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Departments of Nephrology (N.W., D.G.T., M.A., T.M., H.N., K.T., K.K.) and Biopharmaceutics (N.W., M.O.), Kumamoto University Graduate School of Medical and Pharmaceutical Sciences, Kumamoto 860-8556, Japan; Department of Pediatrics, Sapporo Tokushukai Hospital (T.Ok., O.U.), Hokkaido 003-0021, Japan; Department of Pediatrics, Fujita Health University School of Medicine (M.T.), Aichi 470-1192, Japan; Department of Medicine, Kasukabe Shuwa Hospital (S.K.), Saitama 344-0038, Japan; Department of Medicine, Kitasato Medical Center Hospital (Y.Y., H.S.), Saitama 364-0026, Japan; Second Department of Internal Medicine (T.Od., Y.K.) and First Department of Physiology (H.M.), National Defense Medical College, Saitama 359-0042, Japan; and Department of Pharmacology and Toxicology, Kyorin University School of Medicine (M.H., H.E.), Tokyo 181-8611, Japan
Address all correspondence and requests for reprints to: Dr. Kenichiro Kitamura, Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Kumamoto, Kumamoto 860-8556, Japan. E-mail: ken{at}gpo.kumamoto-u.ac.jp.
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Abstract |
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Introduction |
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In 2002, Enomoto et al. (14) isolated human urate transporter 1 (hURAT1) and identified loss of function mutations in hURAT1 in three subjects with idiopathic renal hypouricemia, suggesting that hURAT1 is responsible for the regulation of serum urate levels in humans. Recently, Ichida et al. (15) examined the mutations in hURAT1 in 32 unrelated subjects with idiopathic renal hypouricemia and identified eight new mutations. Komoda et al. (16) reported a high prevalence of the W258X mutation in hURAT1 in the Japanese population.
In the present study, we investigated the clinical features and mutations in the hURAT1 gene in seven families with presecretory reabsorption defect-type familial renal hypouricemia and in one family with postsecretory reabsorption defect-type familial renal hypouricemia. We identified a novel mutation as well as three previously reported mutations in the families with presecretory reabsorption defect, and Xenopus oocytes expressing these mutant hURAT1s showed substantially reduced urate-transporting activity. However, no mutations in the coding region of hURAT1 could be detected in the family with postsecretory reabsorption defect. These findings indicate the possibility that hURAT1 might be the gene responsible for the presecretory reabsorption defect-type renal hypouricemia, and that another transporter(s) might be involved in the postsecretory reabsorption defect-type renal hypouricemia.
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Subjects and Methods |
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Twelve affected subjects and 26 family members belonging to eight unrelated Japanese families diagnosed with familial renal hypouricemia were studied. Hypouricemia was defined as serum urate levels less than 2.0 mg/dl. At least one member of each family had an episode of exercise-induced acute renal failure and underwent both PZA and probenecid (or benzbromarone) loading tests to determine the type of disorder in urate handling in the kidney. Based on the results of these drug loading tests, seven of eight families (families 1–7) were classified as having the presecretory reabsorption defect type, and the other family (family 8) was classified as having the postsecretory reabsorption defect type. The pedigrees are depicted in Fig. 1
. The group of affected subjects was composed of nine men and three women, with a mean age of 23.3 yr. The clinical features of affected subjects in families 2, 4, and 8 were previously reported (17, 18). Written informed consent was obtained from each subject upon enrollment in this study. This study was approved by the institutional review board of Kumamoto University.
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All exons of the hURAT1 gene were screened for mutations in each affected subject and family members. Primers used for the hURAT1 sequence determination were described by Ichida et al. (15). The technique used to detect mutations was the PCR-direct sequencing method. Briefly, genomic DNA from each subject was isolated from peripheral blood cells using the GFX Genomic Blood DNA Purification Kit (Amersham Biosciences, Little Chalfont, UK). Each exon was amplified by PCR using the genomic DNA as a template. The PCR product was purified and subjected to sequencing reaction using the dideoxy chain termination method with fluorescent dye-labeled terminators on an automated sequencer (ABI model 310, Applied Biosystems, Foster City, CA). When two different mutations in hURAT1 gene were identified, we performed PCR in which the PCR product contains both mutations. The PCR product was then subcloned into pGEM-T Easy Vector (Promega Corp., Madison, WI), and several clones were sequenced to determine whether the two mutations are located on the same allele.
Microsatellite genotyping
Genotyping of all family members of family 8 was performed by PCR with primer sets for D11S4191 and D11S4162 (Table 1). The D11S4191 and D11S4162 loci are located upstream and downstream of the hURAT1 gene, respectively (Table 1). In brief, specific segments from genomic DNA were amplified with specific fluorescence-labeled primers for each marker locus on chromosome 11, the site of the hURAT1 gene. Genotypes were analyzed by an ABI373S autosequencer with Genescan software (Applied Biosystems) to measure the nucleotide length of the amplified fragments from each allele. Heterozygosity of the markers used reported by Applied Biosystems was as follows: 0.87 for D11S4191 and 0.64 for D11S4162.
