JP5574357B2 - Renal urate transporter - Google Patents

Description

The invention kidney urate transporter, in particular human GLUT9, i.e. potential driven kidney urate transporter directly controls the blood uric acid levels, and to a novel gene responsible for hypouricemia, this is a promising treatment for gout Proposing to be a target.

Urate (uric acid) is the end product of purine metabolism because of the deficiency of liver uricase in the human body, but it is currently evaluated as an endogenous antioxidant with neuroprotective properties. Low blood uric acid levels, multiple sclerosis is believed in conjunction with Parkinson's disease and Alzheimer's disease (1). Despite its beneficial role, increase in blood uric acid value, gout, hypertension, cardiovascular disease (e.g. myocardial infarction and stroke), and kidney disease (such as acute uric acid nephropathy and nephrolithiasis (1,2) . correlated with kidney, Oite the maintenance of blood uric acid levels through the discharge process, the dominant role:. it it removes approximately 70% of the uric acid production (3) of the day Therefore, large It is important to understand the mechanism of uric acid processing in the kidney, since many hyperuricemia patients show reduced uric acid excretion (4).

Since uric acid is a weak acid at physiological pH (pKa, 5.75), it hardly penetrates the cell membrane in the absence of transport protein (4). Despite the suggestion that the uric acid transporter is a key molecule, the exact mechanism of renal uric acid transport was unclear due to its species differences and its diversity: bidirectional transport of nephron uric acid , “ 4-component model” (5). In 2002, we identified a long- presumed urate anion exchanger, namely URAT1, classified as SLC22A12, located on the proximal renal tubular lumen (6) . To date, URAT1 is the loss of function due to the genetic mutation, physiological role in renal uric acid reabsorption based on the fact that renal hypouricemia (6) occurs has been demonstrated, the only transporter is there. However, it is not known how uric acid that enters tubule cells via URAT1 exits the cells . Furthermore, the existence of a uric acid reabsorption system different from URAT1 is suggested by Ichida and Wakida. They reported patients with renal hypouricemia who had no mutations in SLC22A12 (7, 8) .
Similarly, a recent study using Slc22a12 deficient mice, the majority of reabsorption renal uric acid was found to be maintained (9), physiological of this finding for the uric acid treatment in human kidney The association remains unknown . Following the identification of URAT1, several promising candidate proteins responsible for uric acid transport were proposed: UAT, OAT1, OAT3, OAT4, OATv1 / NPT1, MRP4 , and recently identified OAT10 also mediate uric acid transport in vitro (10-12) .

1. M. K. Kutzing, B. L. Firestein, J. Pharmacol. Exp. Ther, 324, 1-7 (2008) 2． M. A. Becker, M. Jolly, Rheum. Dis. Clin. N. Am. 32, 275-293 (2006) 3． D. A. Sica, A. C. Schoolwerth, in The Kidney (WB Saunders, Philadelphia, ed.), Pp. 680-700 (2000) 4). N. Anzai, H. Endou, Expert Opin. Drug Discov. Vol. 2, 1251-1261 (2007) 5. D. B. Mount, Curr. Opin. Nephrol. Hypertens. Vol. 14, 460-463 (2005) 6). A. Enomoto, H. Kimura, A. Chairoungdua, et al. Nature 417, 447-452 (2002) 7). K. Ichida, M. Hosoyamada, I. Hisatome, et al. J. Am. Soc. Nephrol. 15: 164-173 (2004). 8). N. Wakida, D. G. Tuyen, M. Adachi, et al. J. Clin. Endocrinol. Metab. 90, 2169-2174 (2005). 9. S. A. Eraly, V. Vallon, T. Rieg, et al. Physiol. Genomics, 33, 180-192 (2008). Ten. M. A. Hediger, R. J. Johnson, H. Miyazaki, H. Endou, Physiology Vol. 20, pp. 125-133 (2004). 11. M. A. Rafey, M. S. Lipkowitz, E. Leal-Pinto, R. G. Abramson, Curr. Opin. Nephrol. Hypertens. Vol. 12, 511-516 (2003) 12． A. Bahn, Y. Hagos, S. Reuter, et al. J. Biol. Chem. In press 13. K. Mori, Y. Ogawa, K. Ebihara, et al. FEBS Lett. 417, 371-374 (1997). 14. S. H. Cha, T. Sekine, H. Kusuhara, et al. J. Biol. Chem. 275, 4507-4512 (2000). 15． M. Uldry, B. Thorens, Pflugers Arch.No. 447, No. 480-489 (2004). 16． J. E. Phay, H. B. Hussain, J. F. Moley, Genomics 66, 217-220 (2000). 17． H. Doege, A. Bocianski, H.-G. Joost, A. Schurmann, Biochem. J. 350, 771-776 (2000). 18． R. Augustin, M. O. Carayannopoulos, L. O. Dowd, etal. J. Biol. Chem. 279, 16229-16236 (2004). 19. B. T. Emmerson, N. Engl. J. Med., 334, 445-451 (1996). 20． O. Sperling, Mol. Genet. Metab. 89, 14-18 (2006) twenty one. T.H.Steel, Ann.Intern.Med. 79, 734-737 (1973) twenty two. Y. Kikuchi, H. Koga, Y. Yasutomo, et al. Clin. Nephrol. 53, 467-472 (2000) twenty three. H.-G. Joost, B. Thorens, Mol. Membr. Biol. 18, 247-256 (2001) twenty four. D. M. Papazian, L. C. Timpe, Y. N. Jan, L. Y. Jan, Nature 349, 305-310 (1991) twenty five. S. Li, S. Sanna, A. Maschio, et al. PLoS Genet. Volume 3, 194 (2007) 26. A. Doring, C. Gieger, D. Mehta, et al. Nat Genet. 40, 430-436 (2008) 27. V. Vitart, I. Rudan, C. Hayward, et al. Nat Genet. 40, 437-442 (2008)

