Abstract
ABSTRACT. Mouse renal-specific transporter (RST) cDNA, the amino acid sequence of which has 74% identity with that of human urate transporter 1 (hURAT1), is potentially the mouse homologue of hURAT1, the gene responsible for hereditary renal hypouricemia. The aim of this study is to determine the location and characteristics of RST molecule in mouse kidney and investigate urate transport by RST using the Xenopus oocyte expression system. RST transported 14C-urate in a Michaelis-Menten manner. The Km and the Vmax values of RST-dependent urate transport were 1213 ± 222 μM and 268.8 ± 38.0 pmol/oocyte per hr, respectively (n = 3). RST-dependent urate transport was cis-inhibited significantly by 1 mM probenecid (68.7 ± 9.4%), 50 μM benzbromarone (67.9 ± 6.4%), and 10 mM lactate (50.9 ± 9.5%). However, 1 mM p-aminohippurate (PAH), 1 mM xanthine, and 1 mM oxonate did not inhibit RST-dependent urate transport. Substitution of Cl anion with gluconate in the external solution enhanced RST-dependent urate transport. Pre-injected pyrazinoic acid (PZA) or l-lactate trans-stimulated RST-dependent urate transport. Using immunohistochemistry for mouse kidney, the brush border or intracellular membrane of proximal tubules was stained by an affinity-purified antibody that recognized mouse URAT1 (mURAT1) expressed on Xenopus oocyte. Using Western blotting, anti-mURAT1 antibody detected 70-kD and 62-kD protein bands. The 70-kD protein was n-glycosylated and was identified as a Triton X-100 insoluble brush border membrane protein. RST mRNA and protein levels were higher in male kidneys than female. RST transported urate similar to hURAT1 and, therefore, appears to be mURAT1—the mouse homologue of hURAT1.
Urate is metabolized by uricase for most mammals, and is an intermediate product of purine metabolism. Urate becomes the end product of purine metabolism for higher primates who have lost uricase activity. Therefore, it is important to understand urate handling mechanisms in the kidney because the underexcretion of urate has been implicated in the development of hyperuricemia that leads to gout. However, urate handling mechanisms are complicated; in that, urate is transported bidirectionally, being both reabsorbed and secreted in the kidney. Moreover, the differences in renal urate transport among different species have made it difficult to analyze urate handling mechanisms in the kidney. Nevertheless, renal urate transport in various animals has been investigated for comparison and to elucidate the evolution of renal urate handling mechanisms. Pigs and rabbits excrete more urate than is filtered through the glomerulus. Birds, like humans, have lost uricase activity. However, birds don’t reabsorb urate in the kidney (1). In contrast, rats and mice reabsorb urate in their kidney, like humans, although uricase maintains their plasma urate at a lower level. The cloning and characterization of the urate transporter from mice is significant for understanding urate handling in the human kidney because the renal transport system of urate in mice was considered to be similar to that in humans (2).
As a transporter molecule for urate reabsorption, we recently cloned urate transporter 1 (URAT1), located at the apical membrane in the proximal tubules of human kidney. We demonstrated that human URAT1 (hURAT1) reabsorbs urate by showing that some patients with renal hypouricemia have a hURAT1 gene (SLC22A12) abnormality (3). Because probenecid and benzbromarone inhibit urate transport by hURAT1 expressed on Xenopus oocyte, it has been deduced that hURAT1 is a transporter responsible for urate reabsorption in human kidney. However, we have no information about the localization of hURAT1 in lower nephron segments of human kidney. It was also demonstrated that urate transport by hURAT1 expressed on Xenopus oocyte was trans-stimulated by the preinjection of PZA into oocyte. Therefore, the antiuricosuric effect of PZA may not be caused by inhibition of urate secretion, but by enhancement of urate reabsorption as suggested by vesicle studies (4,5⇓).