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To introduce mutations that were found in the present study into wild-type hURAT1 cDNA, we used the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer’s instructions. Proper construction of the mutated cDNA was confirmed by complete sequencing. Mutant hURAT1 cRNA was synthesized using the mMESSAGE mMACHINE kit (Ambion, Inc., Austin, TX) from each linearized mutant URAT1 cDNA. A polyadenylated tail was added by the Poly(A)+ Tailing kit (Ambion, Inc.), and synthesized cRNA was purified by the MEGAclear Purification kit (Ambion, Inc.). Stage 5–6 Xenopus oocytes were treated with 1 µg/ml collagenase type I (Sigma-Aldrich Corp., St. Louis, MO) for 1 h to remove follicle cell layers and injected with 50 ng cRNA for wild-type or mutant hURAT1 in 25 nl water. Injected oocytes were kept at 19 C in modified Barth’s saline [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 15 mM HEPES, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, and 0.82 mM MgSO4]. Uptake studies were performed 72 h after cRNA injection. Oocytes were incubated with ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) containing 10 µM [14C]urate (1.85–2.22 GBq/mmol; American Radiolabeled Chemicals, Inc., St. Louis, MO) and 100 µM cold urate. Thirty minutes after incubation, oocytes were washed five times with ice-cold ND96, pH 7.4, and were solubilized with 500 µl 10% sodium dodecyl sulfate. Each sample was then mixed with 2.5 ml Emulsifier-safe (Packard Bioscience, Meriden, CT), and the radioactivities were measured using a scintillation counter.
Statistical analysis
Statistical significance was evaluated using ANOVA, followed by the Newman-Keuls method. P < 0.05 was regarded as statistically significant. Values are expressed as the mean ± SE.
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Results |
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The clinical features and genotypes of the hURAT1 gene in the affected subjects and related family members with familial renal hypouricemia are described in Table 2. The affected subjects in families 1–7 were classified into the presecretory reabsorption defect type according to the results from PZA and probenecid (or benzbromarone) loading tests, and the affected subjects in family 8 were classified into the postsecretory reabsorption defect type. The results of PZA and probenecid loading tests were described in Table 3. Serum urate levels in all affected subjects were substantially decreased (0.60 ± 0.16 mg/dl; n = 12), and fractional excretion of urate (FEUA) levels were significantly increased (59.9 ± 12.6%; n = 12). These data are compatible with the patterns observed in renal hypouricemia. Mutational analysis revealed that homozygous or compound heterozygous mutations in the hURAT1 gene were identified in nine of 12 affected subjects. Three subjects who had no mutations in the hURAT1 gene originated from the same family (family 8). In 18 subjects who were identified as having heterozygous mutations in the hURAT1 gene, serum urate levels were relatively lower (2.79 ± 0.54 mg/dl; n = 18), and FEUA levels were relatively higher (14.5 ± 3.7%; n = 18) than the normal range (normal urate level, 2.5–7.0 mg/dl; normal FEUA level, <10.0%). Except for the subjects in family 8, four subjects were shown to have no mutations in the hURAT1 gene, and they had normal urate and FEUA levels (urate, 5.78 ± 1.54 mg/dl; FEUA, 6.5 ± 1.8%; n = 4).
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Mutational analysis revealed that all of the affected subjects and 20 of 24 related family members in the presecretory reabsorption defect-type familial renal hypouricemia group (families 1–7) had mutations in the hURAT1 gene. We detected one nonsense mutation (G774A), two missense mutations (A1145T and T1253G), and one 5-bp deletion mutation (1639–1643 del-GTCCT) in these subjects. The T1253G mutation was a novel mutation, and the other mutations were previously reported (14, 15, 16). None of these mutations was identified in 75 randomly chosen, unrelated control Japanese individuals. Genotyping of the hURAT1 gene in the related family members revealed the segregation of each mutated allele in the affected family and elucidated the autosomal recessive inheritance of this disease. In contrast, no mutations in the hURAT1 gene were identified in three affected subjects and two related family members in the postsecretory reabsorption defect-type familial renal hypouricemia group (family 8). We determined the sequences only of all exons and exon/intron boundaries of the hURAT1 gene, not the entire hURAT1 genome. However, we could not detect any insertion, deletion, or substitution mutations in this family, suggesting that hURAT1 may not be the gene responsible for the postsecretory reabsorption defect type. To elucidate this possibility, we next performed linkage analysis of family 8 using two microsatellite markers (D11S4191 and D11S4162) that are located upstream and downstream, respectively, of the hURAT1 gene (Table 1
). As shown in Fig. 2, segregation patterns of both D11S4191 and D11S4162 markers were totally different among the affected children, although they showed the same phenotypes (hypouricemia). These findings strongly support the possibility that hURAT1 is not responsible for the postsecretory reabsorption defect type.
