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Department of Biochemistry and Center of Novel Functional Molecules, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
(Received July 21, 2006; accepted February 22, 2007)
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). Hyperuricemia is a hallmark of gout (Rott and Agudelo, 2003). Moreover, recent studies suggested that hyperuricemia might also be a cause of other metabolic syndromes including hyperinsulinemia and hypertriglyceridemia, probably due to its ability to inhibit endothelial function (Nakagawa et al., 2006). Previous epidemiologic data also indicated that increased serum uric acid levels were independently and significantly associated with the risk of cardiovascular mortality (Fang and Alderman, 2000).
Seventy percent of the daily output of uric acid is excreted through the kidney (Marangella, 2005). Underexcretion of urate has been implicated in the development of hyperuricemia and 80 to 90% of gouty patients are in fact underexcreters (Liote, 2003; Rott and Agudelo, 2003). Thus, the renal handling of urate transport constitutes an important target in the development of drugs to treat hyperuricemia. Recently, the human urate anion transporter (hURAT1) was identified and demonstrated to mediate urate handling in the human kidney (Enomoto et al., 2002). It is a member of the organic anion transporter family and is expressed only in the kidney, where the protein is located at the apical membrane of the epithelium in the proximal tubules but not in the distal tubules (Enomoto and Endou, 2005). It is encoded by a gene (SLC22A12) located on chromosome 11q13 and is believed to regulate the reabsorption of urate from the lumen to the cytosol in the human kidney proximal tubules (Marangella, 2005).
In our previous study (Yu et al., 2006), we have identified a natural compound, morin (Fig. 1), which exhibits potent inhibitory action on urate uptake in rat renal brush-border membrane vesicles with an IC50 value of 18 µM. Its inhibition on urate uptake is much more potent than that of probenecid, one of the currently prescribed uricosuric agents used in the treatment of hyperuricemia. Our in vivo studies in rats also demonstrated that this natural compound could increase the excretion of urate in the urine and decrease the plasma urate level in experimental animals (Yu et al., 2006). Its potent biological activities in rat, together with its low toxicity (Wu et al., 1994; Cho et al., 2006), has made this compound a potential candidate for further development into a therapeutic for the treatment of hyperuricemia in humans. However, such an extrapolation of animal data to human subjects needs to be substantiated, particularly in view of the fact that species difference does exist regarding the transport of urate across the proximal tubule cells in the kidney (Dantzler, 1996; Hediger et al., 2005). Moreover, even though both human and rat exhibit a net tubular urate reabsorption in their kidneys, the magnitude of the reabsorption varies greatly (Roch-Ramel and Guisan, 1999; Rafey et al., 2003). It is therefore necessary to show that the uricosuric actions of morin that we have documented in rats are also applicable to the human situation. Since the hURAT1 has been cloned and demonstrated to be involved in the urate reabsorption process in humans, we have therefore used a cultured human kidney cell system expressing this transporter to investigate the putative action of morin on urate transport in the human kidney.
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Materials and Methods |
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Cell Culture. Human embryonic kidney 293 (HEK293) cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were kept at 37°C in an incubator with 5% CO2.
Establishment of HEK293 Cells Expressing hURAT1. HEK293 cells were made to express hURAT1 by transient transfection. Transfection by Lipofectamine was performed according to the manufacturer's instructions. In brief, 24 h before transfection, HEK293 cells were plated on 24-well culture plates at a density of 2 x 105 cells/well. Before the seeding of cells, the culture plates were precoated with 0.1 mg/ml poly-D-lysine dissolved in distilled water and air-dried in the culture hood. Cells were maintained normally in DMEM containing regular serum and antibiotics until transfection. Twenty-four hours later, when the culture reached 70 to 80% confluence, cells were washed with phosphate-buffered saline and then the medium was replaced by 0.4 ml of DMEM containing 2.5 µl of Lipofectamine and 0.4 µg of pCMV·SPORT6 harboring the full-length hURAT1 cDNA. No serum and antibiotics were present in the medium at the time of transfection. Cells were incubated at 37°C ina5%CO2 incubator for 5 h, followed by addition of an equal volume of the medium containing two times the normal concentration of serum and antibiotics. The transport activities of the transfected cells were assessed 48 h afterward. The empty vector pCMV·SPORT6 was also transfected into another batch of cells in parallel as a control.
