Isolation and Immunolocalization of a Rat Renal Cortical Membrane ...

Vol. 269, No.9, Issue of March 4, p p , 6759-6764, 1994 Printed in U.S.A.

THEJOURNAL OF BIOLCGICAL CHEMISTRY Q 1994 by The American Society for Biochemistry and Molecular Biology, Ine

Isolation and Immunolocalization of a Rat Renal Cortical Membrane Urate Transporter" (Received for publication, September 23, 1993)

Barbara A. Knorr, Michael S . Lipkowitz, Barry J. Potter*, Sandra K. MasurO, and Ruth G. Abramson From the Renal Division, Department of Medicine and §Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029 and the @Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana 70112-1393

prepared from the renal cortex to physiologically characterize the transport mechanisms that are responsible for urate reabsorption and secretion. Two modalities of urate transport have been reported in rat renal proximal tubule brush-border and basolateral membranes. One, a n anion exchanger, transports urate inexchange for a variety of organic and inorganic anions (4, 5). The second, described by our laboratory, is a voltagea number of characteristics simisensitive urate uniporter with lar to those of the hepatic peroxisomal enzyme uricase (6, 7); both the transporter and uricase are Cu2+-dependent, have virtually the sameaffinity for urate, oxidize urate to allantoin, and are inhibited by oxonate, a specific inhibitor of the oxidative activityof uricase (8).Although uricase that resides in pig liver peroxisomes functions solely as an oxidative enzyme (9111, this protein was found to be capable of functioning as a saturable urate transporter when inserted in a lipid bilayer (12). Oxonate,which inhibited urate transport in the renal membranes, also inhibited transport inuricase-containing proteoliposomes (12). While these studies inproteoliposomes reinforced the hypothesis that the renal urate uniporter has some homology to hepatic uricase,to date uricase has notbeen identified in the ratkidney; in rat, uricase hasonly been localized to liver peroxisomes (9, 11, 13-17). As a first approach to purifying and characterizing theproteins responsible for urate transport in renal cortical plasma membranes, an affinity chromatography system was used to isolate the urate transporteds). Evidence has been obtained indicating that rat renal cortical plasma membranes contain urate-binding protein(s),which have some homology to hepatic peroxisomal uricase. Functional homology was revealed by uricase-like enzymatic activity of the purified renal membrane protein. Immunologic homology was demonstratedby immunoreactivity of the purified renal membrane protein to an antibody tohepaticuricase.Thisantibody also inhibited urate transport in intact membrane vesicles, suggesting that theimIt is now generally accepted that urate is bidirectionally munoreactive purified proteins represent components of the transported in the renal proximal tubule (reviewed in Ref. 1). uricase-like urate transporter. On the basis of immunocytouricase, this Since little,if any, net urateflux has been detectedat nephron chemical labeling with the antibody to hepatic putative urate transporter has been localized in the renalcorsites distal to the pars recta of the proximal tubule (2,3),it has been concluded that the vast majority of urate flux occurs tex to proximal tubules, the siteof urate transport. within the renal cortex (1).As a consequence it has been posMATERIALS AND METHODS sible toutilize brush border and basolateral membrane vesicles Isolation of Plasma Membranes-Renal cortical membrane vesicles modifications of methodspreviously de* This work was supported in part by a National Kidney Foundation werepreparedwithminor Fellowship with matching fundsfrom its affiliate, the National Kidney scribed (6,18). In brief, in each experiment 45-50 male rats (Charles Foundation of New YorWNew Jersey (to B. A. K.),by National Institutes River Breeding Laboratory, Wilmington, MA) were anesthetized with of Health Grants DK08419 (to B. A. K.),EY09244 (to S. K. M.), and an intraperitoneal injection of pentobarbital (45 mgkg body weight). DK37315 (to R. G. A,), and by a grant-in-aid from the American Heart The kidneys were harvested, placed in ice-cold 250 m~ sucrose, 10 m~ Tris buffered to pH 7.4 with HCl (STbuffer), decapsulated, andslices of Association, New York City Affiliate (to R. G. A,). The costs of publicaslices were weighed (79.2 f 3.7 tion of this article were defrayedin part by the payment of page renal cortices were obtained. The cortical charges. Thisarticle must therefore be hereby marked "advertisement" g, wet weight), placed in fresh ST buffer (2 d g tissue) containing 0.2 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. m~ phenylmethylsulfonyl fluoride, finely minced with scissors,and ho-