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To assess the functional significance of the new mutation (T1253G) as well as the three previously reported mutations (G774A, A1145T, and 1639–1643 del-GTCCT), we expressed these mutant or wild-type hURAT1 in Xenopus oocytes and tested for the urate-transporting activities. As shown in Fig. 3, all hURAT1 mutants had significantly reduced urate-transporting activities compared with wild-type hURAT1 (water, 0.03 ± 0.005* pmol/min·oocyte; wild-type, 0.56 ± 0.021; G774A, 0.03 ± 0.005*; A1145T, 0.05 ± 0.009*; T1253G, 0.04 ± 0.009*; 1639–1643 del-GTCCT, 0.06 ± 0.006*; *, P < 0.01 vs. wild type; n = 12), and the transporting activities of the hURAT1 mutant were similar to those of water-injected oocytes. These findings strongly support the idea that the mutations found in the current study account for the loss of function in hURAT1 and the renal hypouricemia phenotype in the affected subjects.
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Discussion |
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As mentioned in the introduction, renal hypouricemia is traditionally classified into five types according to the responses to PZA and probenecid loading tests (5, 6, 7, 8). The presecretory reabsorption defect type is characterized by the attenuated response to both PZA and probenecid. The postsecretory reabsorption defect type is accompanied by significant suppression of urate clearance by PZA and no influence on urate clearance by probenecid. The FEUA of the affected subjects in families 1–7 was approximately 51–88% under basal conditions. Because both PZA and probenecid loading tests had only a modest effect on FEUA, they were diagnosed as having the presecretory reabsorption defect type (17). The FEUA of the affected subjects in family 8 was approximately 47–61% under basal conditions. PZA loading resulted in a significant decrease in FEUA to 4%, whereas probenecid had no effect on FEUA. Thus, this subject was diagnosed as having postsecretory reabsorption defect type (18).
We identified four mutations in the hURAT1 gene in subjects with the presecretory reabsorption defect; functionally, these mutations impaired the urate-transporting activity of hURAT1. G774A, A1145T, and 1639–1643 del-GTCCT were previously reported (14, 15, 16), and T1253G was newly identified. G774A resulted in truncated immature hURAT1 protein, and 1639–1643 del-GTCCT is considered to reduce the routing of the hURAT1 protein to the apical membrane by disrupting the PDZ binding motif (15). Because A1145T and T1253G mutations were located in the 9th and 10th putative transmembrane domains, respectively, these mutations might modulate the urate permeability of hURAT1. As previously reported by Ichida et al. (15), our study also demonstrated a gene dosage effect of hURAT1 on FEUA and serum urate levels. There was a correlation between the number of wild-type hURAT1 alleles and FEUA or serum urate levels, suggesting the significance of hURAT1 in the regulation of serum urate levels.
We were unable to detect any mutation in the hURAT1 gene in the subject with postsecretory reabsorption defect. Sequence analysis was performed only for all exons and exon/intron boundaries, and we could not detect any mutation. Then we performed linkage analysis using microsatellite markers located around the hURAT1 locus. The linkage analysis revealed that segregation patterns of hURAT1 gene were not identical in the affected subjects in the family with the postsecretory reabsorption defect type, although they had similar hypouricemia. These findings strongly suggest that another urate transporter(s) might be involved in the postsecretory reabsorption defect type. Also, the fact that the response to PZA loading test in this subject is completely different from that in subjects with the presecretory defect type supports our hypothesis.
In summary, our study revealed a novel mutation in the hURAT1 gene in the presecretory reabsorption defect type of familial renal hypouricemia. In addition, microsatellite linkage analysis indicated that the hURAT1 locus is not associated with the hypouricemic phenotype in the postsecretory reabsorption defect type. Our findings provide new insight into understanding the structure-function relationship of hURAT1. Future studies are required to identify the gene responsible for postsecretory reabsorption defect-type renal hypouricemia.
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Acknowledgments |
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Footnotes |
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First Published Online January 5, 2005
1 N.W. and D.G.T. contributed equally to this work. 
Abbreviations: FEUA, Fractional excretion of urate; hURAT1, human urate transporter 1; PZA, pyrazinamide.
Received June 11, 2004.
Accepted December 27, 2004.
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and •, Affected subjects; 
and 
, unaffected subjects. Deceased subject (diagonal line) is of unknown phenotype. Below the symbol for each subject, genotypes of URAT1 gene are shown. Mutations identified in each family are indicated below the family number and are superscribed on the genotypes. +, Wild-type allele; –, mutant allele.