Characterization of HEK293-hURAT1 Cells. Reverse transcription-polymerase chain reaction (RT-PCR) techniques were adopted to confirm the transcription of hURAT1 in the transfected HEK293 cells. In brief, total RNA was extracted from HEK293 cells 48 h post-transfection using TriPure isolation reagent (Roche Applied Science, Basel, Switzerland) and dissolved in diethyl pyrocarbonate-treated RNase-free water. One microgram of total RNA was then reverse-transcribed using the Improm-II Reverse Transcription System (Promega, Madison, WI) with oligo(dT)15 as the primer. The synthesized first-strand cDNA was used as the template in the subsequent PCR using Taq DNA polymerase (Invitrogen) to confirm the successful transcription of hURAT1. The PCR involved an initial denaturation at 94°C for 2 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. A final extension at 72°C for 10 min was allowed. The gene-specific primers used in the amplification of hURAT1 were as follows: forward primer, 5'-CCCTGAGGTCTTGCTTTCAG-3'; reverse primers, 5'-ATGTCCACGACACCAATGAA-3'. Amplification of ß-actin was carried out in parallel as the internal control. A negative control was also performed without adding RNA in the initial step of reverse transcription.
Subcellular Localization of hURAT1. To investigate the subcellular localization of the exogenously introduced hURAT1 in the cultured HEK293 cells, the full-length sequence of hURAT1 was also subcloned into pEGFP-N1. The full-length hURAT1 sequence was amplified from the original plasmid using a pair of primers with an XhoI (forward) and BamH I (reverse) restriction site on their 5' ends, respectively, for insertion into the pEGFP-N1 vector. The primer sequences were as follows: XhoI-forward, 5'-GTGATACTCGAGATGGCATTTTCTGAACTC-3'; BamH I-reverse, 5'-GTGATAGGATCCGTAAACTGTGTGGATTTTAG-3'. The High Fidelity Expand DNA polymerase (Roche Applied Science) was used in the amplification with 6 ng of the pCMV·SPORT6 carrying hURAT1 as the template. The PCR involved an initial denaturation at 94°C for 4 min, followed by 20 cycles at 94°C for 30 s, 55°C for 45 s, and 72°C for 90 s. A final extension at 72°C for 7 min was allowed. The amplified fragment was then purified using the QIAGEN Purification Kit (QIAGEN, Clifton Hill, Australia). After double restriction digestion with BamH I and XhoI (New England Biolabs, Ipswich, MA), the PCR product was purified using the QIAGEN Purification Kit and then directionally subcloned into the eukaryotic expression vector pEGFP-N1. The final plasmid was sequence-confirmed.
The pEGFP-N1 vector carrying the hURAT1 was transfected into HEK293 cells using the method described above. Forty-eight hours post-transfection, the subcellular localization of hURAT1 was studied by confocal microscopy. Cells grown on a coverslip were inverted on microscope slides for analysis on a TCS NT confocal microscope (Leica Microsystems GmbH, Heidelberg, Germany) using an argon laser at an excitation wavelength of 488 nm. Fluorescence was detected with the BP525/50 filter set and assigned the color green. Images were captured using the TCS NT software and modified using Photoshop software (Adobe Systems, Mountain View, CA) by autolevel and autocontrast.