As the mouse homologue of hURAT1, Mouse RST has been shown to have significant identity in predicted amino acid sequence (Genbank accession No. AB005451). Similar to hURAT1, expression of RST was demonstrated in the renal proximal tubule by in situ hybridization (6). Nevertheless, there has been no information about urate transport function by RST. The purpose of this study is to clarify the urate transport function of RST and to verify RST as a urate transporter in mouse kidney. Moreover, the localization and characterization of mURAT1 molecule in the kidney is investigated to understand urate handling in the kidney.
Materials and Methods
Construction of RST cDNA
RST cDNA was amplified by the Advantage HF-2 PCR kit (BD Biosciences Clontech Palo Alto, CA) with mURAT1-05 sense primer (5′-TCTGTGAAGTGGAAGCTGCGTGGTGG-3′) and mURAT1-06R antisense primer (5′-TGTTCCTTCTCCAGGCAACTACAG CC-3′) from the EST clone as Genbank accession number AW106663. The cDNA clone was rescued with the TOPO-TA sequencing vector kit (Invitrogen). The sequence of the isolated clone was confirmed by the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).
Functional Analysis of Urate Transport by RST
RST cRNA was synthesized by the mMessage mMachine T7 kit (Ambion, Austin, TX) and poly (A)+ tailing kit. Synthesized cRNA was purified by the MEGAclear kit (Ambion). Fifty nanograms of RST cRNA was injected into Xenopus oocyte, which was defolliculated by 1.5 to 2.0 mg/ml collagenase (Sigma C-9891) treatment for 1.5 to 2.0 h in OR-2 solution (in mM: 82 NaCl, 2.5 KCl, 1.0 MgCl2, 5.0 HEPES) at room temperature. After 2 to 3 d cultures at 18°C, uptake studies were performed at room temperature with 10 μM 14C-urate, and cold urate for 1 h in ND96 solution (in mM: 96 NaCl or Na gluconate, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 HEPES, pH 7.4) and various concentrations of cold urate. For the inhibition studies, 1 mM probenecid, 50 μM benzbromarone, 10 mM L-lactate, 1 mM PAH, 1 mM xanthine, and 1 mM oxonate were added to the uptake solution containing 10 μM 14C-urate. For the preinjection studies, uptake studies followed a 50 nl injection of 100 mM of potassium L-lactate (pH 7.4) or potassium PZA (pH 7.4). Uptake studies were stopped by adding ice-cold 14C-urate free ND96 solution, and oocytes were washed five times. Each oocyte, solubilized with 200 μl of 10% SDS, was mixed with 2.5 ml of Aquasol-2 (Packard, Meriden, Connecticut) for radioactivity determination using a scintillation counter (LC-3010, ALOKA, Japan).
Immunofluorescence of mURAT1 Expressed in Xenopus Oocyte
Anti-mURAT1 polyclonal antibody was obtained as 1.705 mg/ml of IgG fraction by immunization of rabbit with KLH-conjugated synthetic peptide ELLDRVGGLGRF, corresponding to RST amino acid 5-17, and by peptide affinity-purification. Xenopus oocytes, which were injected with 50 ng of RST cRNA and cultured for 3 d at 18°C, were fixed in 4% paraformaldehyde in ND96 overnight at 4°C. Eight μm-frozen sections were made by a Cryostat (MICROM HM500M, Carl Zeiss) and dried by cool air for 30 min. The sections were stained with anti-mURAT1 antibody (1:100), followed by staining with Cy5 conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania). Images were visualized by an Olympus FLUOVIEW FW500 confocal laser microscope.
Immunohistochemistry of mURAT1 in Mouse Kidney
Ten-week-old male mice purchased from Saitama experimental animal supplier company (Saitama, Japan) were anesthetized by pentobarbital injection (50 mg/kg intraperitoneally), perfused with 4% paraformaldehyde/PBS through the heart, and their sliced kidneys were embedded in paraffin. The 2-μm sections were stained with anti-mURAT1 antibody (1:500) followed by staining with Envision kit (DakoCytomation, Glostrup, Denmark). Nuclei were stained with hematoxylin. Images were visualized, at ×40, ×100, and ×400 magnification by an Olympus BX60 microscope.
