Urate Secretion Research Articles

Functional and structural regulation of urate handling by human SLC2A9 isoforms in the Xenopus laevis oocyte expression system

Human GLUT9 (SLC2A9) is a high capacity urate transporter, backed by genome‐wide association scans. This gene's SNPs coincide with urate pathology1. The protein is predominant in liver and kidney, main organs in human urate homeostasis. Two splice variants of the carrier, SLC2A9a (A) and SLC2A9b (B), which differ in their residue composition in the N‐terminus, target differently to cell membranes of polarized epithelium2. Hydrophobic residues in this family of transporters were shown to play a role in hexose selectivity3.OBJECTIVESTo investigate differences in urate transport function and selectivity by SLC2A9 variants, for uptake and efflux modes.METHODhSLC2A9 isoforms and appropriate mutants were overexpressed in X. laevis oocytes. Radiolabelled urate uptake and efflux was measured under varied conditions.PKC agonist affects (A)‐ but not (B)‐mediated urate uptake and efflux. Kinetic experiments reveal that both affinity and capacity of the transporter are altered, without changes in funcional surface expression of the protein. Elevation of intracellular cAMP altered (B)‐ but not (A)‐mediated urate uptake under specific conditions. These observations suggest that urate reabsorption (A) and urate secretion (B) may be differentially regulated in polarized epithelia. Furthermore, hydrophobic residues around exofacial vestibule may act as hexose and urate selectivity determinants.

The Increased Protein Level of URAT1 was Observed in Obesity/Metabolic Syndrome Model Mice

To elucidate the mechanism of obesity/metabolic syndrome-related hyperuricemia, this study aimed to determine the expression levels of transport systems for urate absorption (Urat1, Smct1, Glut9) and urate secretion (Abcg2). The kidneys of two obesity models in mice were used: 1) leptin-deficient mice (ob/ob mice) and 2) Quick fat diet model. 1) 8-week-old male ob/ob mice demonstrated the increased protein levels of Slc22a12 (Urat1), Slc2a9 (Glut9), and Abcg2 (Abcg2) and a decreased protein level of Slc5a8 (Smct1). However, no significant changes in the mRNA levels of these genes were observed. 2) C57BL/6 mice were fed with a Quick fat diet (crude fat content: 13.6%) from the age of 24 to 28 weeks (Quick fat diet group). The average body weights of the Quick fat diet group were heavier than those of the control group fed with a normal diet (crude fat content: 4.8%). The mRNA levels of Slc22a12, Slc2a9, Abcg2, or Slc5a8 did not change significantly in both groups. The protein levels of Slc22a12 (Urat1) and Abcg2 (Abcg2) increased significantly in the Quick fat diet group. Those of Slc2a9 (Glut9) and Slc5a8 (Smct1) were not changed significantly in the Quick fat diet group. In conclusion, the Quick fat diet enhanced the protein levels of Urat1 and Abcg2 without any changes in their mRNA transcription levels. The cause of obesity/metabolic syndrome-associated hyperuricemia appears to be associated with the urate reabsorption transporter Urat1 protein enhanced by fat.

Identification of ABCG2 Dysfunction as a Major Factor Contributing to Gout

The ATP-binding cassette, subfamily G, member 2 gene ABCG2/BCRP locates in a gout-susceptibility locus (MIM 138900) on chromosome 4q. Recent genome-wide association studies also showed that the ABCG2 gene relates to serum uric acid levels and gout. Since ABCG2 is also known as a transporter of nucleotide analogs that are structurally similar to urate, and is an exporter that has common polymorphic reduced functionality variants, ABCG2 could be a urate secretion transporter and a gene causing gout. To find candidate mutations in ABCG2, we performed a mutation analysis of the ABCG2 gene in 90 Japanese patients with hyperuricemia and found six non-synonymous mutations. Among the variants, ATP-dependent urate transport was reduced or eliminated in five variants, and two out of the five variants (Q126X and Q141K) were frequently detected in patients. Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. As Q126X and Q141K are a nonfunctional and half-functional haplotype, respectively, their genotype combinations are divided into four estimated functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those who had dysfunctional ABCG2 had an increased risk of gout, and that a remarkable risk was observed in those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10−21). In 2,150 Japanese individuals, the frequency of those with dysfunctional ABCG2 was more than 50%. Our function-based clinicogenetic analysis identified the combinations of dysfunctional variants of ABCG2 as a major contributing factor in Japanese patients with gout.

