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Promsuk Jutabha - One of the best experts on this subject based on the ideXlab platform.
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contribution of rare variants of the slc22a12 gene to the missing heritability of serum Urate levels
Genetics, 2020Co-Authors: Promsuk Jutabha, Eikan Mishima, Kazuharu Misawa, Yosuke Kawai, Kaname Kojima, Motoshi Ouchi, Takanori Hasegawa, Masafumi MatsuoAbstract:Gout is a common arthritis caused by monosodium Urate crystals. The heritability of serum Urate levels is estimated to be 30‒70%; however, common genetic variants account for only 7.9% of the variance in serum Urate levels. This discrepancy is an example of "missing heritability." The "missing heritability" suggests that variants associated with uric acid levels are yet to be found. By using genomic sequences of the ToMMo cohort, we identified rare variants of the SLC22A12 gene that affect the Urate transport activity of URAT1. URAT1 is a transporter protein encoded by the SLC22A12 gene. We grouped the participants with variants affecting Urate uptake by URAT1 and analyzed the variance of serum Urate levels. The results showed that the heritability explained by the SLC22A12 variants of men and women exceeds 10%, suggesting that rare variants underlie a substantial portion of the "missing heritability" of serum Urate levels.
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Characterization of Urate Transport System in JAR and JEG-3 Cells, Human Trophoblast-derived Cell Lines
獨協医学会, 2017Co-Authors: Kaori Kiuchi, Promsuk Jutabha, Motoshi Ouchi, Naoyuki Otani, Asuka Morita, Masakatsu Nohara, Ichio Fukasawa, Tomoe Fujita, Naohiko AnzaiAbstract:Urate (uric acid) is the major inert end product of purine metabolism in humans. Since it is water soluble, it requires a membranous protein called transporter for its permeation across the plasma membrane. Increased blood Urate level is often seen in preeclampsia, but its precise mechanism remains unknown. Syncytiotrophoblasts function as a barrier between maternal blood and fetal one so called “blood-placental barrier”. So far, the expression of several Urate transporters was identified in these cells, but it is still unclear about their contribution to Urate handling in blood-placental barrier. In this study, we investigated the expression of Urate transporters and the properties of ［14C］Urate transport in both JAR and JEG-3, human choriocarcinoma cells as a model of human placenta. Conventional PCR analysis revealed that both JAR and JEG-3 cells express strongly breast cancer resistance protein (BCRP/ABCG2) mRNA. Uptake of ［14C］Urate by these cells is time-dependent with Na＋- and Cl－-independent and voltage-insensitive manner and is not inhibited by benzbromarone, a representative renal Urate transport inhibitor. Then, we focused on BCRP which shows strong mRNA expression and found that these cells have Urate efflux property that is sensitive to fumitremorgin C (FMC), a BCRP inhibitor. These results suggest that BCRP is one of the important components for Urate handling in human placenta in pathophysiological condition such as preeclampsia
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Molecular Mechanism of the Urate-lowering Effects of Calcium Channel Blockers
2016Co-Authors: Go Tsuchiya, Promsuk Jutabha, Motoshi Ouchi, Naoyuki Otani, Takayuki Hori, Nobuyuki Onizawa, Sawako Tanaka-nakadate, Tatou Iseki, Keitaro Hayashi, Toru ObaAbstract:Hyperuricemia has recently been recognized as one of the risk factors for cardiovascular diseases. Some calcium channel blockers(CCBs), commonly used in the treatment of hypertension, have been reported to decrease serum Urate level. Here, we tried to elucidate the molecular mechanism of the Urate-lowering effects of CCBs. We performed [^C]Urate uptake in cells stably expressing human Urate transporter 1, a major contributor of renal Urate reabsorption and a major target of uricosuric drugs such as benzbromarone and losartan(HEK-URAT1), together with mock(HEK-mock)cells to analyze the uricosuric action of CCBs. We also measured the activity of human xanthine oxidase(XO)to determine whether CCBs have inhibitory effects on Urate production. The CCBs tested were nifedipine, nilvadipine, nitrendipine, benidipine, nisoldipine, nicardipine, efonidipine, amlodipine, azelnidipine, verapamil and diltiazem. We found for the first time that at least seven CCBs in the dihydropyridine subgroup interacted with URAT1-mediated Urate uptake in HEK-URAT1 cells. Among these CCBs, nifedipine, nilvadipine and nitrendipine strongly inhibited URAT1-mediated Urate uptake. Their IC_s were 15.8, 0.018 and 0.40?μM, respectively. In contrast, Urate production mediated by XO was weakly inhibited by nifedipine and nisoldipine. In summary, URAT1 interacted with various CCBs differently, whereas XO, a major enzyme for Urate production in the liver, did not interact with most of CCBs. Although CCBs were not excreted from the urine basically, their Urate-lowering effects may be associated with the inhibition of renal Urate reabsorption mediated by renal Urate transporters such as URAT1 with their metabolites, and the results for structure-activity information in this study will provide a clue for developing new uricosuric drugs targeting URAT1
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apical voltage driven Urate efflux transporter npt4 in renal proximal tubule
Nucleosides Nucleotides & Nucleic Acids, 2011Co-Authors: Promsuk Jutabha, Hitoshi Endou, Naohiko Anzai, Michael F Wempe, Shin Wakui, Hiroyuki SakuraiAbstract:Uric acid (Urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered Urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of Urate into allantoin, results in a higher plasma Urate concentration in humans compared to other mammals. A major determinant of plasma Urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal Urate absorption in proximal tubular epithelial cells occurs mainly via apical Urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven Urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal Urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a Urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net Urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with Urate underexcretion abolished Urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).