Western Blotting of Mouse Kidney
Samples for Western blot analysis were prepared as follows. After adding 4 vol/wt of PBS containing protease inhibitors (Complete, Roche) to 1 vol/wt of mouse kidney tissue, the mixture was homogenized in a Potter-Elvehjam homogenizer on ice. The homogenate was centrifuged at 6000 × g for 15 min at 4°C, and the resultant supernatant was centrifuged at 100,000 × g for 30 min at 4°C. The precipitate was resuspended with the PBS containing protease inhibitors as a crude membrane fraction, or resuspended with extraction buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0) followed by 30-min shaking on ice and 100,000 × g centrifugation for 30 min at 4°C. The resultant supernatant was used as the Triton X-100 extracts of mouse kidney. Protein concentrations of samples were determined with a BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
For preparation of brush border membrane, mouse kidney cortex was homogenized on ice in 10 vol/wt of homogenate solution (10 mM mannitol and 2 mM Tris-HCl, pH 7.1). The homogenate was centrifuged for 2 min at 200 × g to remove unbroken cells. Solid MgCl2·6H2O was added to the resultant supernatant to give a concentration of 10 mM and then shaken for 15 min in an ice bath. After centrifugation for 12 min at 1500 × g, the resultant supernatant was centrifuged for an additional 12 min at 15,000 × g. The precipitate was resuspended in the homogenate solution with 10 mM MgCl2 and centrifuged for 12 min at 2200 × g. The resultant supernatant was centrifuged for 12 min at 15000 × g. The precipitate was washed three times and resuspended in the homogenate solution without MgCl2 as a brush border membrane fraction (7). Alkaline phosphatase activity of the samples was determined with the Alkaline Phospha K kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
For the deglycosylation of crude membrane fraction, 100 μg of crude membrane fraction, which had been boiled for 1 h in 20 mM phosphate buffer (pH 7.4) containing 0.25% SDS and 125 mM 2-mercaptoethanol, was deglycosylated with 5 mU PNGase F and 12% NP-40 (Bio-Rad Laboratories, Hercules, CA) at 37°C overnight.
Western blotting analysis was performed as follows: 10 μg of the samples, the crude membrane fraction, the Triton X-100 extracts, the brush border membrane fraction, and the deglycosylated crude membrane fraction were separated with 10% polyacrylamide gel by Laemmli method and semidry blotted on a nitrocellulose filter (Hybond-ECL, Amersham Biosciences UK Ltd, Buckinghamshire, UK). The blotted filter was shaken for 1 h at room temperature in blocking solution (5% blocking agent of the ECL kit, Amersham Biosciences, in the TBS with 0.02% Tween 20), and was washed three times with washing solution (150 mM NaCl, 10 mM Tris-HCl, pH 7.8, 0.1% Tween 20). The blocked filter was stained overnight at 4°C using affinity-purified anti-mURAT1 antibody (1:900) with or without 10 μg/μl antigen peptide in the TBS-T solution containing 1% bovine serum albumin. The detection was performed according to the manufacturer’s instructions with the ECL kit (Amersham Biosciences).
Sex Difference of the mURAT1 mRNA and Protein Levels
Three 10-wk male and female mice were anesthetized by pentobarbital injection (50 mg/kg intraperitoneally), and their kidneys were excised. Ten micrograms of crude membrane fraction prepared from a kidney was analyzed by Western blotting. The total RNA was extracted from another kidney using ISOGEN kit (Nippon Gene Co. Ltd., Tokyo, Japan) and purified with RNeasy kit (QIAGEN K.K., Tokyo, Japan) and RNase-free DNase. Two micrograms of total RNA was separated with denatured agarose gel and transfered to a positive charged nyron membrane (Hybond-N+, Amersham Biosciences UK Ltd, Buckinghamshire, UK) by alkali blotting (3 M NaCl, 0.01 N NaOH). Probes for mURAT1 mRNA was prepared by PCR DIG probe synthesis kit (F. Hoffmann-La Roche Ltd, Basel, Switzerland) with mURAT1–07 sense primer (5′-ATGACCTTGAACGCCTTGGGCTTCAG-3′) and the mURAT1–06R antisense primer (5′-TGTTCCTTCTCCAGGCAACTACAGCC-3′) from mouse RST cDNA. Probes for mouse β-actin mRNA were prepared with mBACT-01 sense primer (5′- ACCTCATGAAGATCCTGACCG-3′) and the mBACT-02R antisense primer (5′- TGCTTGCTGATCCACATCTGC-3′) from mouse β-actin cDNA that was cloned by RT-PCR from mouse kidney. Hybridization and 0.1× SSC washing were conducted at 65°C. Detection of mURAT1 mRNA was performed with DIG Luminescent Detection kit (Roche).
