Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

The effect of female hormones upon urate transport systems in the mouse kidney

Increased levels of serum urate in postmenopausal women are thought to be caused by a change in renal urate elimination associated with the loss of female hormones. In this study, we investigated the regulation of renal urate transporter expression by female hormones using ovariectomized mice with or without hormone replacement. Estradiol suppressed the protein levels of urate reabsorptive transporters urate transporter 1 and glucose transporter 9 (Urat1 and Glut9), and that of urate efflux transporter ATP-binding cassette sub-family G member 2 (Abcg2). Progesterone suppressed protein levels of sodium-coupled monocarboxylate transporter 1 (Smct1). However, neither estradiol nor progesterone influenced the respective levels of mRNA.     

Functional analysis of phenolsulfonphthalein transport system in Long-Evans Cinnamon rats

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.     

Functional analysis of phenolsulfonphthalein transport system in Long-Evans Cinnamon rats

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.     

The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.     

Carrier-mediated transport of urate by chicken (Gallus domesticus) renal brush-border membrane vesicles

1. Urate is transported by an anion exchange system in chicken renal brush-border membrane vesicles. An outward chloride gradient stimulates an overshoot in urate uptake. 2. The anion exchanger is similar to those described for the kidneys of rats and dogs except that the chicken exchanger has a higher specificity for urate. 3. p-Aminohippurate has only a weak affinity, if any, for the urate exchanger. 4. There were no apparent qualitative differences in urate transport between brush-border membrane vesicles from genetically normouricemic and hyperuricemic chickens.     

Galectin 9 is the sugar-regulated urate transporter/channel UAT

UAT, also designated galectin 9, is a multifunctional protein that can function as a urate channel/transporter, a regulator of thymocyte-epithelial cell interactions, a tumor antigen, an eosinophil chemotactic factor, and a mediator of apoptosis. We review the evidence that UAT is a transmembrane protein that transports urate, describe our molecular model for this protein, and discuss the evidence from epitope tag and lipid bilayer studies that support this model of the transporter. The properties of recombinant UAT are compared with those of urate transport into membrane vesicles derived from proximal tubule cells in rat kidney cortex. In addition, we review channel functions predicted by our molecular model that resulted in the novel finding that the urate channel activity is regulated by sugars and adenosine. Finally, the presence and possible functions of at least 4 isoforms of UAT and a closely related gene hUAT2 are discussed.     

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.     

ABCG2 Dysfunction Increases Serum Uric Acid by Decreased Intestinal Urate Excretion

ATP-binding cassette transporter G2 (ABCG2), also known as breast cancer resistance protein (BCRP), is identified as a high-capacity urate exporter and its dysfunction has an association with serum uric acid (SUA) levels and gout/hyperuricemia risk. However, pathophysiologically important pathway(s) responsible for the ABCG2-mediated urate excretion were unknown. In this study, we investigated how ABCG2 dysfunction affected the urate excretion pathways. First, we revealed that mouse Abcg2 mediates urate transport using the membrane vesicle system. The export process by mouse Abcg2 was ATP-dependent and not saturable under the physiological concentration of urate. Then, we characterized the excretion of urate into urine, bile, and intestinal lumen using in vivo mouse model. SUA of Abcg2-knockout mice was significantly higher than that of control mice. Under this condition, the renal urate excretion was increased in Abcg2-knockout mice, whereas the urate excretion from the intestine was decreased to less than a half. Biliary urate excretion showed no significant difference regardless of Abcg2 genotype. From these results, we estimated the relative contribution of each pathway to total urate excretion; in wild-type mice, the renal excretion pathway contributes approximately two-thirds, the intestinal excretion pathway contributes one-third of the total urate excretion, and the urate excretion into bile is minor. Decreased intestinal excretion could account for the increased SUA of Abcg2-knockout mice. Thus, ABCG2 is suggested to have an important role in extra-renal urate excretion, especially in intestinal excretion. Accordingly, increased SUA in patients with ABCG2 dysfunction could be explained by the decreased excretion of urate from the intestine.     

Urate Transport Across the Apical Membrane of Renal Proximal Tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or “urate transportsome,” that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Galectin 9 is the sugar-regulated urate transporter/channel UAT

UAT, also designated galectin 9, is a multifunctional protein that can function as a urate channel/transporter, a regulator of thymocyte-epithelial cell interactions, a tumor antigen, an eosinophil chemotactic factor, and a mediator of apoptosis. We review the evidence that UAT is a transmembrane protein that transports urate, describe our molecular model for this protein, and discuss the evidence from epitope tag and lipid bilayer studies that support this model of the transporter. The properties of recombinant UAT are compared with those of urate transport into membrane vesicles derived from proximal tubule cells in rat kidney cortex. In addition, we review channel functions predicted by our molecular model that resulted in the novel finding that the urate channel activity is regulated by sugars and adenosine. Finally, the presence and possible functions of at least 4 isoforms of UAT and a closely related gene hUAT2 are discussed. Published in 2004.     