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increased expression of slc2a9 decreases Urate excretion from the kidney
Nucleosides Nucleotides & Nucleic Acids, 2011Co-Authors: Toru Kimura, Promsuk Jutabha, Kimiyoshi Ichida, Naohiko Anzai, Ai Tsukada, Toshiyuki Fukutomi, Sirirat Amonpatumrat, Thanapol Thammapratip, Hiroyuki SakuraiAbstract:Urate is the final metabolite of purine in humans. Renal Urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a Urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven Urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal Urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on Urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma Urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wil...
Ruth G. Abramson - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and functional reconstitution of a Urate transporter channel
Journal of Biological Chemistry, 1997Co-Authors: Edgar Lealpinto, Wenjing Tao, Jay Rappaport, Max Richardson, Barbara A. Knorr, Ruth G. AbramsonAbstract:Maintenance of Urate homeostasis requires Urate efflux from Urate-producing cells with subsequent renal and gastrointestinal excretion. The molecular basis for Urate transport, however, has not been identified. A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (Urate transporter), has been cloned from a rat renal cDNA library by antibody screening. UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues. Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective Urate transporter/channel in planar lipid bilayers. The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked Urate channel activity. Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for Urate flux across cell membranes, allowing Urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal Urate excretion.
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classical and channel like Urate transporters in rabbit renal brush border membranes
Kidney International, 1994Co-Authors: Barbara A. Knorr, Jeanne C Beck, Ruth G. AbramsonAbstract:Classical and channel-like Urate transporters in rabbit renal brush border membranes. The precise mechanism by which Urate is transported across rabbit renal proximal tubule luminal membranes has not been defined. To determine whether Urate flux across this membrane represents simple diffusion or transport on specific carriers, Urate uptake was examined in brush border membrane vesicles that were prepared by a Mg ++ -aggregation technique and then exposed to CuCl 2 . Na + -independent, voltage sensitive Urate transport was demonstrated in these Cu ++ -exposed vesicles. Transport was trans-stimulated by Urate and cis inhibited by pyrazinoic acid and oxonate. A small fraction of transported Urate and Urate in the extravesicular fluid was oxidized to allantoin. Kinetic analysis revealed the presence of two kinetically distinct transporters; a channel-like carrier that was inhibited by pyrazinoic acid and oxonate, and a high-affinity, classical, saturable carrier that was inhibited by higher concentrations of oxonate. These studies provide the first direct evidence for carrier-mediated Urate transport in rabbit renal brush-border membranes and demonstrate that the rabbit transporter(s) share a number of properties with the Urate uniporter in rat proximal tubule cell membranes.
Chris I. Cheeseman - One of the best experts on this subject based on the ideXlab platform.
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identification of key residues for Urate specific transport in human glucose transporter 9 hslc2a9
Scientific Reports, 2017Co-Authors: Wentong Long, Rashmi Panigrahi, Pankaj Panwar, Kenneth Wong, Debbie O Neill, Xingzhen Chen, Joanne M Lemieux, Chris I. CheesemanAbstract:Human glucose transporter 9 (hSLC2A9) is critical in human Urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, Urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 &SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that Urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for Urate binding. Our functional studies confirmed that N429 is a key residue for both Urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating Urate transport. Additional data from chimaeric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for Urate transport but not sufficient to allow Urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that Urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.