Results
Urate Transport by RST/mURAT1
Figure 1A indicates that 14C-urate uptake by mouse RST/URAT1 cRNA-injected oocytes increased linearly in a time-dependent manner for 75 min and was significantly different from intrinsic 14C-urate uptake by non-injected oocytes after 15 min. Therefore, we observed 14C-urate uptake for either 30 or 60 min in the following experiments.
Figure 1. (A) Time course of 10 μM 14C-urate and 100 μM cold urate uptake in non-injected (open circles) or mouse RST/URAT1 RNA-injected (filled circles) Xenopus oocyte under ND96 (pH 7.4). Points shown are means ± SEM (n = 8). (B) Typical concentration dependence of urate transport by mouse RST/URAT1 expressed in Xenopus oocyte under ND96 solution. The Eadie-Hofstee plot is shown in the inset. Points shown are means ± SEM (n = 8). From three independent experiments, the Km value was 1213 ± 222 μM, and average Vmax was 268.8 ± 38.0 pmol/oocyte per hr (mean ± SEM, n = 8, n = 3).
Figure 1B indicates the typical concentration dependence of RST-dependent urate uptake determined by the subtraction of 14C-urate transport of non-injected oocytes from the 14C-urate transport of RST cRNA-injected oocytes. From this figure, RST-dependent urate uptake was determined to be Michaelis-Menten type, and the Eadie-Hofstee plot, shown in the inset, was linear. The average Km value from three independent experiments was 1213 ± 222 μM, and the average Vmax was 268.8 ± 38.0 pmol/oocyte per hr (mean ± SEM, n = 8, n = 3). These results demonstrated that RST transported urate and verified that RST was mURAT1, the mouse homologue of hURAT1.
Figure 2 shows the inhibition profiles of urate secreting and retaining drugs, indicated as the percent of urate uptake observed in the presence of each drug, compared with mURAT1 uptake in the absence of drugs as the control. One mM probenecid (68.7 ± 9.4%), 50 μM benzbromarone (67.9 ± 6.4%), and 10 mM lactate (50.9 ± 9.5%) significantly cis-inhibited mURAT1-dependent urate transport. On the other hand, 1 mM PAH, xanthine, and oxonic acid did not inhibit urate transport by mURAT1.
Figure 2. Inhibition of mouse RST/URAT1 dependent 10 μM 14C-urate transport under ND96 solution by 1 mM probenecid, 50 μM benzbromarone, 10 mM L-lactate, 1 mM PAH, 1 mM xanthine, and 1 mM oxonic acid. From three independent experiments, values are shown as means ± SEM of percent urate uptake by mouse RST/URAT1 in the presence of drugs compared with mURAT1 uptake in the absence of drugs as the control (n = 8, 1.00 ± 0.10 pmol/oocyte per hr). **P > 0.01.
Figure 3 shows that urate transport by mURAT1 was trans-stimulated by Cl anion, L-lactate, and PZA. Substitution of NaCl in ND96 solution with Na gluconate enhanced urate transport by mURAT1. This suggests that mURAT1 is a urate/Cl− exchanger like hURAT1. Preinjected L-lactate and PZA trans-stimulated urate influx by mURAT1. Therefore, mURAT1 appears to be a urate/lactate and urate/PZA exchanger just like hURAT1.