Here we report a previously unknown urate transporter localized to the basal side of the renal proximal tubule. It will exert its physiological role in the human body with URAT1 and tandem for uric acid reabsorption .

To identify novel renal urate transporters, we are known to mediate uric acid transport against NCBI's Swissprot protein database, human and mouse URAT1 / Urat1 (SLC22A12 / Slc22a12) and human A homology search (blastp) was performed using the OAT4 (SLC22A11) sequence (6, 13, 14). Surprisingly, we have found that some of the facilitating glucose transporter (SLC2) family (GLUT6, 9, 10, 12, and 14) have a remote similarity to SLC22A11. Among these molecules , we have one of the members of the expanded (Class II) SLC2 family, GLUT9 encoded by SLC2A9 is mainly localized to the kidney as well as the liver, so that Decided to do the analysis (15). Human GLUT9 was initially identified as a gene of unknown function (16). Despite the demonstrated glucose transporter activity of GLUT9, it does not appear to be as efficient as the classical (class I) glucose transporter GLUT4 (17, 18). Human GLUT9 has two splice variants with different amino termini: Isoform 1 (Transcript Variant 1: Genbank A click session NM_020041) and, isoform 2 (Transcript Variant 2: Genbank A click session NM_001001290). The difference between these two isoforms is the first exon of isoform 1 alone, which is the targeting difference in polarized MDCK cells.

First, we examined the plasma membrane expression and urate transport activity of both isoforms of GLUT9 using the Xenopus egg cell expression system. Cell membrane expression of both isoforms of GLUT9 is the oocytes injected with GLUT9 complementary RNA (cRNAs), was confirmed by immunofluorescence analysis (Fig. 4A). After confirming that both its isoforms showed similar urate transport activity (FIG. 4B), we used isoform 2 for further characterization . The [ ¹⁴ C] uric acid uptake rate of GLUT9-expressing oocytes was 9 times higher than that of control oocytes. On the other hand, very low uptake rates of [ ¹⁴ C] glucose and [ ¹⁴ C] fructose were observed. GLUT9 is a typical organic anionic substrate such as PAH (paraaminohippuric acid ), estrone sulfate, salicylic acid, and classic lactic acid, nicotinic acid, β-hydroxybutyric acid, salicylic acid (FIG. 4C). There was no significant uptake of interactors with the renal urate transport system (including URAT1). Thus, GLUT9 had a narrower substrate specificity than URAT1.

Next, we investigated the uric acid transport properties of GLUT9. Xenopus oocytes injected with GLUT9 cRNA showed time-dependent [ ¹⁴ C] uric acid transport activity (FIG. 1A) . The uric acid uptake mediated by GLUT9 showed saturation kinetics and followed the Michaelis-Menten equation.

