest significant uptake of AT substrates, including te fluorescent organic anions 5CF and 6CF, wit tis uptake being inibitable by te classic AT inibitor probenecid. Moreover, te individual contribution of eiter AT (AT1 or AT3) in isolation as also been studied by performing experiments in kidney organ cultures derived from mice null for te oter AT (transport of AT substrates in oat1 knock-out tissue sould be largely due to AT3 and vice versa) (3). In tis study, we used te oat knock-out kidney organ culture assay to analyze antiviral interactions wit AT1 and AT3. Wen tested in oat1 K-derived tissues (function of AT3), te uptake of 1 M 6CF (Fig. 5A) was blocked by 2 mm probenecid (Fig. 5, B and H) and, to a varying degree, by te five antivirals: zidovudine (100 M) (Fig. 5, C and H), acyclovir (200 M) (Fig. 5, D and H), 100 M tenofo- JAUARY 7, 2011 VLUME 286 UMBER 1 JURAL F BILGICAL CHEMISTRY 247

6 Role of AT1 and AT3 in Antiviral Handling FIGURE 5. Inibition of 6CF uptake by antivirals in cultured oat1 knockout embryonic kidneys in organ culture (function of AT3). Fluorescent potomicrograps sow 6CF (1 M) uptake in cultured oat1 knock-out embryonic kidneys in te presence of a veicle control (A), 2 mm probenecid (B), 100 M zidovudine (C), 200 M acyclovir (D), 100 M tenofovir (E), 200 M lamivudine (F), and 1 mm stavudine (G). Images are representative of triplicate embryonic kidney cultures. Te bar grap (H) sows relative fluorescent signal strengt of 6CF in cultured embryonic kidneys derived from quantitative image analysis. WEK, wole embryonic kidney. Scale bar 0.2 mm. *, p vir (100 M) (Fig. 5, E and H), lamivudine (200 M) (Fig. 5, F and H), and stavudine (1 mm) (Fig. 5, G and H). Similarly, tracer uptake by cultured kidneys derived from oat3 knock- 248 JURAL F BILGICAL CHEMISTRY FIGURE 6. Inibition of 6CF uptake by antivirals in cultured oat3 knock-out embryonic kidneys in organ culture (function of AT1). Fluorescent potomicrograps sow 6CF (1 M) uptake in cultured oat3 knock-out embryonic kidneys in te presence of a veicle control (A), 2 mm probenecid (B), 100 M zidovudine (C), 200 M acyclovir (D), 100 M tenofovir (E), 200 M lamivudine (F), and 1 mm stavudine (G). Te bar grap (H) sows relative fluorescent signal strengt of 6CF absorption in cultured embryonic kidneys derived from quantitative image analysis. WEK, wole embryonic kidney. Scale bar 0.2 mm. *, p outs (function of AT1) was also inibited by te antivirals (Fig. 6, A H). Tus, our results suggest tat tese antivirals interact wit AT1 and AT3 transporters in te cultured embryonic kidneys from te knock-out tissues. VLUME 286 UMBER 1 JAUARY 7, 2011

7 However, te degree of antiviral inibition was muc greater for 6CF tracer uptake in oat3 K (function of te AT1 transporter) kidney organ cultures (Fig. 6) tan for tat in oat1 K kidney organ cultures (Fig. 5), representing AT3-mediated interaction. Altoug all five antivirals could inibit AT3-mediated 6CF interaction (oat1 K cultures) by 50%, tenofovir, lamivudine, and zidovudine inibited AT1-mediated transport (oat3 K kidney organ cultures) by 90% (Fig. 5, E, F, and C, respectively), wereas acyclovir inibited it by 80% (Fig. 5D). Stavudine was te only compound for wic inibition of transport was comparable in oat1 and oat3 K kidney organ cultures (Figs. 5G and 6G). Tese findings suggest a greater affinity of antivirals for AT1 tan for AT3 in te embryonic kidney. Additionally, it is clear tat tere are significant differences between te adult and embryonic kidney tree-dimensional assays. Tese differences, wic may be clinically important, could be due to variations in post-translational modifications and migt also represent tissue- and/or developmental stage-specific modulation of AT function. Altoug te fluorescent substrate 5CF could also be andled by oter transporters (including ABCC2 (MRP2), ABCC4 (MRP4), MRP5, ABCG2, and ABCB1 (P-glycoprotein)), tese proteins are expressed, owever, on te luminal (apical) surface of renal tubular cells. Altoug tey could possibly ave an effect on te overall andling of te drug, tey would not be expected to be probenecid-sensitive, and furtermore, we ave supported our findings in knock-out tissue. Te basolateral import transporters (AT1 and AT3) seem to determine te rate-limiting step in te clearance of organic anionic drugs from te blood, across te tubule, and into te urine. Additionally, we previously publised a study reporting te expression profiles of abcc2 (mrp2) and abcc4 (mrp4)inoat1 and oat3 knock-out mice in comparison wit