Figure 3. Enhancement of mouse RST/URAT1 dependent 10 μM 14C-urate transport by 5 nmol preinjected L-lactate and pyrazinoic acid (PZA) under ND96-NaCl and that of mouse RST/URAT1 dependent 10 μM 14C-urate uptake under ND96-Na gluconate. From three independent experiments, values shown are means + SE of % uptake of mouse RST/URAT1 dependent urate uptake without preinjection under ND96-NaCl as a control (n = 8, 0.37 ± 0.07 pmol/oocyte per 30 min). **P > 0.01.
Immunohistochemistry of mURAT1 in the Kidney
Figure 4A is an immunofluorescence of mURAT1 stained with mURAT1 antibody. Because noninjected oocyte was not stained by this antibody (Figure 4B), the antibody was verified to recognize mURAT1 protein.
Figure 4. Immunofluorescence of mouse URAT1 cRNA-injected oocyte (A) and non-injected oocyte (B) using affinity-purified anti-mURAT1 antibody. 2.55 μg/ml antibodies were applied at 4°C overnight and stained in the red fluorescence with Cy5 conjugated anti-rabbit IgG. Immunohistochemistry of mouse kidney using affinity-purified anti-mURAT1 antibody in 40× (C), 100× (E), and 400× (F) magnification. 0.85 μg/ml antibodies were applied at 4°C overnight and stained in dark brown with diaminobentizine. (D) antibody absorption test using 100 μg/ml of antigen peptide corresponding to panel C.
Figures 4C to 4F are immunohistochemistries of mouse kidney using the affinity-purified mURAT1 antibody. Staining of mURAT1 was restricted to the proximal tubule and is not present in the medulla (Figure 4C), and the staining was absorbed by 100 μg/μl antigen peptide (Figure 4D). Localization of mURAT1 was observed from the exit of Bowman’s capsule and mixed in the proximal tubule cells, with dotlike patterns in the cytoplasm and linear patterns in the brush border membrane (Figure 4, E and F). Therefore, it was difficult to indicate the relationship between the localization pattern of mURAT1 and the segments of proximal tubule.
Western Blotting of mURAT1
Figure 5 shows the western blot analysis of mouse kidney using the affinity-purified mURAT1 antibody. Two bands of 70-kD and 62-kD protein were detected in the crude membrane sample (lane 1). These bands completely disappeared following the addition of 10 μg/μl of antigen peptide (lane 5). The 62-kd protein was in the Triton X-100 soluble fraction (lane 2), and the band was completely eliminated by the addition of 10 μg/μl of antigen peptide (lane 6). Because the upper 70-kD band disappeared in the Triton X-100 extracts from the crude membrane sample, the 70-kD protein was in the Triton X-100 insoluble fraction. However, only the upper 70-kD band was detected in the brush border membrane fraction (lane 3), the alkaline phosphatase activity of which was 25.5 times higher than that of the initial homogenate. The upper 70-kD band relocated to the lower 62-kD band following deglycosylation of crude membrane fraction using PNGase F (lane 4). Therefore, the N-glycosylated 70-kD form of mURAT1 was sorted in the brush border membrane, and the native 62-kD form of mURAT1 was soluble with Triton X-100.
Figure 5. Western blot analysis of 10 μg of protein prepared from mouse kidney with 0.19 μg/ml affinity-purified anti-mURAT1 antibody. Lane 1, crude membrane fraction of mouse kidney. Lane 2, Triton X-100 extract from the crude membrane fraction of mouse kidney. Lane 3, brush border membrane fraction from mouse kidney. Lane 4, deglycosylated sample of the Triton X-100 extract by PNGase F. Lanes 5 and 6, antibody absorption tests by the 10 μg/ml antigen peptide corresponding to lanes 1 and 2, respectively.