Two modalities of urate transporthave beenreported in rat kidney, a uratdanion exchanger and a potential sensitive, uricase-like uniporter. As an initial attempt to isolate and charac4erize the responsible transport protein(&, rat renal cortical membranes were harvested, solubilized, and subjected to affinity chromatography with urate or xanthine as the affinity ligand. Pig liver peroxisomal uricase was purified with the same system, and the enzymatically active protein was used to generate polyclonal antibodies in rabbit. Silver stain of SDSpolyacrylamide gel electrophoresis gels of the eluted fraction containing the affinity-purified renal membrane protei&) demonstrated bands at 25,32,36,and 41 m a . On Western blot, two of these bands (32and 36 kDa) were immunoreactive tothe polyclonal antibody to pig liver uricase. In 6 of 10 studies, the affinity-purified renal membrane protein(s) also oxidized urate. Anti-pig liver uricase produced a selective and dose-dependent inhibition of the uricase-like urate uniporter in renal membrane vesicles, but did not affect the uratelanion exchanger or the sodium-dependent glucose transporter. Immunocytochemical studies of rat renal cortex with the same antibody indicated that theimmunoreactivity was localized to proximal tubules. These studies demonstrate that the renal cortical plasma membranes contain urate-binding proteins, which have some functional and immunological homologyto the hepatic peroxisomal core protein, uricase. Within the renal cortex, these proteins are localized to proximal tubules, the site of urate transport. Since the antibody that reacts with the affinity-purified urate-binding proteins on Western blot selectively inhibits urate transport in intactmembrane vesicles, it is concluded that at least one of the affinity-purifiedurate-binding proteins is a uricase-like urate transporter.

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Isolation Immunolocalization and