Urate Uptake Inhibitory Assay. Uptake assays were performed on the transfected cells following the method described by Anzai et al. (2004). In brief, 48 h post-transfection, the culture medium was removed and the cells were incubated in 0.4 ml of serum-free and chloride-free Hanks' balanced salt solution (HBSS) containing 125 mM sodium gluconate, 4.8 mM potassium gluconate, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM calcium gluconate, 5.6 mM glucose, and 25 mM HEPES (pH 7.4) for 10 min. Then, the HBSS was removed and replaced by another 0.18 ml of fresh HBSS. Uptake was initiated by the addition of 20 µl of HBSS containing radioactively labeled urate with or without the test compound. The final concentration of urate in the assay medium was 50 µM. The reaction mixture was allowed to stand for 1 min in the standard assay and then the assay medium was immediately removed by suction. The cells were quickly washed with 0.5 ml of prechilled HBSS twice, and then 0.2 ml of 0.1 M NaOH was added to lyse the cells. After at least 20 min, the cell lysate was quantitatively collected into a scintillation vial and the radioactivity therein was counted in a liquid scintillation counter after the addition of 5 ml of scintillant. The test compound was originally dissolved in dimethyl sulfoxide and diluted by the appropriate assay medium before the assay. The final concentration of dimethyl sulfoxide in the assay was not more than 0.2%. This concentration of dimethyl sulfoxide was determined in initial experiments to exert minimal effects on the uptake assay.
Statistical Analyses. Experimental values were expressed as mean values ± S.E.M. IC50 values were determined from the concentration dependence curves. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni's multiple comparison test to determine the level of significance. P values of <0.05 were taken as statistically significant.
The successful transcription of hURAT1 in the transfected HEK293 cells was confirmed by RT-PCR using a pair of gene-specific primers unique to hURAT1 (Fig. 2). A single band of the expected size (222 bp) could be visualized in HEK293 cells 48 h after transfection with pCMV·SPORT6 carrying the entire coding sequence of hURAT1. This transcript of consistent band intensity was observed invariably in the hURAT1-transfected cells from different experiments. On the other hand, no bands were observed in the control parental cells, which were not transfected or transfected with the empty vector pCMV·SPORT6 alone, as in the case of the negative control, in which no RNA was incorporated in the RT reaction. The results indicated that the exogenously introduced hURAT1 construct was successfully expressed in the transfected cells. Amplification of ß-actin was carried out in parallel. In all the cells, a single band was found with the expected size of 620 bp.
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) and functions as a plasma membrane protein for the uptake of urate. Radioactively labeled urate was used in urate uptake assays of the transfected cells. In a time course study, urate uptake proceeded rapidly in a linear manner within at least 1 min and then gradually leveled off (Fig. 4A). Thus, in subsequent experiments, urate uptake was determined at 1 min to measure the initial velocity of the uptake process. Urate uptake was also demonstrated to be proportional to the initial concentration of urate. At 50 µM urate, its uptake value was calculated to be 2552 ± 246 cpm per well (n = 8) for a 24-well culture plate at 1 min. This value was significantly increased by about 5-fold in HEK293 cells transfected with hURAT1 compared with the nontransfected parental cells or cells transfected with the empty vector (Fig. 4B). In subsequent uptake assays, the concentration of urate was fixed at 50 µM.