Edie - Hof stays plot against uric acid, next (mean ± SE of five independent experiments) Km 365 ± 42μM and _{V max 5,521 ± 291pmol / hr /} oocyte, GLUT9 is higher than the uric acid in the same manner as URAT1 It was suggested to have affinity (FIG. 1B). The fact that removal of extracellular Na ⁺ did not affect uric acid transport by GLUT9 indicated that it was not involved in direct Na ⁺ -uric acid cotransport.

GLUT9 activity was affected by membrane potential , since an increase in extracellular fluid K ⁺ (depolarizing the plasma membrane of Xenopus laevis cells) promoted uric acid uptake (FIG. 1C). This potential drivability works favorably for excretion of uric acid from the tubule cell to the outside due to electronegative inside the cell. We then outward Cl ^- to determine the effect of the uric acid uptake gradient (from inside to outside of cells). The complete removal of, Cl ^{- -} external Cl imposing gradients without accelerating the uric acid uptake via GLUT9, GLUT9 inorganic Cl observed in URAT1 ^- showed to have no exchange mechanism for (FIG. 1C). In addition, unlike URAT1, it was observed in GLUT9 that uric acid transport activity is dependent on extracellular pH (FIG. 1D).

To further investigate the substrate selectivity of GLUT9, inhibition studies were performed using oocytes expressing GLUT9. Mediated GLUT9 ^[14 C] cis inhibitory effect of various substances 1mM for uric acid (10 [mu] M) uptake (only benzbromarone 50 [mu] M) (i.e., the inhibitor was added to the same side as the uric acid identified with a radioactive isotope was.) were examined (Table 1). GLUT9 again exhibited pharmacological properties different from those of URAT1 (6). We found that uric acid transport via GLUT9 was strongly suppressed by uric acid and weakly suppressed by estrone sulfate.

Hexoses such as glucose and fructose, lactate, monocarboxylic acids such as nicotinate, orotate and ketone bodies (e.g., acetoacetate and β- hydroxybutyrate) inhibit uric acid uptake through GLUT9 There wasn't. These results are consistent with earlier observations that uric acid is the only substrate for GLUT9, as shown in FIG. 4C. We also tested the effects of uric acid excretory substances (3, 19). Probenecid and benzbromarone (50 μM) moderately inhibited uric acid uptake, whereas phenylbutazone and sulfinpyrazone weakly inhibited it. Diuretics did not inhibit uric acid uptake through GLUT9. Among non-steroidal anti-inflammatory drugs, indomethacin strongly inhibited uric acid uptake in cis and naproxen did weakly. Interestingly, Pirajinoato (PZA) (widely been used as an anti-tuberculosis drugs, known in which pyrazinamide active metabolite as interactors against the URAT1 (6)), to the uric acid uptake via GLUT9 Did not have any inhibitory effect. Losartan, an angiotensin II receptor blocker (ARB), moderately cis inhibited uric acid uptake. These results indicate that uric acid is virtually the only GLUT9 transport substrate and that GLUT9 has a different reactivity to various drugs that potentially affect renal uric acid treatment compared to URAT1. We further demonstrated that GLUT9 could be a potential target for novel uric acid excretion drugs .

To elucidate the transport mode of GLUT9, we performed a trans-promoting experiment (ie, the substrate was injected into the oocyte prior to [ ¹⁴ C] uric acid uptake). Uric acid (100 mM) loaded intracellularly significantly enhanced [ ¹⁴ C] uric acid uptake via GLUT9 (FIG. 5A). As expected from results showing no cis inhibition of uric acid uptake by glucose, fructose, lactate, nicotinate, PZA, orotate, β-hydroxybutyrate and salicylate , these substances enter the cell Loading did not show a trans-promoting effect (FIG. 5A).

Given the potential driving transport of uric acid represented in FIG. 1C, uric acid, therefore the electrical gradient of the physiological conditions (i.e. negative inside the cell), can also be transported from the intracellular compartment into the extracellular compartment. As shown in FIG. 2A, when cultured in a standard uptake solution, GLUT9-expressing oocytes pre- loaded with [ ¹⁴ C] uric acid have a time-dependent radioactivity efflux. Indicated. This functional feature is well suited for the pathway of uric acid excretion from cells. In view of previous reports (which showed that human GLUT9 is localized in the proximal basal membrane of proximal tubule cells (18)), we resorbed GLUT9 after an initial step mediated by URAT1 We propose to play the role of moving uric acid from the cells to the tubulointerstitium as the second step (Fig. 2B).