Urate Dansporter of a

mogenized. The homogenate was then subjected to differential centrifuphoresis revealed a single band with the purified protein. EnzymegationusingaSorvall model RC-5B refrigeratedcentrifugewith a linkedimmunosorbentassayrevealedantibodytiters of atleast SS-34 rotor(6,19). The final membrane pellet was suspended 280 mM in 1/12,000 t o the monomeric formof porcine uricase. TheIgG fraction of sucrose, 8.5 mM Tris acetate buffered to pH 7.4 with NaOH involume a the polyclonal antibody to porcine uricase was purified usinga protein estimated to yield a protein concentrationof 8-10 mg/ml. Brush border A antibody purification kit (RepliGen, Cambridge, MA). The IgG fracand basolateral membranes were subsequently separated and isolated tion was then concentrated with a Centricon 30 concentrator (Amicon, by free flow electrophoresis (model FF5, Garching Instruments, Bender Beverly, M A ) to 10 mg/ml. Additionally, aliquots of preimmune and and Hobein, Munich, Germany) using methods identical t o those preimmune sera were purified against the 33-kDa monomer of porcine viously described (6, 19). The electrophoretic fractions containing brush uricase as previously described (25,261. Briefly, reduced porcine uricase border and basolateral membranes were identifiedon the basis of the was electrophoresedon a preparative 10% SDS-PAGE gel. The gel was marker enzymes alkaline phosphatase (determined with a Sigma test stained with Rapid Reversible Stain (Diversified Biotech, Newton Cenkit) and potassium-activated para-nitrophenyl phosphatase (20), re- tre, MA). The 33-kDa monomer was cut of outthe gel and subsequently spectively. Thereafter, the purified brush border and basolateral mem- electroeluted fromthe gel slice with the LittleBlue Tank (Isco, Lincoln, brane fractions were recombined, diluted ain n equal volume of 100 mM NE). The electroeluted monomer was electrophoresed onsecond a 10% mannitol, 20 UM CuCI,, 10 mM HEPES buffered with Tris to pH 7.4, SDS-PAGE gel and transferred to nitrocellulose filters using the Mulhomogenized, and centrifuged at 32,000 x g for 30 min. The resulting tiphor I1 Nova Blot semidry transfer unit (Pharmacia). Undiluted prepellet was solubilized in a volume of 0.1 M borate, 0.5% CHAPS,' 10% immune or immune serum was incubated with the transferred antigen glycerol a t pH 9.0 estimated to yield a detergent:protein ratio of ap- and subsequently eluted with 1 M potassiumthiocyanate(26).The proximately 1:2 w/w. Following 30 min on ice the solubilized mem- eluted antibody was desalted on a Sephadex G25 column equilibrated branes were centrifugedat 32,000 x g for 30 min. The supernatant was with phosphate-buffered saline (PBS) and stored with 2% albumin. aspirated and subsequently subjected to affinity chromatography. Western Blot Analysis-Samples were suspended in Laemmli sample Pilot studies demonstrated that identical results were obtainedbuffer when(0.125M Tris-HC1, 4% SDS, 20% glycerol, 10% mercaptoethanol, either solubilized, electrophoretically purified membrane fractions or pH 6.81, resolved on 10% SDS-polyacrylamidegels (27) using a Mighty less pure, combined cortical membrane suspensions (pre-electrophore- Small I1 gel electrophoresis unit (Hoefer, San Francisco, CA) and elecsis) were applied to the affinity gel. Thus, in subsequent studies the trophoreticallytransferredtonitrocellulosefilters.Theblotswere membrane suspensions were not subjected to free-flow electrophoresis. blocked with 2.0% milk in PBS, pH 7.4, for 1 h a t room temperature, In these studies CuCl, was added to the membrane suspension t o yield incubated overnight at 4 "C in either a1/250 dilution of serum, a 11500 approximately 70 nm Cu2+/mg protein. After30 min on ice, the mem- dilution of the IgG fraction of anti-porcine uricase, or undiluted antigenbranes were centrifugedat 32,000 x g for 30 min, the supernatant was purified anti-uricase antibody in the milkPBS solution. The blots were discarded, and the pellet was solubilized and subsequently handled in washed at room temperature with milWBS followed by PBS, then a manner identical to that detailed above. In all studies the protein incubated a t room temperature with a 1/250 dilution of peroxidase concentrations of themembranesuspensionsand solubilized mem- labeled goat anti-rabbit IgG in the PBS solution. After washing with branes were determined by the method of Lowry et al. (21) using bovine PBS, the blots were developed with the 4 CN Peroxidase substrate serum albumin as the standard. system kit (Kirkegaard & Perry, Gaithersburg, MD). In some studies, Afinity Chromatography-An affinity gel, in which the ligand urate solubilized membrane samples were examined with and without deglywas linked t o epoxy-activated Sepharose 6B (PharmaciaLKB Biotech- cosylationusinga De-N-Glycosylation kit (Oxford GlycoSystems, nology Inc.), was preparedby a modification of the method of BatistaRosedale, N Y ) prior to Western blot analysis. Viera et al. (22). The affinity gel was hydrated, suspended in 50 mM Urate Oxidation-Since hypoxanthine is a uricase inhibitor, hypourate in1% KOH, pH 12.0 (5 