The inhibitory actions of morin on urate uptake via the human urate transporter were demonstrated using the transfected human kidney cells in culture (Fig. 5). Inhibition by probenecid, sulfinpyrazone, and benzbromarone were performed in parallel as the positive controls. At a concentration of 2 µM, morin could inhibit urate uptake into the transfected HEK293 cells by 50% (Fig. 5A). But at this concentration of the inhibitor, probenecid gave only about 30% inhibition (Fig. 5B) and sulfinpyrazone showed no significant inhibition at all (Fig. 5C), whereas benzbromarone elicited an inhibition of about 70% (Fig. 5D). None of the four compounds exhibited any significant effect on urate uptake in cells transfected with the empty vector alone. Figure 5 shows the dose-dependent inhibition of morin and the other three drugs on urate uptake in the cultured HEK293 cells bearing hURAT1. The observed 50% inhibition (IC50) values were 2.0 µM for morin, 50 µM for probenecid, 100 µM for sulfinpyrazone, and 0.3 µM for benzbromarone. Subsequent Lineweaver-Burk transformation of the uptake kinetics data revealed that morin inhibited urate uptake via hURAT1 in a competitive manner (Fig. 6). Thus, the equation Ki = Km [I]/(Kmi - Km) applies, where Ki is the inhibition constant, Km is the Michaelis constant, Kmi is the apparent Km in the presence of the inhibitor, and [I] is the concentration of the inhibitor (Kong et al., 2000). The Ki value of morin was determined to be 5.74 µM.
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Discussion |
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). Probenecid and sulfinpyrazone are the most commonly used of these drugs (Fam, 2001). In particular, uricosuric agents are the preferred drugs of choice in allopurinol-allergic patients and in urate underexcretors who have normal renal function with no history of nephrolithiasis (Fam, 2001). However, these therapeutic options are sometimes contraindicated, ineffective, toxic, or poorly tolerated. The inapplicability of probenecid in patients with renal calculi (Harris et al., 1999) and the limited application of sulfinpyrazone because of its adverse effects (Schlesinger, 2004) are examples of such limitations. Benzbromarone is a very strong uricosuric agent that can lower serum urate level and increase urinary urate excretion in normal, hyperuricemic, and gouty subjects (Heel et al., 1977). If available (as in Europe, South Africa, and Japan), benzbromarone may be used on patients with gout and mild-to-moderate renal insufficiency. However, benzbromarone can cause fulminant hepatotoxicity and this, in fact, had made it not readily available in the United States (Schlesinger, 2004) and had led to its withdrawal from the French market in 2001 (Bieber and Terkeltaub, 2004). Thus, in patients allergic to allopurinol, the limitation to the treatment of hyperuricemia is still the availability of equally effective urate-lowering alternatives.
In our laboratory, morin has been previously identified to be a potent urate-lowering agent with a dual action. This compound exhibits potent inhibition on urate uptake and moderate inhibition on xanthine oxidase (Yu et al., 2006). In a rat renal brush-border membrane vesicle system, morin inhibited urate uptake in a dose-dependent manner with a potency better than that of probenecid. This finding indicated that morin probably acts on the kidney to inhibit urate transport via an organic anion transporter that contributes to urate renal reabsorption. Thus, morin might promote urate renal excretion by inhibiting its renal reabsorption. Our subsequent animal study in an acute hyperuricemic rat model substantiated this proposed mechanism. Morin was indeed shown to exhibit an in vivo uricosuric action in rats accompanied by a lowering in serum urate level (Yu et al., 2006).
Before such favorable biological actions of this compound could be extended to human subjects, it is imperative to demonstrate that morin could also inhibit urate transport in human kidney cells, particularly in view of the diverse species variation in the renal reabsorption of urate (Dantzler, 1996; Hediger et al., 2005). The availability of a cloned human urate transporter (Enomoto et al., 2002; Anzai et al., 2004) and the successful functional expression of this human transporter on cultured human kidney cells in the present study provides a convenient cell-based system to substantiate the actions of morin as an effective uricosuric agent in humans.