For GLUT9 to determine whether the antiporter or promote transporters, ^[14 C] the outflow of radioactivity from oocytes uric acid is preloaded, extracellular uric acid, absence of glucose and fructose and in the presence, compared. The outflow of radioactivity was affected by extracellular uric acid but not by extracellular glucose or fructose (FIG. 5B), suggesting that the substrate binding site of uric acid is different from that for glucose and fructose. did. Therefore, uric acid transport through the GLUT9 is, by blood levels of these sugars, will not change.

Idiopathic renal hypouricemia (or, simply, renal hypouricemia: MIM220150) is caused by congenital defects in transport of uric acid in renal proximal tubules, the bulimia renal uric acid clearance It is a characteristic genetic disease (21). Mutation of SLC22A12 despite it is well known to be associated with renal hypouricemia, there are renal hypouricemia patients with no mutation of SLC22A12 (7,8). GLUT9 may act Te after the other uric acid reabsorption pathway smell URAT1, patients of this kind may have a SLC2A9 gene mutation. Therefore, we searched for a sequence variation of SLC2A9 in a Japanese patient with renal hypouricemia without a mutation in the coding region of SLC22A12. One of these patients (a 43-year-old man) had a blood uric acid concentration of 2.7 mg / dl (normal range is ˜5 mg / dl). This was in the same range as a renal hypouricemia patient with a URAT1 heterozygous mutation (eg, W258X) . The fraction had a uric acid excretion rate of 14.6% (usually <10%) and because the patient had a normal response in a test loaded with pyrazine amide (21, 22), he was known for PZA It shows that there was no functional change in the target URAT1. Analysis of the polymerase chain reaction (PCR) product of the GLUT9-coding region from patient genomic DNA revealed that nucleotide 1138 within exon 10 of SLC2A9 (FIG. 3A), where arginine 380 (CGG) was changed to tryptophan (TGG). Showed the presence of heterozygotes migrating from C to T. The residue (R380) is located in the loop 8 that menses on the cytoplasmic side (FIG. 6), were among the conserved motifs of all SLC2 family members (23) (D / EXXGRR) . We speculated that loss of positive charge (R380W) alters the uric acid transport function of GLUT9 (voltage- driven transporter). And the arginine residue, the arginine residue S4 portions at the potential-dependent Shaker K ⁺ channel, it may be interesting comparison can be seen. It shows that arginine acts as a potential sensor (24) . This mutation, in personal Japan's control that was selected in 50 people of random, was not confirmed. To confirm the function of mutant GLUT9, we analyzed the uric acid transport activity after injecting wild type or mutant GLUT9 cRNA into oocytes, and this substitution (R380W) was found on the cell membrane. without changing the expression, it was found that the loss of urate transport activity (Figure 3B, C). These in vivo and in vitro data, the mutation of GLUT9, showed that renal hypouricemia of the patient occurs. Based on our model shown in Figure 2B, since both transporters act by the same route in tandem, we fully functional normal uric acid reabsorption in the kidney proximal tubules of URAT1 and GLUT9 Predict that it is necessary. Thus, normal URAT1 may not be able to compensate for the loss of GLUT9 function and vice versa. Our data, and demonstrate the heterogeneity of gene renal hypouricemia, it showed a significant role of GLUT9 in renal reabsorption of uric acid in vivo.

Recently reported a relationship between blood uric acid concentration and polymorphisms of a common gene in the GLUT9 (SLC2A9) gene region on chromosome 4. SNPs in that region were linked to glucose metabolism and uric acid synthesis and / or affect the renal reabsorption, suggesting that influence the uric acid concentration in blood (25). Our findings are consistent with that third idea that GLUT9 affects renal uric acid excretion. In addition, our results, the blood uric acid concentration prove very close to or SLC2A9 correlate with SNPs, very strong to the whole of the relevant study two new genomes (pathological) physiological Histological It will be a boost . Taken together, the protein encoded by the SLC2A9 is the resorbed uric acid from the tubular cells operated to function as urate transporter for discharging in the kidney, also those urate transport activity affect blood uric acid levels I was given. Therefore, we, the protein SLC2A9 can you code, instead of GLUT9, proposes that hump and URATv1 (potential driving urate transporter 1).