Apical Voltage-Driven Urate Efflux Transporter NPT4 in Renal Proximal Tubule

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).

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.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

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.

Morin Improves Urate Excretion and Kidney Function through Regulation of Renal Organic Ion Transporters in Hyperuricemic Mice

Morin (2′,3,4′,5,7-pentahydroxyflavone), a plant-derived flavonoid, has beneficial effects on hyperuricemia and renal dysfunction in animals. Since the decreased renal excretion of uric acid is the hallmark of hyperuricemia, here we studied the effects of oral morin administration on renal organic ion transporters in potassium oxonate-induced hyperuricemic mice. Hyperuricemia in mice was induced by potassium oxonate. Uric acid and creatinine concentrations in urine and serum, and fractional excretion of uric acid (FEUA) were performed to evaluate renal urate handling. Changes in expression levels of renal organic ion transporters were detected by Western blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) methods. Morin treatment significantly increased urinary uric acid/creatinine ratio and FEUA, resulting in reduction of serum uric acid levels in hyperuricemic mice. And kidney conditions were also improved after morin treatment in this model. Protein and mRNA levels of glucose transporter 9 (mGLUT9) and urate transporter 1 (mURAT1) were significantly decreased, and of organic anion transporter 1 (mOAT1) were remarkably increased in the kidney of morin-treated hyperuricemic mice. Morin treatment also blocked down-regulations of renal organic cation and carnitine transporters (mOCT1, mOCT2, mOCTN1 and mOCTN2) in hyperuricemic mice. These results suggest that morin exhibits the uricosuric effects via suppressing urate reabsorption and promoting urate secretion in the kidney of hyperuricemic mice and may help to attenuate deleterious effects of hyperuricemia with renal dysfunction.

Breast cancer resistant protein (Bcrp, Abcg2) contributes to active urate secretion in mouse and bird renal proximal tubule

Unlike most mammals, higher primates, humans, and birds lack uricase and must rely primarily on urinary excretion for the elimination of urate, the intermediate product of purine metabolism. Health complications such as gout, kidney stones, cardiovascular disease, type II diabetes, and metabolic syndrome are associated with hyperuricemia. In the renal proximal tubule, urate is strongly reabsorbed, and the bulk of that excreted is due to active tubular secretion. Following the entry step at the peritubular membrane on organic anion transporters 1 and 3 (OAT1/3), urate is transported to the lumen by a process involving multi‐drug resistance protein 4 (Mrp4, ABCC4). Recently another ATP‐binding cassette family transporter, BCRP, was identified as a possible apical membrane urate efflux transporter. In this study, primary cultures of mouse (mPTCs) and chicken (cPTCs) renal proximal tubule cell monolayers were used to further examine the role of Bcrp in urate secretion. Unidirectional urate flux measurements across mPTCs and cPTCs mounted in Ussing chambers revealed net active urate secretion in both models, and in both, urate secretion was inhibited by luminal exposure to Fumitremorgin C (FTC), a known BCRP inhibitor. Peritubular exposure to FTC did not inhibit urate secretion in mPTCs or cPTCs. Supported by NSF.

Inhibition of AMP‐ activated kinase (AMPK) mitigates zinc‐induced inhibition of urate secretion

Plasma urate level is primarily regulated by urinary excretion, and ~70% of urate excretion is due to tubular secretion. Birds, which only secrete urate, and humans, which secrete and reabsorb, maintain similar normal urate concentrations (~300 μM). Tubular secretion includes basolateral entry via Oat1/3‐like transport and apical membrane exit via Mrp4 transport, but regulating factors in the pathway remain uncertain. In other systems, cellular stress has been shown to affect transport properties by altering both stress protein levels and transporter expression or activity. We have shown that prolonged zinc‐induced cell stress inhibited active transepithelial urate secretion by chicken renal proximal tubule epithelial cell monolayers (cPTCs) with no change in Mrp4 expression, glucose transport, or transepithelial resistance. Acute exposure of cPTCs to zinc had no effect, nor was there an effect of zinc on urate uptake by isolated brush border membrane vesicles, suggesting involvement of a cellular stress adaptation. AMPK, which responds to cellular stress by shutting down non‐vital ATP‐utilizing processes, may be linked to this stress adaptation. Activation of AMPK by AICAR decreased urate secretion by cPTCs similar to the effect seen with zinc. The AMPK inhibitor, Compound C, prevented both AICAR and zinc inhibition of urate secretion. Supported by NSF.