wild-type mice (16). Te expression of tese transporters was not significantly altered by te deletion of eiter oat1 or oat3. We ave extended tese results to include abcb1 and abcg2 in oat3 knock-outs (Fig. 7). Te renal expression of tese transporters was also not significantly altered in oat3 knock-outs in te same microarray analysis. However, it is evident from te expression data reported (16) tat te expression of oat1 was reduced by 40% in te oat3 knock-out, and conversely, oat3 expression was reduced by 40% in te oat1 knock-out. Tis is peraps a consequence of te oat1 and oat3 genes being located adjacent to eac oter. It is important to note tat, despite te observed cange in transcription, te renal transport of AT1 substrate in te oat3 knockout and te renal transport of AT3 substrate in te oat1 knock-out were unaffected. Terefore, te cange in RA levels did not appear to be reflected in a functional difference in transport in te intact animal. DISCUSSI AT1 (initially identified by us as KT (4)) is te prototypical organic anion transporter and, along wit AT3, is considered a primary component of te classic renal excretion patway. AT1 was initially proposed to be eiter an organic anion transporter or an organic cation transporter (4), and it Role of AT1 and AT3 in Antiviral Handling FIGURE 7. Expression data in oat3 knock-out mice sowing renal expression of ABCB1 and ABCG2. Gene expression profiling in oat3 knockout kidney using Affymetrix mouse GeneCip 230 was performed (16), and te expression levels of ABCB1 (A) and ABCG2 (B) were determined. is now clear tat AT1 and AT3 can be bot, altoug organic anions remain te preferred substrates. Bot of tese major transporters are located on te basolateral membrane of te renal epitelial tubule and account for te rate-limiting step in te excretion of many drugs from te blood into urine. Togeter, tese two ATs initiate te vectorial movement of organic anions across te proximal tubular cell by taking tem up from te blood. Meanwile, MRP, ABCG2, and oter transporters, located on te luminal (apical) side of te cell, are responsible for excretion. Tus, te overall distribution of substrates between blood and urine is te result of te coordinated activities of bot apical and basolateral localized transporters. However, it is generally accepted tat uptake via AT1 and AT3 is te rate-limiting step for most organic anion drugs transversing tis patway (1). Most of te current data come from in vitro transport experiments in microinjected frog oocytes. How drugs, toxins, and metabolites distribute into different body compartments and te rate-limiting patways involved are a major biocemical, pysiological, and parmacological problem. Moreover, little is known about te interplay between multiple fluid compartments, suc as blood and urine, and te role of AT isoforms, given overlapping substrate specificities, in te distribution and elimination of drugs, toxic compounds, and metabolites (7). Indeed, it as been proposed tat ATs and oter drug transporters are part of a systemic remote sensing and signaling network (1, 7, 8). Here, we ave used novel approaces employing oat1 and oat3 knock-out tissues (to assess AT3 and AT1 function, respectively) (14, 15) to analyze, in a tree-dimensional con- JAUARY 7, 2011 VLUME 286 UMBER 1 JURAL F BILGICAL CHEMISTRY 249

8 Role of AT1 and AT3 in Antiviral Handling text, te rate-limiting patways involved in te andling of antivirals used in cases of HIV infection and proposed for use for small pox propylaxis. As discussed below, te results not only define te transporter (AT1 or AT3) preferences in a tree-dimensional context, tey provide te first functional evidence tat altoug AT1 and AT3 overlap in te proximal tubule, tere is major AT3 activity distally. Tese patways govern te distribution of antivirals between blood and urine. Te issue is important not only from te perspective of treatment but also toxicity. For example, drug-induced neprotoxicity is a significant problem in ospitalized patients. In te case of HIV patients, low-level antiviral drug-induced neprotoxicity is increasingly reported (27). Most of te nucleoside reverse transcriptase inibitor antivirals ave neprotoxic side effects, wic can be potentiated in patient groups (suc as HIV patients) wo are treated wit a combination of tese antiviral terapies. Because renal excretion plays a major role in te elimination of tese drugs, te kidney is routinely exposed to ig concentrations of tese antivirals and teir metabolites. Accumulation and toxicity of drugs and