Protein Data For immunocytochemistry , cRNAs-injected Xenopus oocytes are fixed in paraformaldehyde, cultured with anti-GLUT9 antibody (alfaDiagnostics) (1: 500), followed by Alexa546-labeled goat anti-rabbit immunoglobulin ( IgG) (Wako; diluted 1: 200). The compartments were observed under a confocal laser scanning microscope (Fluoview FV500, Olympus). Alexa546 fluorescence was excited by a green HeNe laser beam with a wavelength of 543 nm.

Expression and transport testing
CDNA of isoform 1 and 2 of GLU9 were purchased from the cage Gene Technologies (OriGene Technologies) (isoform 2) and open Biosystems (Open Biosystems) (isoform 1). In vitro transcription and injection of capped cRNA (30-50 ng per oocyte) into egg cells was performed as previously reported (1, 2). Oocytes were maintained in bath buffer 12 baths at 18 ° C. for 2-3 days prior to use. ND96 buffer 12 is 96 NaCl, 2 KCl, 1
MgCl ₂ , 1.8 CaCl ₂ and 5 HEPES buffer (pH 7.4) (unit: mM) were included. In the Cl-bath, Cl- was replaced with an equimolar amount of gluconate. Evaluation of kinetic parameters of uric acid uptake via transport studies and GLUT9 was performed as previously reported (1, 2). Experiments were performed using 3 batches of oocytes and results from representative experiments are expressed as mean ± SEM. Statistical significance was determined by Student's t-test.

Mutation analysis and structure of mutant cDNA
For the determination of the GLUT9 sequence we used the primers described by S. Li with slight modifications (Table 2). Some primer sequences were newly selected according to the genomic structure of human GLUT9 (see Figure 6). High molecular weight genomic DNA was extracted from peripheral whole blood cells (3, 4) (3,4) and amplified by PCR. PCR products were sequenced from both directions using a 3130xl gene analyzer (Applied Biosystem) (3). Mutation of exon 10 of GLUT9 (R380W) was detected by restriction enzyme treatment of PCR products (Table 2) with BtsCI for analysis selected randomly from 50 control Japanese individuals . The mutagenic oligonucleotide primers for generation of the R380W mutant were 5′-GGTGTGCGGGACTG (A) ACACTGCTGCAGC-3 ′ and 5′-GGGGCCTGCAGGA (T) CTGTGGAGGGTGGCTG-3 ′. The proper structure of the mutated cDNA was confirmed by complete sequencing.

References in Examples
1. A. Enomoto, H. Kimura, A. Chairoungdua, et al. Nature 417, 447-452 (2002)
2. Jutabha P, Kanai Y, Hosoyamada M, et al. J. Biol. Chem. 278, 27930-27938 (2003)
3. H. Matsuo, K. Kamakura, S. Matsushita, et al. Am. J. Med. Genet. Vol. 88, 733-737 (1999)
4. H. Matsuo, K. Kamakura, M. Saito, et al. Arch. Neurol. Volume 56, 721-726 (1999)