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.     

ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

ABCG2 is a High-Capacity Urate Transporter and its Genetic Impairment Increases Serum Uric Acid Levels in Humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3′-azido-3′-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10^?5). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.     

Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase

Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion.     

Expression,Purification, and Structural Insights for the Human Uric Acid Transporter,GLUT9, Using the Xenopus laevis Oocytes System

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.     

Expression of a human NPT1/SLC17A1 missense variant which increases urate export

ABSTRACT

Human sodium-dependent phosphate cotransporter type 1 (NPT1/SLC17A1) is one of the urate transporters in the kidney. Our recent study revealed that a common missense variant, I269T (rs1165196), of NPT1 decreases the risk of renal underexcretion gout. Moreover, we demonstrated that human NPT1 is localized to the apical membrane of the renal proximal tubule, and that I269T is the gain-of-function variant which increases the NPT1-mediated urate export. However, the mechanism by which I269T variant increases the urate export remains to be clarified. Thus, we performed immunostaining and functional analysis of human NPT1 using the Xenopus oocyte expression system. For comparison of human NPT1 expression levels of oocyte membrane between 269I (wild type) and 269T (variant), immunostaining was performed with anti-human NPT1 antibodies. As a result, we showed that NPT1 I269T variant did not change the human NPT1 membrane expression levels, although NPT1 I269T variant increased the urate transport compared with NPT1 wild type. Combined with the previous report that I269T variant did not induce Km changes but increased the Vmax of urate transport in a proteoliposome system, our findings suggest that I269T variant increases NPT1-mediated urate export without increase of NPT1 expression levels on the membrane. Thus, I269T, a common missense variant of NPT1, might have faster conformation changes than NPT1 wild type in terms of the alternating-access model of transporters, and increases renal urate export in humans.     

Urate/alpha-ketoglutarate exchange in avian basolateral membrane vesicles

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/alpha-ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [(14)C]urate and [alpha-(3)H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of alpha-ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of alpha-ketoglutarate but not in its absence, suggesting urate/alpha-ketoglutarate exchange. alpha-Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of alpha-ketoglutarate. alpha-Ketoglutarate-coupled urate uptake was specific for alpha-ketoglutarate, Cl dependent, and insensitive to membrane potential. alpha-Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and alpha-ketoglutarate-coupled PAH uptake was observed. alpha-Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, alpha-ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.     

Normal urate transport into erythrocytes in familial renal hypouricemia and in the Dalmatian dog.

It has been hypothesized that humans with familial renal hypouricemia may have a generalized defect of urate transport across cell membranes due to the genetic deletion of a specific carrier, a defect similar to that reported in the Dalmatian dog. In this study the transport of urate labelled with carbon 14 by the erythrocytes of four patients with familial renal hypouricemia was identical to that of five healthy controls. The addition of hypoxanthine to the incubation medium inhibited the transport to a similar extent in the two groups of patients, demonstrating the presence of a carrier specific for urate. This carrier was also found to be present in the erythrocytes of Dalmatian and mongrel dogs. Thus, the renal anomaly causing the hypouricemia in both species is not related to a generalized deletion of a urate-transporting protein on cell membranes.     

Mechanism of neutrophil activation by an unopsonized inflammatory particulate. Monosodium urate crystals induce pertussis toxin-insensitive hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Monosodium urate crystals are believed to trigger acute inflammation via the direct stimulation of leukocytes. Unopsonized urate crystals activate neutrophil (PMN) membrane G proteins in a pertussis toxin (PT)-sensitive manner, but induce PT-insensitive cytosolic [Ca2+]i elevation. Thus, we have further defined the mechanism of PMN responsiveness to urate crystals in this study. Though urate crystals can increase membrane permeability by lytic effects, we observed elevation of PMN cytosolic [Ca2+]i in the absence of extracellular [Ca2+]i. In addition, the early, crystal-induced cytosolic [Ca2+]i transient was buffered in cells loaded with a [Ca2+]i-chelator. This suggested mobilization of internal [Ca2+]i stores, which was supported by demonstrating rapid phosphatidylinositol bisphosphate (PIP2) hydrolysis, and the formation of inositol (1,4,5) trisphosphate (as well as phosphatidic acid) in a PT-insensitive manner. Importantly, PMN activation by urate crystals was discriminatory, as evidenced by the absence of phosphatidylinositol trisphosphate formation, a PT-sensitive event triggered by chemotactic factors. Urate crystal-induced PIP2 hydrolysis was not a nonspecific consequence of the early cytosolic [Ca2+]i transient itself, and it did not require phagocytosis. However, crystal-induced O2- release was markedly inhibited by buffering of the early cytosolic [Ca2+]i transient under conditions where crystal phagocytosis and PMA-induced O2- release were unaffected. We conclude that urate crystals activate PT-insensitive PIP2 hydrolysis, resulting in IP3 generation, and early urate crystal-induced mobilization of cytosolic [Ca2+]i. This pathway appears to modulate crystal-induced O2- release.