Common Defects of ABCG2, a High-Capacity Urate Exporter, Cause Gout: A Function-Based Genetic Analysis in a Japanese Population

Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout

Genome-wide association studies (GWAS) have successfully identified common single nucleotide polymorphisms (SNPs) associated with a wide variety of complex diseases, but do not address gene function or establish causality of disease-associated SNPs. We recently used GWAS to identify SNPs in a genomic region on chromosome 4 that associate with serum urate levels and gout, a consequence of elevated urate levels. Here we show using functional assays that human ATP-binding cassette, subfamily G, 2 (ABCG2), encoded by the ABCG2 gene contained in this region, is a hitherto unknown urate efflux transporter. We further show that native ABCG2 is located in the brush border membrane of kidney proximal tubule cells, where it mediates renal urate secretion. Introduction of the mutation Q141K encoded by the common SNP rs2231142 by site-directed mutagenesis resulted in 53% reduced urate transport rates compared to wild-type ABCG2 (P < 0.001). Data from a population-based study of 14,783 individuals support rs2231142 as the causal variant in the region and show highly significant associations with urate levels [whites: P = 10(-30), minor allele frequency (MAF) 0.11; blacks P = 10(-4), MAF 0.03] and gout (adjusted odds ratio 1.68 per risk allele, both races). Our data indicate that at least 10% of all gout cases in whites are attributable to this causal variant. With approximately 3 million US individuals suffering from often insufficiently treated gout, ABCG2 represents an attractive drug target. Our study completes the chain of evidence from association to causation and supports the common disease-common variant hypothesis in the etiology of gout.

Renal proximal tubule secretion of urate is regulated by a stress‐induced pathway

Urate is an important antioxidant at normal plasma concentrations (~ 300 µM) in both birds and humans, and is the major nitrogenous waste in birds. Plasma urate concentrations are primarily regulated by urinary excretion, and ~70% of renal urate excretion is due to tubular secretion. The steps in tubular secretion include basolateral entry via Oat1/3‐like transport and apical membrane exit through Mrp4 transport. Factors that regulate this pathway remain uncertain. Cellular stress has been shown to affect transport properties in other systems by altering both stress protein levels and transporter expression or activity. Here, the effect of zinc‐induced cell stress on active transepithelial urate secretion by chicken renal proximal tubule epithelial cell monolayers was determined. Direct measure of net secretion following a six‐hour conditioning stress with 250 µM ZnCl2, then 1.5 hour zinc‐free recovery, inhibited urate transport with no change in Mrp4 expression, as shown by fluorescence immunohistochemistry and real‐time PCR, and no change in glucose transport or transepithelial resistance. Acute exposure to zinc had no effect suggesting involvement of a cellular adaptation to stress. Preliminary data show an AMP kinase activator (AICAR, 0.1 mM) strongly inhibited urate secretion. We tentatively conclude that cellular stress may inhibit active renal urate secretion. Supported by NSF.

A sugar transporter regulates serum urate levels: Implications for prevention and management of hyperuricemia in gout