Functional expression of GLUT9 in Xenopus egg cells. (A) Time course of uric acid uptake by GLUT9. Uptake of 20 μM [ ¹⁴ C] uric acid in water-injected oocytes (control) and GLUT9-expressing oocytes was measured during a 120 minute incubation. (B) GLUT9 transport dynamics. Uric acid transport was measured in control oocytes and oocytes expressing GLUT9 in the uric acid concentration range of 10-1,500 μM. The GLUT9- specific transport activity of oocytes expressing GLUT9 was calculated by subtracting the substrate transport activity of control oocytes. These GLUT9 specific transport activities were used for kinetic analysis. Inset , Eadie-Hofstee plot. (C) [ ¹⁴ C] uric acid uptake by GLUT9 is promoted by K ⁺ substitution of extracellular fluid Na ⁺ . The transport rate of [ ¹⁴ C] uric acid (20 μM) in control oocytes and oocytes expressing GLUT9 was measured for 1 hour in the presence and absence of extracellular Na ⁺ , K ⁺ and Cl ⁻ It was done. ***, P <0.001 compared to ND96. (D) pH dependence of uric acid transport mediated by GLUT9 was observed. Uric acid transport rate (20 [mu] M), in the control oocytes using ND96 solution (open squares) and GLUT9 oocytes expressing (closed squares) was determined. In order to prepare uptake solutions with different pH values, MES-NaOH, HEPES-NaOH and Tris-HCl were used as buffer systems. The net uric acid uptake rate increased at the extracellular acidic pH of the ND96 solution . ***, P <0.001 compared to pH7.4. All data are mean ± SEM (error bar) with n = 8−10. (A) Mediation of [ ¹⁴ C] uric acid excretion by GLUT9. The outflow of radioactivity from oocytes expressing GLUT9 ( black circles ) was measured in a standard culture solution compared to controls ( white circles ). The value is expressed as a percentage of the total radioactivity packed in the oocyte. (B) Proposed model for transcellular transport of uric acid via URAT1 and URATv1 (renamed from GLUT9). Monocarboxylates (MCs) containing pyrazinecarboxylic acid (PZA) are located on the luminal (vertical) side of the proximal tubule and depend on sodium-dependent monocarboxylic acid transporter 1 (SMCT1) And 2 (SMCT2) is stored in the cell. Uric acid, the PZA the exchange for the including intracellular MCs through URAT1 located luminal side and into the cell. Uric acid which is re-absorbed through the URATv1 (GLUT9) present in basolateral surface, by electrochemical gradient, leaving the cell. Mutations in the SLC2A9 is associated with idiopathic renal hypouricemia. (A) Electropherogram of partial sequence of exon 10 of SLC2A9 showing atypical point mutation (R380W) found in a patient (43 year old male). The R380W mutation in patients presenting with clinical features with similarity to renal hypouricemia, were held. (B) Disease-related GLUT9 variants from patients completely lose uric acid transport activity. Uric acid uptake (20 μM) was measured in oocytes injected with wild-type or mutated cRNA. Data are mean ± SEM of 8 or 10 oocytes per group. (C) Intracellular localization of wild type or R380W mutant oocytes . Immunodetection of anti-GLUT9 antibody showed that wild type and R380W protein was expressed in the cell membrane, but no fluorescence level was detected in oocytes injected with water. (A) Immunohistochemical analysis of oocytes injected with two isoforms of water and GLUT9 cRNA. Control (injected water), showing the oocytes isoform 1 and isoform 2 is implanted GLUT9 cRNA. (B) Comparison of uric acid transport activity of isoform 1 and isoform 2 of GLUT9. (C) Incorporation of compounds identified with various radioisotopes by GLUT9 isoform 2. ***, P <0.001 compared to control. (A) GLUT9 trans promoting effects of organic anions which are preloaded against uric acid uptake mediated by. Oocytes expressing control or GLUT9 were injected using 50 nl water or unlabeled test anion (100 mM, pH 7.4) using a fine-tip micropipette and then 20 μM [ ¹⁴ C] Transferred to ND96 solution containing uric acid and 1 hour uptake rate was determined. *, P <0.05 compared with water injected. (B) Trans-promoting effect of extracellular uric acid , glucose and fructose on uric acid excretion mediated by GLUT9. Oocytes expressing control or GLUT9 are injected with 50 nl [ ¹⁴ C] uric acid and in the presence or absence of unlabeled compounds (0.5 and 1 mM uric acid and 0.5 and 3 mM glucose and fructose) It was transferred to the lower ND96 solution and incubated at room temperature. The [ ^14C ] uric acid excretion for 60 minutes is shown as a percentage of the preloaded amount. **, p <0.01, ***, p <0.001 compared to control. All data are mean ± SEM with n = 8−10. (A) Location of mutations in the predicted membrane-topology model of SLC2A9. (B) Genomic structure of GLUT9 / SLC2A9 gene. (C) Exon-intron boundary of GLUT9 / SLC2A9 gene. Array 1 Array 2 Array 3 Array 4

Claims (3)

A screening method for a substance having an action of promoting uric acid excretion from the kidney , characterized by measuring whether a substance added to GLUT9-expressing cells promotes uric acid excretion by GLUT9-induced uric acid potential-driven transport . A screening method for a substance having an action of reducing blood concentration of uric acid , characterized by measuring whether or not a substance added to a GLUT9-expressing cell suppresses uric acid excretion by uric acid potential-driven transport by GLUT9. A screening method for a substance having an action of promoting uric acid excretion from the kidney by voltage-driven transport of uric acid by GLUT9 ,
a) preparing a cell in which GLUT9 is expressed,
b) raising the extracellular potassium concentration of the cell and culturing the cell in the presence of a substance and uric acid in a state where the ionic environment inside and outside the cell is reversed ,
c) A screening method for determining whether or not the substance has an action of promoting uric acid excretion from the kidney by GLUT9 based on the amount of uric acid taken up by cells.

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