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critical roles of two hydrophobic residues within human glucose transporter 9 hslc2a9 in substrate selectivity and Urate transport
Journal of Biological Chemistry, 2015Co-Authors: Wentong Long, Kate Witkowska, Pankaj Panwar, Kenneth Wong, Xingzhen Chen, Joanne M Lemieux, Debbie Oneill, Chris I. CheesemanAbstract:High blood Urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates Urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity Urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports Urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for Urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for Urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with Urate during transport. Together, these studies confirm that hSLC2A9 transports both Urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates Urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes.
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Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of Urate with different characteristics in the presence of hexoses.
American Journal of Physiology-renal Physiology, 2012Co-Authors: Kate Witkowska, Kyla M. Smith, Amy M. L. Ng, Debbie O'neill, Edward Karpinski, James D. Young, Chris I. CheesemanAbstract:Human SLC2A9 (GLUT9) is a novel high-capacity Urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of Urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of Urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na+ transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl−, but there was no evidence for exchange of the two anions. [14C]Urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in Urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to Urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of Urate suggest that these transporters play key roles in the regulation of plasma Urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.
Naohiko Anzai - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Urate Transport System in JAR and JEG-3 Cells, Human Trophoblast-derived Cell Lines
獨協医学会, 2017Co-Authors: Kaori Kiuchi, Promsuk Jutabha, Motoshi Ouchi, Naoyuki Otani, Asuka Morita, Masakatsu Nohara, Ichio Fukasawa, Tomoe Fujita, Naohiko AnzaiAbstract:Urate (uric acid) is the major inert end product of purine metabolism in humans. Since it is water soluble, it requires a membranous protein called transporter for its permeation across the plasma membrane. Increased blood Urate level is often seen in preeclampsia, but its precise mechanism remains unknown. Syncytiotrophoblasts function as a barrier between maternal blood and fetal one so called “blood-placental barrier”. So far, the expression of several Urate transporters was identified in these cells, but it is still unclear about their contribution to Urate handling in blood-placental barrier. In this study, we investigated the expression of Urate transporters and the properties of ［14C］Urate transport in both JAR and JEG-3, human choriocarcinoma cells as a model of human placenta. Conventional PCR analysis revealed that both JAR and JEG-3 cells express strongly breast cancer resistance protein (BCRP/ABCG2) mRNA. Uptake of ［14C］Urate by these cells is time-dependent with Na＋- and Cl－-independent and voltage-insensitive manner and is not inhibited by benzbromarone, a representative renal Urate transport inhibitor. Then, we focused on BCRP which shows strong mRNA expression and found that these cells have Urate efflux property that is sensitive to fumitremorgin C (FMC), a BCRP inhibitor. These results suggest that BCRP is one of the important components for Urate handling in human placenta in pathophysiological condition such as preeclampsia
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functional cooperation of urat1 slc22a12 and uratv1 slc2a9 in renal reabsorption of Urate
Nephrology Dialysis Transplantation, 2013Co-Authors: Takeo Nakanishi, Naohiko Anzai, Kouhei Ohya, Sho Shimada, Ikumi TamaiAbstract:BACKGROUND: Serum Urate (SUA) level is affected by alteration in urinary reabsorption caused by clinically important drugs; however, there are no experimental models suitable to assess their effect on renal reabsorption. We, therefore, aimed to establish an experimental system co-expressing the Urate transporters URAT1 (SLC22A12) and URATv1 (SLC2A9) (designated UUv cells) at the apical and basolateral membranes, respectively. METHODS: Apical uptake and vectorial transport of [(14)C]Urate in the apical-to-basolateral direction in UUv cells were measured in the presence or absence of uricosuric benzbromarone or anti-uricosuric trans-stimulators. RESULTS: The Urate permeability in the apical-to-basolateral direction remarkably increased by 7.0-fold in UUv cells, compared with non-transfected mock cells. The apical-to-basolateral transport was cis-inhibited by benzbromarone, but trans-stimulated by pyrazinecarboxylic acid and monocarboxylates such as nicotinate and lactate. Furthermore, salicylate showed both trans-stimulation and cis-inhibition in the Urate transport at low and high concentrations, respectively. Finally, coexpression of URAT1 and URATv1 in human kidney epithelial cells was exhibited immunohistochemically. CONCLUSIONS: It is demonstrated that functional cooperation of URAT1 and URATv1 is essential for renal reabsorption of Urate, and in the established system influence of drugs on SUA is reflected in the alteration of Urate permeability across the renal tubular epithelial cells.