ofg geVlO ml of potassium urate), rotated xanthine was removed from the eluate fractions by desalting with dein the darkat 37 "C for 24 h to link urate to the spacer arms and thentergent andglycerol-free 0.1 M borate buffer on Sephadex G25 columns rinsed sequentially with 1 liter of 0.1 M borate, pH 8.0, distilled water, (Pharmacia). Solubilized membranes were similarly treated. [2-l4C10.1 M acetate, pH4.0, and distilled water.To block free spacer arms, the Urate (49.1mCi/mM, Amersham Carp.) was added to a final concentragel was then suspended in 100 ml of 2.0 M glycine, rotated in the dark tion of 2 p~ to aliquotsof the solubilized membranes and hypoxanthinea t room temperature for 15h, and suspended in 0.1 M borate pH 9.0 (23). free eluate fractions and incubated for 60 min at room temperature. The The gel was stored at 4 "C until use. In additional studies xanthinefraction of [2-l4C1uratethat wasoxidized t o [2-14Clallantoin was deterSepharose (Sigma) was substitutedfor the urate-Sepharose. All other mined by separating urate and allantoin with a previously described aspects of the affinity chromatography were identical t o those detailed column chromatographic technique (28). below. 'DunsportStudies-Membrane vesicles wereisolated and treated Water-jacketed columns that were maintained at 4 "C were packed with CuCl, as detailed above. Paired vesicles were preincubated for 30 with gel and rinsed with 0.1 M borate, pH 9.0. Solubilized membranes min with non-immune IgG or the IgG fraction of anti-porcine uricase (60-117 mg), which were also maintainedat 4 "C, were then applied to (0.01-1 pg of IgG/pg of vesicle protein). Thereafter, the pairedvesicles the gel. Unbound proteins were washed out of the gel with the same were incubatedfor 5 s in medium containing PM 50 [2-l4C1urate, 100 mM borate buffer, which contained CHAPS and glycerol at 1/10,000 of the mannitol, 100 mM NaC1, 10 mM Tris-Hepes, at pH 7.5. In a subset of concentrations used tosolubilize the membranes. Subsequently, bound experiments the uptake of ~-[6-~Hlglucose (6.4 pCiinmo1; DuPont NEN) protein was specifically eluted with 10 mM hypoxanthine (a uricase was examined; paired vesicles that were preincubated with non-iminhibitor) in the same buffer at 22 "C; 5-ml eluate fractions were col- mune or immune IgG were then incubated 10 and 30 s in media conlected on ice. taining 50 p~ D-glucose, 100 mM mannitol, 100 mM NaC1, 10 mM TrisProtein Electrophoresis-Aliquots of protein samples that were apHepes a t pH 7.5. Uptake was terminated by the addition of 4 ml of plied t o and eluted from the affinity gel were subjected t o sodium do- ice-cold stop solution (154mM NaCl, 10m~ Tris-Hepes, pH 7.5) followed decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8-25% by filtration through 0.65-pm Millipore filters. The tubes and filters gradient gels usingthePhastSystem(Pharmacia)(24). All protein were subsequently rinsed with a n additional 12 ml of the same solution. samples were reduced with P-mercaptoethanol. Gels were subsequently In additional studies, brush-border membranevesicles were prepared silver stained using a Pharmacia kit. The molecular weights of the by magnesium aggregation (4). Urate uptake was examined under conproteins detected on SDS-PAGE were estimated usingan UltrascanXL ditions identical t o those previously described (4); pairedvesicles were laser densitometer (PharmaciaLKB). prepared in 201 mM mannitol, 49 mMK,' 80 mM HEPES, 10m~ MgSO,, Rabbit Anti-pigLiver Uricase-Commercial pig liver uricase (Sigma) pH 7.5, and then preincubated 30 min with non-immune or I& immune was affinity-purified with the urate affinity gel described above. The (1pg of IgG/pg of vesicle protein) that was diluted in the same buffer. specifically eluted protein,which was shown to have enzymatic activity, Paired vesicles were subsequently incubated in 155 mM mannitol, 116 was used t o generate polyclonal antibodies in rabbits (Pocono Rabbit mM K+, 59 mM PO,, 16 m~ HEPES, and 2mM MgSO, a t pH 7.5 or 204 Farm & Laboratory, Inc., Canadensis, PA). Assays for antibody speci- mM mannitol, 68 mM K', 58 mM PO,, 16 mM HEPES, and 2mM MgSO, ficity indicated that the antibody wasmonospecific to the antigen and a t pH 6.0 (4). Uptake was stopped as detailed above, however the stop that the purifiedantigenwas a singleprotein;Ouchterlonydouble solution contained 160 mM MgSO,, 1mM Tris-HEPES, and250 p~ DIDS diffusion analysis revealed a single precipitin line, and immunoelectro- at pH 7.5. All filters were dried and dissolved in 10 ml of scintillation fluid. The radioactivity of each filter was measured in a Packard TriThe abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimeth- Carbspectrometer(model 4430; PackardInstrument Co., Downers ylammoniol-1-propanesulfonic acid; PAGE, polyacrylamide gel electro- Grove, IL). Uptake was calculated from the disintegrations per minute that accumulated on the filters, thespecific activity of the substrate in phoresis; PBS, phosphate-buffered saline; DIDS, 4,4'-diisothiocyanothe medium, and the protein concentrationof the vesicles. stilbene-2,2'-disulfonicacid.