Sex Difference of mURAT1 Expression
Figure 6 shows the sex difference of mURAT1 protein (6A) and mRNA (6B) expression. There was a significant sex difference in body weight (male, 37.7 ± 0.33 g; female, 27.3 ± 0.17 g) and kidney weight (male, 0.57 ± 0.05 g; female, 0.40 ± 0.01 g); and male mURAT1 protein levels in 10 μg crude membrane fraction were higher than those in females. Comparing mouse β-actin mRNA levels, male mURAT1 mRNA levels in 2 μg total RNA from kidney were also higher than those of females. The male/female ratio of the intensity of mURAT1 mRNA band was 2.3, normalized by the intensity of mouse β-actin mRNA band. Therefore, mURAT1 transcription was sex-dependent, being greater in males than females.
Figure 6. Sex difference of the mURAT1 expression. (A) Western blot analysis of 10 μg of protein of crude membrane fractions prepared from three male and three female mouse kidneys with 0.19 μg/ml affinity-purified anti-mURAT1 antibody. (B) Northern blot analysis of 2 μg of total RNA purified prepared from three male and three female mouse kidneys using mURAT1 probe and mouse β-actin probe. The ratio of band intensity of mURAT1 mRNA/β-actin mRNA was 1.17 ± 0.05 in male and 0.50 ± 0.10 in female (mean ± SD).
Discussion
Structure of mURAT1
RST was reported as a 553-amino-acid protein with 30% identity with the rat organic cation transporter 1 at the amino acid level. RST cDNA and hURAT1 cDNA contained 1659-bp coding regions that were 78.1% identical at the nucleotide level and 74.0% identical at the expected amino acid level. In comparison, human organic anion transporter 4 (OAT4) was only 37.4% identical with RST at the expected amino acid level. Therefore, RST was verified as the mouse homologue of hURAT1 based on molecular structures.
The identity between RST and hURAT1 at the expected amino acid level was lower than the 87.8% identity between rat OAT1 (8) and human OAT1 (9), which has been reported to be a urate transporter (10). Mouse Slc22a12, contained in a genomic clone from mouse chromosome 19 (Genbank accession No. AC124394), was separated to ten exons by nine introns at the same exon-intron boundaries of human SLC22A12 (Table 1). The nucleotide identities between each exon of human SLC22A12 and that of mouse gene were distributed from 67.2% (exon 10) to 83.8% (exon 9). Some N-glycosylation sites and protein kinase A-dependent phosphorylation sites are identical between two cDNA clones indicated as Figure 7.
Table 1. Sequence homology between mouse and human SLC22A12
Figure 7. Deduced amino acid sequence of mURAT1 compared with that of hURAT1. Dots in the hURAT1 sequence represent identity to mOAT1. *Predicted N-glycosylation sites; #predicted protein kinase A-dependent phosphorylation sites. Predicted membrane spanning domain are underscored. GenBank accession numbers: AB005451, RST/mURAT1; AB071863, hURAT1.
Urate Handling in Mouse Kidney
Plasma concentrations of urate in mice of the DBA/2N strain and ddY strain were reported as 0.192 ± 0.017 mg/dl and 0.173 ± 0.016 mg/dl, respectively (11). These values correspond to 10 μM urate and are much lower than the Km value of mURAT1. Therefore, the plasma urate level is not dependent on the urate transport activity of mURAT1 in normal mice, but rather on the urate breakdown by uricase. However, the plasma urate concentration of uricase-deficient hyperuricemia model mice (11.0 ± 1.7 mg/dl) closely approximated the Km value of mouse URAT1 (12). Consequently, mURAT1 may regulate the plasma urate level in uricase-deficient mice, similar to hURAT1.
The fractional excretions of urate (CUA/CCr) in mice of the DBA/2N and ddY strains were 0.278 ± 0.020 and 0.382 ± 0.021, respectively (11). Therefore, the mouse is one of the mammals that are categorized as net urate-reabsorption animals, like humans, rats, and dogs. Like hURAT1, urate transport by mURAT1 was trans-stimulated by L-lactate. Therefore, the physiologic concentration gradient between the lower concentration of L-lactate in the luminal urine and the higher intracellular concentration of the proximal tubule cell can function as a driving force for reabsorption of urate.