Introduction: Gout is increasing in prevalence in the United States. More patients with refractory tophaceous disease are being seen, including older patients and those with multiple comorbidities such as diabetes, hypertension, chronic kidney disease, and congestive heart failure. There is also growing but as yet unresolved evidence for the potential of hyperuricemia to directly impact on blood pressure and cardiovascular diseases. New treatments, such as febuxostat and pegloticase, are emerging for refractory hyperuricemia. However, in most gout patients hyperuricemia is caused by long-standing renal underexcretion of uric acid, an abnormality that is often precipitated or exacerbated by diuretic therapy, insulin resistance, hypertension, and development of even moderate degrees of renal insuffi ciency [1]. This scenario points to opportunities to intervene at the level of both prophylaxis and treatment of hyperuricemia by more advanced uricosuric therapies. This will be aided by recent clinical-translational advances in understanding renal uric acid disposition. The primary uricosuric drugs (probenecid, benzbromarone, sulfi npyrazone) and the weaker secondary uricosuric drug (losartan) work in large part by inhibiting the urate anion exchanger URAT1 (SLC22A12) at the apical (brush border) membrane of the renal proximal tubule epithelial cell [2]. Furthermore, several URAT1 single nucleotide polymorphisms (SNPs) were linked with prominent regulatory effects on serum urate and susceptibility to gout in Asian and European cohorts [2]. Although URAT1 appears to be the linchpin for urate reabsorption at the apical surface of the renal proximal tubule epithelial cell, URAT1 knockout mice showed an unexpectedly small loss in renal urate reabsorption [3]. Secondary changes in renal urate secretion were believed to be partly responsible. SLC2A9 (also known as GLUT9, URATv1) promotes transport of hexose sugars and is a potent electrogenic transporter of urate anion [4]. Recently, probenecid and benzbromarone (and also indomethacin) were discovered to inhibit the activity of SLC2A9 at the basolateral membrane of the proximal renal tubule epithelial cell [4], the interface where urate is reabsorbed into the peritubular interstitium and ultimately back into the venous circulation. Not only URAT1, but also SLC2A9 mutations, have now been implicated in the etiology of the uncommon disorder idiopathic renal hypouricemia. Furthermore, SLC2A9 mutations were linked to hyperuricemia and bladder uric acid calculi in dalmatian-breed dogs [5]. This was a stunning observation that indicates how robust the role for SLC2A9 can be in renal urate handling, as dogs, like other lower mammals, are generally protected from hyperuricemia via uricase expression. More recently, several SLC2A9 SNPs have been linked by genome-wide association (GWA) studies with hyperuricemia, altered renal uric handling, and susceptibility to gout [6–9].

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.

Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture

Birds are uricotelic, and because they excrete urate by renal tubular secretion, they provide a convenient model for examination of this process. Primary monolayer cultures of the isolated renal proximal tubule epithelium from the domestic chicken, Gallus gallus L., were mounted in Ussing chambers where several substrates/inhibitors of renal organic anion transporters were tested for the sidedness and specificity of their effects on transepithelial urate transport. Transepithelial electrical resistance, electrical potential and sodium-dependent glucose current were monitored to detect nonspecific effects. Under control short-circuited conditions the ratio of unidirectional fluxes of [(14)C]urate was found to be 3:1. Active net secretion was specifically inhibited by 1 mmol l(-1) probenecid and 10 mmol l(-1) para-aminohippuric acid (PAH). Bromocresol Green, cimetidine, nocodozole, cytochalasin D and ouabain also inhibited secretion but were toxic. Interstitial-side lithium (5 mmol l(-1)) and glutarate (1 mmol l(-1)) specifically blocked transport, but 10-100 micromol l(-1) glutarate had no effect. Interstitial estrone sulfate (ES) stimulated urate secretion at 10 micromol l(-1) but was inhibitory at 500 micromol l(-1). Active PAH secretion (5:1 flux ratio) was inhibited 34% by 330 micromol l(-1) urate. ES (500 micromol l(-1)) blocked the remainder. From the lumen side, glucose-free, Cl(-)-free and high K(+) (30 mmol l(-1)) solutions, or an alkaline pH of 7.7 had no effect on urate transport and neither did several compounds known to be uricosuric. Lumen-side methotrexate (500 micromol l(-1)) and MK571 (20 micromol l(-1)) strongly inhibited urate secretion. MK571 had no effect from the interstitial side. RT-PCR revealed mRNA for OAT1-, OAT3-, MRP2- and MRP4-like organic anion transporters in chicken proximal epithelium.