Isolation and ImmunolocalizationUrate Dansporter of a

6761

4 I'

94.0

L

14.4

L 1

2

3

4

FIG.1. Silver stain of a %26% SDSPAGE gel. Lane 1, molecular weight markers; lane2, 1.5 pg of solubilized membranes; lane3, 1.0 pg of nonspecifically eluted protein; lune 4, purified protein specifically eluted from the affinity gel.

1 2 FIG.2. Western blot wing rabbit anti-pig liver uricase as the first antibody and a peroxidase-labeled goat anti-rabbit IgG as the second antibody.Lane 1, 500 pg of solubilized renal membranes; lune 2, less than 0.3 pg of purified protein specifically eluted from the affinity gel.

ent size bands do not reflect different degreesof glycosylation of the sameprotein(s). Two of the immunoreactive bands (32 and 36 kDa) that were present in solubilized membranes were deImmunocytochemistry-Rat kidneys were perfused in situ with nor- tected in the purified protein fraction (Fig. 2); the 27-kDa immal saline followed by 4% paraformaldehyde. Slices(2-3 mm thick) of munoreactive band that was seen in solubilized membranes kidney were then dehydrated, cleared, and embedded in paraffin. Secthe tions (3 pm) were cut and placedonpolylysine-coatedslides. f i r was not detected in the purified protein fraction. Since deparaffinization, immunoperoxidase staining was performed usinga smallest of the immunoreactive membrane proteins was not detected in the purified protein fraction, it may have been Super Sensitive Multilink Immunodetection System (BioGenex, San Ramon, CA) in which the antibody is localized via a biotin-streptavidin degraded or denatured after solubilization, obviating purificahorseradish peroxidase complex using diaminobenzidine as substrate. tion via the affinity gels employed. Thereafter the tissue was counterstained with Hams hematoxylin. The Functional Assessment of Urate-binding Proteins-The uriprimary antibody was a l/6400 dilution of the IgG fraction of anticase-like activity of the purified proteins and solubilized memporcine uricase or undiluted antigen-purified antibody. A 116400 diluby measuring theability of these proteins tion of non-immune IgG or undiluted antigen-purified preimmune se- branes was assessed fraction containing rum served as the controls.The tissue was examinedwith a Zeiss to oxidize urate. In6 of 10 experiments, the Axiomat using light and differential interferencecontrast microscopy. the purifiedprotein oxidized 8.0 2 2.6%of 2 [2-14C]urate; activity was not detected in the remaining 4 studies. In the RESULTS same six experiments in which the purified proteins oxidized Purification of Urate-binding Proteins-The extent of purifi- urate, the solubilized membranes oxidized a similar percent cation of renal urate-binding proteins that wasachieved with (9.25 2 2.9%). It is of note that the assays with solubilized the above described affinity chromatographysystem wasevalu- membranes contained 0.74 0.18 mg of protein, yielding a ated by SDS-PAGE (Fig. 1).As anticipated, the numberof pro- specific activity of 161 * 64 pmolof urate oxidizedlmg of tein bands in both the sample of solubilized membranes that proteinh, whereas assays with thepurified protein contained was applied to the affinity gel and in the fraction containing an amountof protein that was below the lower limit (1.0 pg) of unbound protein were too numerous to count. In contrast, sil- detection with the Pierce Micro BSA protein assay. Although ver stain of the protein fraction that was specifically eluted the specific activity could not be determined, thefinding that a with hypoxanthine revealed two to four protein bands. In vir- comparable amount of urate was oxidized by such disparate tually all studies bands were seen at 25 and 36 kDa; less fre- amounts of purified protein and solubilized membrane protein quently bands were detected at 32 and 41kDa. Protein bands indicates that the specific activity of the purified urate-binding of comparable size were obtained when xanthine was substi- protein was markedly enriched (at least 740-fold) relative to tuted for urate as the affinity ligand. SDS-PAGE of hepatic that in the membrane from which it derived. Since uricase uricase that waspurified with the same affinity systemyielded functions as an enzyme that oxidizes urate, thefinding that the a protein band that approximated 33 kDa. Despite the differ- purified renal urate-binding protein(s) also oxidized urate inence in size in thepurified renal proteins and hepaticuricase, dicates that thisprotein has some functional homology to hethese proteins bound to the same ligands and eluted with the patic peroxisomal uricase. same substrate.Additional studies were therefore performed to Since a very limited amount of purified urate-binding protein assesswhethertheseproteins displayed any immunologic was recovered, it wasnot possible to assess transportfunction andlor functional homology. by the technique of reconstituting thepurified proteins in lipoImmunoreactivity of Urate-binding Proteins-Western blot somes. The strategy that wasused to assess the role of these analysis with rabbitanti-porcine hepatic uricaserevealed that proteins in urate transport wasbased on the assumption that the solubilized renal membranes and theaffinity-purified pro- transport might be inhibited by a n interaction between antitein were highly reactive to the antibody (Fig. 2), but non- uricase and proteins in the intact plasma membraneif one or non-immune rabbit IgG (not more of the immunoreactive membrane protein(s) (Fig. 2) is reactive to preimmune serum and depicted). Identical immunoreactive bands were detected inde- involved in urate transport.However, two modalities of urate pendent of whether immune serum, the purified IgG fraction of transport have been detected in rat renal brush border memimmune serum, or a n antigen-purified antibody was used. Al- branes; the uricase-like uniporter has been observed in memthough innumerable protein bands were evident on thesilver branes exposed to trace amountsof Cu2+(6,7), while the anion stain of solubilized renal membranes(Fig. l),only three bands exchanger has been evident in membranes preparedwith Mg2+ at 27, 32, and 36kDa were immunoreactive (the 36-kDa band (4, 5). may represent a doublet) (Fig. 2). The sizes of these immunoTo assess the effect of anti-uricase on each mode of urate reactive bands were indistinguishable before and after treat- transport, vesicles were prepared and urate uptake was examment with peptide-N-glycosidase:'F,suggesting that thediffer- ined under conditions in which either the uniporter or ex-