Benzbromarone and probenecid inhibited urate transport by mouse URAT1 expressed on Xenopus oocytes. The uricosuric activity of benzbromarone and probenecid was demonstrated by in vivo studies with mice (11). Luminal perfusion of probenecid partially inhibited net uric acid reabsorption in microperfusion studies with the mouse proximal tubule (13). Thus, mURAT1 could function as a transporter for urate reabsorption in mouse kidney. Localization of mURAT1 was observed from the exit of Bowman’s capsule coinciding with physiologic studies of the reabsorption site of urate in early proximal tubule.
Pyrazinamide is rapidly hydrolyzed to the active antiuricosuric agent PZA. Subsequently PZA is oxidized by xanthine oxidase to 5-hydroxy PZA, which is devoid of antiuricosuric effects (14). Because pyrazinamide in the bath solution abolished the secretion of urate in the microperfusion studies with mouse proximal tubule (13), PZA generated in proximal tubule cells might trans-stimulate urate transport by mURAT1. Although trans-stimulation of urate transport by mouse and human URAT1 advocates that the anti-uricosuric effect of pyrazinamide is through enhancement of urate reabsorption in kidney, UAT expressed at the brush border membrane was also reported to have a PZA-sensitive urate channel activity (15,16⇓). Therefore, the possibility of inhibition of urate secretion by PZA must be investigated in vivo.
Oxonate has been used as an uricase inhibitor to produce a hyperuricemia model in rodents (17,18⇓). Since oxonate did not inhibit urate transport by mURAT1, oxonate-treated mice were suitable for studying the function of mURAT1 in vivo.
URAT1 Expression in Mouse Kidney
It was demonstrated that the RST gene was expressed as a 2.3-kb transcript only in the kidney using Northern blotting. Moreover, in situ hybridization analysis using a cRNA probe constructed from a 1.5-kb 5′-RACE fragment showed that RST gene expression is restricted to the renal proximal tubule (6). It was also demonstrated that some proximal tubule cells expressed mURAT1 at the brush border membrane similar to human URAT1 (3). Moreover, other proximal tubule cells expressed mURAT1 as a dotlike pattern in the cytoplasm, which indicated that mURAT1 localized on the intracellular organelles. From Western blotting, the N-glycosylated 70-kD form corresponds to mURAT1 at the brush border membrane, and the native 62-kD form corresponds to the mURAT1 at the intracellular organelles. The molecular weight of the deglycosylated 62-kD protein was very close to 61 kD, the expected molecular weight from the amino acid sequence of mURAT1.
Because the N-glycosylated 70-kD form was in the Triton X-100 insoluble fraction, mURAT1 protein might be localized in the raft component in the brush border membrane. Although some intestinal brush border proteins were demonstrated to localize in the Triton X-100 insoluble fraction (19), renal brush-border proteins in vivo have not been demonstrated in the Triton X-100 insoluble fraction yet. The brush border protein Na+/H+ exchanger 3 was reported to be expressed primarily in the detergent-insoluble fraction in lipid rafts of the apical surface of proximal tubule-derived OK cells (20).
Sex differences in urate levels in human blood is one of the typical sex differences in endocrinological data, and is caused by a sex-dependent difference in the probenecid-sensitive urate reabsorption in human kidney (21). Because URAT1 is responsible for the probenecid-sensitive urate reabsorption, the sex-dependent difference of mURAT1 expression level is coincident with pharamacological data from humans. Further investigation is needed to detail hormonal regulation of URAT1 transcription and promoter analysis.
In summary, RST transported urate (based on characteristics of molecular structure, transport function, and cellular localization) is apparently the mouse homologue of hURAT1. The 70-kD N-glycosylated form of mURAT1 protein was expressed in the brush border membrane of proximal tubule. Moreover, a sex-dependent difference of mURAT1 transcription level was demonstrated. Therefore, mouse URAT1 is an appropriate experimental model for studying the mechanism of membrane sorting of the URAT1 protein and the hormonal regulation of its expression.
Acknowledgments
The authors thank Ms. Akie Toki for her technical assistance. This study was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture (#s 11770048 and 13770048).
- © 2004 American Society of Nephrology
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