Although kidney is an important therapeutic target in hyperuricemic patients (Rott and Agudelo, 2003), a full understanding of the renal handling of urate in humans is still lacking. Not only does urate transport vary among species, but there also exists a bidirectional transport across renal tubule cells. Normally, less than 5% of circulating urate is bound to plasma proteins and, thus, most of the urate can be freely filtered through the renal glomerulus (Rafey et al., 2003). More than 90% of the filtered urate is reabsorbed in adult human (Maesaka and Fishbane, 1998). Although some genes have been reported to affect renal urate handling, including the UMOD gene, which encodes for the Tam-Horsfall/uromodulin protein (Dahan et al., 2003), and the UAT gene, which encodes for the urate transporter/channel (Leal-Pinto et al., 1997), their exact biological functions are uncertain and whether they specifically regulate serum urate levels by mediating renal urate handling directly awaits further investigations. UMOD has been reported to interfere with the basolateral pathways of organic anion transport or in the more distal postsecretory reabsorption of urate (Marangella, 2005), whereas the natural function of UAT is believed to export excess urate out of cells so that the intracellular urate concentration is kept below the solubility limit to prevent the formation of urate crystals intracellularly (Leal-Pinto et al., 1997; Marangella, 2005).
Recently, the long-hypothesized urate anion transporter was identified in the human kidney. This transporter (hURAT1) is responsible for the reabsorption of urate in human from the lumen to the cytosol in cells along the renal proximal tubule but not in the distal tubule. Genetic defects of hURAT1 cause idiopathic renal hypouricemia. It is believed that this urate-anion exchanger is involved in the regulation of blood urate level by mediating urate reabsorption in the human kidney. Immunohistochemical analysis revealed that the hURAT1 protein resides in the epithelial brush-border membranes of the proximal tubules in the human kidney cortex (Enomoto et al., 2002).
Our RT-PCR data indicated the consistent transcription of hURAT1 in cultured HEK293 cells transfected with the pCMV · SPORT6 plasmid harboring the hURAT1 cDNA. Subsequent functional assays measuring urate uptake in the transfected cells demonstrated a significant increase of urate uptake in the hURAT1-expressing cells, indicating the successful expression of the exogenously introduced urate-specific transporter. To further validate this system, we have studied the subcellular localization of hURAT1 in the transfected HEK293 cells by fusing hURAT1 with EGFP in the C-terminal of hURAT1. Thus, the subcellular localization of hURAT1 could be studied by the fluorescence signal under confocal microscopy. From the microscopic imaging, we have demonstrated the different patterns of the fluorescence signal in cells expressing the fusion protein and in cells expressing EGFP alone. A sharp circle of green fluorescence signal was observed surrounding the periphery of cells expressing the fusion protein, indicating that hURAT1 was localized on the cell surface. Perinuclear localization of the expressed protein is unlikely because the discernible nucleus is a much smaller organelle inside the cell. On the other hand, a diffuse and uniform distribution of the green fluorescence signal in the entire cell was observed in cells expressing EGFP alone. The urate uptake assay performed on cells expressing the hURAT1-EGFP fusion protein also indicated a significant increase in urate uptake efficiency similar to that shown in Fig. 4, but not in cells transfected with the empty vector. These results indicated that a cell-based assay had been successfully established for the investigation of putative urate transport inhibitory agents in humans.
The validity of this transient transfection system can be appreciated at several levels. First, RT-PCR gave a band of similar intensity in our hURAT1-transfected cells from different experiments, indicating successful and consistent introduction of the exogenous transporter into the cultured cells. Second, the urate uptake values in the transfected cells (in the absence of transport inhibitors) from different experiments were essentially the same, again indicating the consistency of the transfection procedure. In fact, we have controlled the transfection conditions carefully in terms of the cell density, volume of transfection, amount of DNA used, amount of transfection agent used, and time of transfection to reach this consistency. In our urate uptake assays, experiments were performed a number of times as individual independent experiments on different days, and each assay was performed in triplicates. Our data are the statistical averages from these different experiments. The small error bars shown in the figures indicate that this transient transfection system is of sufficient reproducibility and reliability to demonstrate the inhibitory actions of the test drugs on the urate transport activities of the transfected cells.