6762

porter is functional (Fig.3). Finally, the immunoreactive uratebinding protein(s), the putative urateuniporter, has been img 80 munolocalized within the renal cortex to proximal convoluted and straight tubules(Fig. 4, B-F). 60 Non-immune IgG In addition to the two immunoreactive proteins that were P Immune IgG purified from the renal membranes, two other proteins (25 and 40 41 kDa)were purified that were non-reactiveto anti-uricase.As 9 20 all of the assayswere performed under reduced conditions, it is E possible that the four protein bands represent subunits of a 3 0 0.01 0.1 0.5 1.0 single urate-binding protein in which some subunits contain, Kg IgG/pg protein but others lack, epitopes recognized by anti-uricase. AlternaFIG.3. Effect of varying amounts of rabbit non-immune IgG tively, as two distinct modalities of urate transport have been and rabbit anti-pig liver uricase IgG on 6-surate uptake.Asterisk described in rat kidney, a n anion exchanger (4, 5) anda potenindicates uptake wassignificantly inhibited by immune IgG relative to tial sensitiveuricase-like uniporter ( 6 , 7), the non-reactive and uptake in thepresence of non-immune I g G ( p < 0.025; n = 3). immunoreactive proteins may represent different urate-binding proteins witheach responsiblefor one mode of urate transchanger would beoperative. As demonstrated inFig. 3, in three port. Based on the immunologic and functional homology bepaired studies in Cu2+-exposed vesicles the purified IgG frac- tween the purified renal proteins and porcine liver uricase, as tion of rabbit anti-porcine uricase produced a dose-dependent well as the fact that anti-uricase inhibited urate uptake in inhibition of urate uptake in renal cortical membrane vesicles. intact membranes under conditions in which only the urate In contrast, in these samevesicles Na+-dependentglucose up- uniporter is detected (Fig. 31, it is concluded that the purified take (at 10 and 30 s) was unaffected by immune IgG (not immunoreactive proteins are components of the uricase-like depicted). Non-immune rabbit IgG did not alter urate (Fig. 3) or transporter. The failure for anti-uricase to inhibit transport glucose uptake. In vesicles prepared with Mg2+,urate uptake under conditions in which only the urate/anion exchanger is was 5-fold greater at 10 and 30 s in thepresence of a n outward observed implies that uratelanionexchange occurs on a protein OH- gradient (pHin 7.5 > pH,,, 6.0) than in its absence (pHin that is different from the uricase-like urate uniporter. If such is 7.5 = pHout 7.5); anti-uricase did not inhibit basal urate uptake the case, then the purified urate-binding proteins that do not or the enhanced uptake that occurred on the urate/anion ex- react to anti-uricase are likely to be components of the uratel changer. These findingsindicate that the effect of anti-pig liver anion exchanger. However, the present studies do not totally uricase on urate transport is quite specific and suggest that at exclude the possibility that thetwo modes of transport occur on least one or more of the affinity-purified immunoreactiveurate- a single protein whose structural configuration is altered when binding proteinsis a component of the uricase-like urate experimental conditions are varied; a change in theconfigurauniporter, but not the uratelanion exchanger. tion of a single urate transport protein that interferes with Immunocytochemistry-Immunoperoxidase staining was ab- binding of the antibody could obviate an effect of the antibody sent from cortical sections incubated with non-immune IgG on urate transport via the exchanger. A definitive conclusion (Fig. 4A). In the presence of either the IgG fraction of anti- regarding this issue must