metabolites are influenced by specific transport patways tat lead to site-specific toxicity in te nepron. Tus, te identification of te key transporters for individual antivirals could be crucial to understanding te patopysiology of te neprotoxicity due to tese compounds wen used alone or in combination. Furtermore, te use of fluorescent tracers as described ere allows for spatial/anatomical demarcation of te domains of AT function (as proximal for AT1 and bot proximal and distal for AT3), enabling one to examine patterns of organic anion transport in te context of te native intrarenal environment. Tis functional knowledge is crucial for devising combination terapies so tat tey do not compete for ATmediated transport during renal elimination or for avoiding furter toxicity to te same segment of te renal tubule. Previous immunolocalization studies ave been somewat discrepant. For example, it was sown tat uman AT1 and AT3 expression is concentrated in te proximal tubules of te adult kidney (28). n te oter and, altoug localization of rat AT1 as been reported in a manner consistent wit proximal tubules (4), rat AT3 as also been localized to connecting tubules and cortical and medullary collecting ducts (17). However, te differential expression pattern of te two oat isoforms, not to mention differential functional expression, as been far from clear. Tis study provides te first clear functional evidence. We determined uptake of te fluorescent AT substrates 6CF and 5CF in adult kidneys from te wild type and oat1 (function of AT3 transport) and oat3 (function of AT1 transport) Ks as a novel metod for te direct visualization of te anatomical domains of AT function. D. biflorus and Lotus lectins were used to differentiate te collecting duct system (D. biflorus lectin-positive) from te tubular structures (Lotus lectin-positive) to identify te functional localization of AT1 (in oat3 Ks) and AT3 (in oat1 Ks). Te results indicate tat altoug localization of bot ATs was specific to te tubular portions of te nepron, te patterns of AT1 function appeared distinct from tose of AT3 function. Te latter (AT3) was localized in bot proximal and distal tubules, wereas te former (AT1) was more proximal tubule-specific. o uptake of 6CF or 5CF was detected in te glomeruli or te collecting duct system, indicating tat, in contrast to wat was suggested by certain immunolocalization studies (17), functional localization is restricted to te tubular portions of te nepron. Altoug te generation of an oat1/oat3 double knock-out would be useful in furter evaluating te function of transporters, tis as so far been difficult because tese two genes are separated by only 8 kb on cromosome 19. It is well establised tat oat expression dramatically increases during late renal development and ten again after birt (13), and altoug oat1 and oat3 are expressed primarily in te kidney, tey are also expressed in oter barrier epitelial tissues. Because te perinatal period represents a critical environmental transition, te marked up-regulation of renal oat genes after birt seems consistent wit te need to adjust te omeostasis. Peraps tis reflects sifting transport and elimination, wic were andled prenatally by te placenta, to te kidney, postnatally. In addition, te immature organs of te premature infant are often exposed to drugs in intensive care units and oter settings. Because antiviral terapy in pregnancy (e.g. maternal HIV) is also a potential issue (29), we investigated te interaction of te aforementioned antivirals wit ATs in embryonic kidney organ cultures from oat1 and oat3 knock-outs. An interesting finding is tat te developing and adult kidneys may utilize different ATs for antiviral andling: te data suggest a predominant role for AT1 (as compared wit AT3) in interaction wit antivirals (wit te exception of stavudine) in te developing kidney, but tis difference may not be present in te adult kidney. 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10 Analysis of Tree-dimensional Systems for Developing and Mature Kidneys Clarifies te Role of AT1 and AT3 in Antiviral Handling Mega A. agle, David M. Truong, Ankur V. Dnyanmote, Sun-Young An, Satis A. Eraly, Wei Wu and Sanjay K. igam J. Biol. Cem. 2011, 286: doi: /jbc.M originally publised online ctober 4, 2010 Access te most updated version of tis article at doi: /jbc.M Alerts: Wen tis article is cited Wen a correction for tis article is posted Click ere to coose from all of JBC's alerts Tis article cites 29 references, 12 of wic can be accessed free at ttp://