Challenges and intriguing problems in comparative renal physiology

The comparative approach has proved important many times in understanding renal function and continues to offer possible approaches to unsolved problems today, in three general areas. (1) Quantification of glomerular ultrafiltration. In contrast to the complex capillary network in the mammalian glomerulus, the glomerulus of the superficial loopless (reptilian-type) avian nephrons consists of a single capillary loop. This structure, in an avian species where it can be approached directly, should for the first time permit accurate determinations of the pressure profiles and the capillary area involved in glomerular ultrafiltration in an animal with high arterial pressure. (2) Fluid reabsorption by proximal renal tubules. In some reptilian proximal renal tubules, isolated and perfused in vitro, isosmotic fluid reabsorption can occur at control rates when lithium replaces sodium or when some other substance replaces sodium or chloride or both in the perfusate and bathing medium simultaneously. Reabsorption at the control rates, regardless of the composition of the perfusate and bathing medium, can be at least partially inhibited by cold and cyanide, but not by blockers of Na(+)-K(+)-ATPase. It is also independent of the buffer system used, but it is reduced about 20% by removal of colloid from the peritubular fluid. During the substitutions, the surface area of the proximal tubule cells increases dramatically and might permit some insignificant force to be more effective in the reabsorptive process. Understanding the process involved in this, apparently unique coupling of solute and fluid transport, certainly would be very valuable in understanding coupled transport of solutes and water across epithelia in general. (3) Urate secretion by proximal renal tubules. Urate is the major excretory end product of nitrogen metabolism in birds, most reptiles, and a few amphibians. It undergoes net secretion by the renal tubules. It has been possible to learn much about the physiology of net secretion in reptiles and birds and this process appears to be similar to the much less significant secretory flux in some mammals. However, nothing is known about the molecular basis of the transport system and understanding these processes may provide important information for mammals as well as reptiles and birds. These are some examples of intriguing possibilities for comparative studies of renal physiology.

Uric acid transport.

The goal of this article is to review the physiology and describe newly defined molecular mechanisms that are responsible for renal urate transport. Four complementary DNAs have recently been cloned whose expressed proteins transport urate. Two of these proteins have been localized to the apical membrane of proximal tubular cells: one, a urate transporter/channel, a galectin, is an electrogenic transporter (an ion channel); the second is a urate-anion electroneutral exchanger, a member of the organic anion transporter family. The other urate transport proteins, organic anion transporters 1 and 3, are also members of the organic anion transporter family. These proteins have been localized to the basolateral membrane of proximal tubular cells: organic anion transporter 1 is an electroneutral organic anion exchanger; the mechanism of urate transport on organic anion transporter 3 remains to be determined. The molecular definition and localization of four urate transport proteins provides a basis for developing a molecular model of the bi-directional transport of urate in renal proximal tubules. It seems likely that the urate-anion exchanger is responsible for luminal reabsorption while the urate transporter/channel permits secretion of urate from the cell into the lumen. Since organic anion transporters 1 and 3 reside in the basolateral membrane, one or both may be relevant in the reabsorptive flux of urate into the peritubular capillary as well as in the cellular uptake of urate from the peritubular space, the first step in the process of urate secretion. Knowledge of the molecular basis of urate transport should provide greater insights into states of altered transport as well as assist in development of drugs to modify urate flux.

Human Organic Anion Transporter 3 (hOAT3) can Operate as an Exchanger and Mediate Secretory Urate Flux

Background/Aims: Renal secretion of organic anions is critically dependent on their basolateral uptake against the electrochemical gradient. Due to their localization, two transporters are likely involved, namely OAT1 and OAT3. While OAT1 as an exchanger clearly operates in the secretory direction, OAT3 in its previously supposed mode as a uniporter should move anionic substrates from cell to blood. It would thus dissipate gradients established by OAT1 of common OAT1/OAT3 substrates. In the present study we therefore reinvestigated the driving forces of human OAT3. Methods: The human OAT3 obtained from the Resource Center/Primary Database was made functional by site-directed mutagenesis. Using the Xenopus laevis oocyte expression system, hOAT3-mediated transport of estrone sulfate (ES) and dicarboxylates was assayed for cis-inhibition and/or trans-stimulation in both the uptake and efflux direction. Results: hOAT3-mediated efflux of glutarate (GA), can be significantly trans-stimulated by a variety of ions with high cis-inhibitory potency, including GA (282%), α-ketoglutarate (476%), p-aminohippurate (179%), and, most notably, urate (167%). Urate cis-inhibited ES uptake with an IC₅₀ close to normal serum urate concentrations. Conclusion: These data indicate that OAT3 does not represent a uniporter but operates as an organic ion%dicarboxylate exchanger similar to OAT1, and may mediate renal urate secretion.

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.

Facilitated diffusion of urate in avian brush-border membrane vesicles.

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward alpha-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.