awaitcomparison of the protein seuricase or antigen-purified antibody, immunoperoxidase label- quences of the exchanger and uniporter. ing was confined to proximal tubules within the renal cortex It is of interest thatprior studies failed to detect immunore(Fig. 4, B-F). Blood vessels, glomeruli,and cortical segments of activity to anantibody to uricase in the rat kidney (13,15). The the distal nephronwerenon-reactive to anti-uricase. Three marked immunoreactivity that was demonstrated in the curpatterns of immunolabeling of proximal tubules were observed. rent studies may, in part, be consequent to the fortuitous genimmu- eration of a high titer, high affinity antibody. It may also be In the very early portions of the proximal tubule (SI), nolabeling appeared tobe localized to the brush borders (Fig.4, consequent to the fact that the protein that was used togenB and C ) . In somewhat more distal segments of the proximal erate antibody was not denaturedas demonstrated by its abiltubule, immunoreactivity was primarily observed in subapical ity to oxidize urate. However, the relevance of using an intact regions (Fig. 4, B and D ) . Within proximal tubule segments protein is uncertainsince the biological activity of the antigens near the corticomedullary junction (S3), the cytoplasm ap- used to generateantibodies in previous studies (13,15)was not peared to be diffusely stained, but the brushborders were un- commentedupon. Perhaps more significantly, polyclonal or stained (Fig. 4, E and F ) . monoclonal antibodies were raised to rat uricase in prior studies (13, 15)whereas the present studiesemployed a polyclonal DISCUSSION antibody that was raised to porcine uricase. Porcine uricase The present studies have demonstrated that urate-binding was selected for two reasons. First, studies inproteoliposomes proteins that were purified from brush border and basolateral demonstrated that porcine uricase, like the renal membrane membrane vesicles of rat renal cortex display some homology to protein, is capable of transporting urate when inserted into a been the hepatic peroxisomal protein uricase. Immunologic homol- lipid bilayer (12).Second, when antibodies to uricase have ogy was revealed by the immunoreactivity of two of the purified raised in rabbit, porcine uricase has been shown to be more antigenic thandog, cow, horse, house musk shrew, and guinea proteins with anti-porcine uricase on Western blot (Fig. 2). Functional homology was evidenced by uricase-like enzymatic pig uricase (29). Thus, in choosing the source of antigen the activity. In additionto demonstrating shared properties of the possibility was considered that porcine protein may also be more antigenic than the rat preparation. Regardless of the purified renal proteins and hepatic uricase, the present studies also suggest that one or more of the purified renal cortical reason(s) for the difference in immunoreactivity of the antibody membrane urate-binding proteins is a component of a urate used in this and prior studies, the finding that anti-porcine transporter. This conclusion is based on the finding that anti- uricase produced a profound inhibition of urate uptake in rat porcine uricase not only reacts with immobilized purified pro- renal cortical membrane vesicles (Fig. 3) provides strong suptein on Western blot (Fig. 2), it also reacts with intact mem- port for the use of this antibody as a marker of a urate transbrane proteins toact as a potent and specific inhibitor of urate port protein. Since the antibody that was employed was a selective and transport under conditions in which the uricase-like trans.- 100

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Isolation and Immunolocalization of a Urate Dansporter