Morin could significantly inhibit urate uptake in this human kidney cell system in a dose-dependent manner. This action of morin could not be attributed to the possible toxic effects of the compound. In fact, within the concentration range of the compound used, there was no observed toxicity on the cultured cells as indicated by initial studies of the compound by cell viability assays. Also, morin has been used on other cultured cells and no cytotoxic effects have been reported (Wu et al., 1994; Zeng et al., 1998; Mazzio et al., 2001; Ibarretxe et al., 2006). As for the three positive controls, they all showed inhibitory actions in the uptake assay. The inhibition potency of morin (IC50 = 2.0 µM) was much stronger than that of probenecid (IC50 = 50 µM) and sulfinpyrazone (IC50 = 100 µM), and weaker than that of benzbromarone (IC50 = 0.3 µM). Among the three positive controls, benzbromarone showed the strongest inhibitory action, followed by probenecid and sulfinpyrazone. In our assay, probenecid exhibits a stronger inhibitory action than sulfinpyrazone, and this appears to contradict with the better clinical efficacy of sulfinpyrazone (100-400 mg orally daily) than of probenecid (500-2000 mg orally daily) (Terkeltaub, 2003). This result is probably due to the fact that sulfinpyrazone undergoes p-hydroxylation in the human body to form a uricosuric metabolite that contributes to the drug's prolonged activity and potency relative to probenecid (Craig and Stitzel, 2004). Benzbromarone is such a potent uricosuric agent that there has been a revival of this drug in Europe since Perez-Ruiz et al. (2002) showed that it could decrease hyperuricemia with a similar or better clinical efficacy than allopurinol. This is in line with our observation that benzbromarone is the strongest urate uptake inhibitor among the three clinically used agents. The inhibitory action of morin compares very favorably with that of benzbromarone, particularly at higher doses. At 10 µM, benzbromarone inhibited about 75% of urate uptake, whereas morin at this dose gave an inhibition of about 70%. The potent action of morin in the present system is encouraging, particularly in view of the nontoxic nature of this natural compound (Wu et al., 1994; Cho et al., 2006) and the reported toxicity problems associated with the use of benzbromarone. Our results therefore strongly suggest that morin is a potential candidate worthy of further clinical evaluation, with the objective of establishing its efficacy and effective dosage range on patients. Together with the other favorable characteristics of morin of being an antioxidant (Wu et al., 1994, 1995; Kok et al., 2000) and a xanthine oxidase inhibitor (Yu et al., 2006), its clinical trials on human subjects are highly warranted.
In the present study we have focused our attention on hURAT1 because it is probably the most important urate transporter in controlling renal urate handling in humans. However, other organic anion transporters might also contribute toward control over urate level in the body. The specificity of action of morin toward these other organic anion transporters would constitute worthwhile subsequent studies to further understand the action of this compound. Again, this cell-based assay system offers such a possibility because different specific transporters can be individually introduced into the cultured cells for detailed investigations. Despite a recent report (Xie et al., 2006) indicating that morin would bind to human serum albumin, the relatively low binding affinity (11.3 µM) of morin toward albumin would not affect the validity of the conclusions drawn in the present study, particularly in view of the low concentration of the protein in the renal ultrafiltrate (Gekle, 2005).
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ABBREVIATIONS: DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; HBSS, Hanks' balanced salt solution; HEK293, human embryonic kidney cell line 293; hURAT1, human urate anion transporter 1; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s).
Address correspondence to: Professor Christopher H.K. Cheng, Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. E-mail: chkcheng{at}cuhk.edu.hk
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) or 50 µM (
) urate. Each data point is the average value ± S.E.M. from four determinations. B shows a significant increase of urate uptake into HEK293 cells transfected with hURAT1 (HEK293-hURAT1) compared with nontransfected parental cells (HEK293) or HEK293 cells transfected with the empty vector only (HEK293-pCMV·SPORT6). For HEK293, the value shown is the mean value ± S.E.M. from four determinations. For HEK293-pCMV·SPORT6 and HEK293-hURAT1, the values shown are the mean values ± S.E.M. of eight independent experiments each performed in triplicates.