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apical voltage driven Urate efflux transporter npt4 in renal proximal tubule
Nucleosides Nucleotides & Nucleic Acids, 2011Co-Authors: Promsuk Jutabha, Hitoshi Endou, Naohiko Anzai, Michael F Wempe, Shin Wakui, Hiroyuki SakuraiAbstract:Uric acid (Urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered Urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of Urate into allantoin, results in a higher plasma Urate concentration in humans compared to other mammals. A major determinant of plasma Urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal Urate absorption in proximal tubular epithelial cells occurs mainly via apical Urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven Urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal Urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a Urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net Urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with Urate underexcretion abolished Urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).
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increased expression of slc2a9 decreases Urate excretion from the kidney
Nucleosides Nucleotides & Nucleic Acids, 2011Co-Authors: Toru Kimura, Promsuk Jutabha, Kimiyoshi Ichida, Naohiko Anzai, Ai Tsukada, Toshiyuki Fukutomi, Sirirat Amonpatumrat, Thanapol Thammapratip, Hiroyuki SakuraiAbstract:Urate is the final metabolite of purine in humans. Renal Urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a Urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven Urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal Urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on Urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma Urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wil...
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human sodium phosphate transporter 4 hnpt4 slc17a3 as a common renal secretory pathway for drugs and Urate
Journal of Biological Chemistry, 2010Co-Authors: Promsuk Jutabha, Naohiko Anzai, Toru Kimura, Kenichiro Kitamura, Atsuo Taniguchi, Shuji Kaneko, Hideomi Yamada, Hidetaka Shimada, Tomohisa Katada, Toshiyuki FukutomiAbstract:The evolutionary loss of hepatic Urate oxidase (uricase) has resulted in humans with elevated serum uric acid (Urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum Urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum Urate levels are largely determined by Urate reabsorption and secretion in the kidney. Renal Urate reabsorption is controlled via two proximal tubular Urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal Urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven Urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced Urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of Urate secretion in the renal tubular cell, where intracellular Urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.
Barbara A. Knorr - One of the best experts on this subject based on the ideXlab platform.
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molecular cloning and functional reconstitution of a Urate transporter channel
Journal of Biological Chemistry, 1997Co-Authors: Edgar Lealpinto, Wenjing Tao, Jay Rappaport, Max Richardson, Barbara A. Knorr, Ruth G. AbramsonAbstract:Maintenance of Urate homeostasis requires Urate efflux from Urate-producing cells with subsequent renal and gastrointestinal excretion. The molecular basis for Urate transport, however, has not been identified. A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (Urate transporter), has been cloned from a rat renal cDNA library by antibody screening. UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues. Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective Urate transporter/channel in planar lipid bilayers. The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked Urate channel activity. Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for Urate flux across cell membranes, allowing Urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal Urate excretion.
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classical and channel like Urate transporters in rabbit renal brush border membranes
Kidney International, 1994Co-Authors: Barbara A. Knorr, Jeanne C Beck, Ruth G. AbramsonAbstract:Classical and channel-like Urate transporters in rabbit renal brush border membranes. The precise mechanism by which Urate is transported across rabbit renal proximal tubule luminal membranes has not been defined. To determine whether Urate flux across this membrane represents simple diffusion or transport on specific carriers, Urate uptake was examined in brush border membrane vesicles that were prepared by a Mg ++ -aggregation technique and then exposed to CuCl 2 . Na + -independent, voltage sensitive Urate transport was demonstrated in these Cu ++ -exposed vesicles. Transport was trans-stimulated by Urate and cis inhibited by pyrazinoic acid and oxonate. A small fraction of transported Urate and Urate in the extravesicular fluid was oxidized to allantoin. Kinetic analysis revealed the presence of two kinetically distinct transporters; a channel-like carrier that was inhibited by pyrazinoic acid and oxonate, and a high-affinity, classical, saturable carrier that was inhibited by higher concentrations of oxonate. These studies provide the first direct evidence for carrier-mediated Urate transport in rabbit renal brush-border membranes and demonstrate that the rabbit transporter(s) share a number of properties with the Urate uniporter in rat proximal tubule cell membranes.