6763

FIG. 4. Inmunoperoxidme labeling of rat renal cortex examined with differential interference microscopy using non-immune IgG (A) and rabbit anti-pig liver uricase IgG (R-F).A, the glomerulus and all cortical nephron segmentsarenon-reactive to non-immune IgG. E , brush-border (arrow)and subapical regions (arrowhad) of outer cortical proximal tubulesareimmunolabeled in the presence of immune IgG. Glomerulua (g)and distal tubule (*) are nonreactive. Noteimmunostaining of brush-border (arrow)in initial segment of proximal tubule (S,)at its origin in right lower comer of glomerulus. C, higher magnificationof proximal tubulethat has immunostaining in the brush-bordermembrane (arrow). D , higher magnification of proximal tubule that has immunostaining in subapical region of cell (arrowhead),but not in brush-border membrane (arrow). E, proximal tubules in inner cortex that have diffuse cytosolic immunostaining (arrowhead).F, higher magnification of innercorticalproximaltubule that has diffuse cellular immunostaining (arrowh a d ) and clear brush-border membrane (arrow). Magnification is the same in B and E , magnification is the same in C, D , and F.

potent inhibitorof urate transport, immunocytochemistry was used to localize the nephron sites of the urate transporter within the renalcortex. In previous studies in rat, it has been suggested that the majority of urate transport occurs within the proximal tubule (1-3). The finding that immunoperoxidase labeling was confined to proximal tubules (Fig. 4, B-F) is thus consistent with the physiologic data. At the cellular level, the demonstration of immunolabeling of the brush border (Fig. 4, B and C) is in accord with the fact that urate is transported across this membrane. Since this luminal membrane transporter must be delivered to the brush border from intracellular organelles, the subapical staining pattern(Fig. 4, B and D ) may represent protein in vesicles involvedin membrane trafficking or protein free within the cytoplasm. However, the precise intracellular localization that is represented by subapical labeling cannot be determined at the light microscopic level. Similarly, light microscopy is inadequate to determine the intracellular constituents that arelabeled in thediffusely immunostained cells in S3segments of the proximal tubule (Fig. 4, E and F).Since urate is also transported across the basolat-

era1 membrane, it would not be surprising if some of the diffuse staining was localized within this membrane, but the present study does not reveal enhanced staining of the basolateral membrane relative to the remainder of the cell. The failure to detect specific labeling of the basolateral membrane may indicate that reactive antigenic sites in thebasolateral membrane to the uricase-like urate transporter are masked or urate is transported across this membrane by a different protein. Resolution of these issueswill require further analysis withimmunoelectron microscopy. Although thecurrent studies indicate thattheurate uniporter has homology to hepatic peroxisomal uricase, it seems clear that the transporter and uricase are not identical proteins. First, pig hepatic uricase and the immunoreactive renal membrane urate-binding proteins have slightly different molecular weights. Second, the uricase-like enzyme activity of the purified membrane proteins appears to be quite weak relative to that of hepatic uricase. Finally, as Northern blot analysis of rat kidney RNA probed with the cDNA for rat hepatic it can be concluded uricase failed to detect transcripts (15,301,

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that themRNA for uricase, per se, is absent or present in very low copy number. However, some epitopes must be conserved between hepatic uricase and the renal membrane transport protein; anti-porcine uricase not only recognizesurate-binding proteins purified from renal membranes, but specificallyinhibits the urate uniporter without affecting other transporters(ie. Na+-dependent glucose transporter and the uratelanion exchanger). A determination of the actual extent of homology between these proteins must await cloning of the renal urate uniporter and subsequent comparison of the nucleotidesequences of the renal and hepatic proteins. The availability of a high titer polyclonal antibody to hepatic uricase may facilitate the cloning of the renal transporter. Acknowledgments-We express our appreciation to Drs. Neil Theis and Steven Dikman for assistance and advice regarding the immunocytochemical studies. REFERENCES 1. Abramson, R. G., and Lipkowitz, M. S. (1990) Evolution of the Uric Acid lhansport Mechanisms in Vertebrate Kidney, Comparative Physiology, pp. 115-153, Karger, Basel, Switzerland 2. Gregor, R., Lang, F., and Deetjen, P. (1974) ptlugers Arch. 362, 115-120 3. Kramp, R. A,, and Lenoir, R. (1975)Am. J. Physiol. 288,875-883 4. Kahn, A. M., Branham, S., and Weinman, E. J. (1983) Am. J. Physiol. 246, Flfil-F15R -- - - -- -

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