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).     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na⁺-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na⁺-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

slc26a3 (dra)-deficient mice display chloride-losing diarrhea, enhanced colonic proliferation, and distinct up-regulation of ion transporters in the colon

Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.     

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.     

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).     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Increased expression of SLC2A9 decreases urate excretion from the kidney

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 wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

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 wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3--buffered Cl--free media. These findings were used to develop a turnover cycle for Cl- and HCO3- transport by slc26a3. Cl- and HCO3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl--mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl- absorption and HCO3- secretion.     

Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K(m): 365+/-42 microm). The transport was Na(+)-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; urate [corrected] is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).     

Comparative analysis of two slc11 (Nramp) loci in Takifugu rubripes

To study the evolution of the solute carrier family 11 (slc11; formerly Nramp) protein, we isolated and characterized two paralogs from the pufferfish Takifugu rubripes (Fugu). These teleost genes, designated Fugu slc11a-a and Fugu slc11a-b, comprise open reading frames of 1743 nucleotides (581 amino acids) and 1662 nt (554 aa), respectively. The proteins are 81% similar, and both exhibit signature features of the slc11 family of proteins including 12 transmembrane domains, a conserved transport motif and a glycosylated loop. Both Fugu paralogs are more Slc11a2-like based on sequence homology and phylogenetic studies. Analysis of gene environment placed both in the proximity of multiple loci syntenic to human chromosome 12q13, that is, within a SLC11A2 gene environment. However, Fugu slc11a-a also gave one match with chromosome 2q35, where human SLC11A1 resides. Functional diversification was suggested by differences in tissue distribution and subcellular localization. Fugu slc11a-a exhibits a restricted expression profile and a complex subcellular localization, including LAMP1 positive late endosomes/lysosomes in transiently transfected mouse macrophages. Fugu slc11a-b is expressed ubiquitously and localizes solely to late endosomes/lysosomes. This comparative analysis extends our understanding of the evolution and function of this important family of divalent cation transporters. [Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ496547/8/9 and AJ496550.]     

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 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.     

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.     

Mutations in Glucose Transporter 9 Gene SLC2A9 Cause Renal Hypouricemia

Renal hypouricemia is an inherited disorder characterized by impaired renal urate (uric acid) reabsorption and subsequent low serum urate levels, with severe complications such as exercise-induced acute renal failure and nephrolithiasis. We previously identified SLC22A12, also known as URAT1, as a causative gene of renal hypouricemia. However, hypouricemic patients without URAT1 mutations, as well as genome-wide association studies between urate and SLC2A9 (also called GLUT9), imply that GLUT9 could be another causative gene of renal hypouricemia. With a large human database, we identified two loss-of-function heterozygous mutations in GLUT9, which occur in the highly conserved “sugar transport proteins signatures 1/2.” Both mutations result in loss of positive charges, one of which is reported to be an important membrane topology determinant. The oocyte expression study revealed that both GLUT9 isoforms showed high urate transport activities, whereas the mutated GLUT9 isoforms markedly reduced them. Our findings, together with previous reports on GLUT9 localization, suggest that these GLUT9 mutations cause renal hypouricemia by their decreased urate reabsorption on both sides of the renal proximal tubules. These findings also enable us to propose a physiological model of the renal urate reabsorption in which GLUT9 regulates serum urate levels in humans and can be a promising therapeutic target for gout and related cardiovascular diseases.     

Pathogenic GLUT9 Mutations Causing Renal Hypouricemia Type 2 (RHUC2)

Renal hypouricemia (MIM 220150) is an inherited disorder characterized by low serum uric acid levels and has severe complications such as exercise-induced acute renal failure and urolithiasis. We have previously reported that URAT1/SLC22A12 encodes a renal urate-anion exchanger and that its mutations cause renal hypouricemia type 1 (RHUC1). With the large health-examination database of the Japan Maritime Self-Defense Force, we found two missense mutations (R198C and R380W) of GLUT9/SLC2A9 in hypouricemia patients. R198C and R380W occur in highly conserved amino acid motifs in the “sugar transport proteins signatures” that are observed in GLUT family transporters. The corresponding mutations in GLUT1 (R153C and R333W) are known to cause GLUT1 deficiency syndrome because arginine residues in this motif are reportedly important as the determinants of the membrane topology of human GLUT1. Therefore, on the basis of membrane topology, the same may be true of GLUT9. GLUT9 mutants showed markedly reduced urate transport in oocyte expression studies, which would be the result of the loss of positive charges in those conserved amino acid motifs. Together with previous reports on GLUT9 localization, our findings suggest that these GLUT9 mutations cause renal hypouricemia type 2 (RHUC2) by their decreased urate reabsorption on both sides of the renal proximal tubule cells. However, a previously reported GLUT9 mutation, P412R, was unlikely to be pathogenic. These findings also enable us to propose a physiological model of the renal urate reabsorption via GLUT9 and URAT1 and can lead to a promising therapeutic target for gout and related cardiovascular diseases.     

Pathogenic GLUT9 mutations causing renal hypouricemia type 2 (RHUC2)

Renal hypouricemia (MIM 220150) is an inherited disorder characterized by low serum uric acid levels and has severe complications such as exercise-induced acute renal failure and urolithiasis. We have previously reported that URAT1/SLC22A12 encodes a renal urate-anion exchanger and that its mutations cause renal hypouricemia type 1 (RHUC1). With the large health-examination database of the Japan Maritime Self-Defense Force, we found two missense mutations (R198C and R380W) of GLUT9/SLC2A9 in hypouricemia patients. R198C and R380W occur in highly conserved amino acid motifs in the "sugar transport proteins signatures" that are observed in GLUT family transporters. The corresponding mutations in GLUT1 (R153C and R333W) are known to cause GLUT1 deficiency syndrome because arginine residues in this motif are reportedly important as the determinants of the membrane topology of human GLUT1. Therefore, on the basis of membrane topology, the same may be true of GLUT9. GLUT9 mutants showed markedly reduced urate transport in oocyte expression studies, which would be the result of the loss of positive charges in those conserved amino acid motifs. Together with previous reports on GLUT9 localization, our findings suggest that these GLUT9 mutations cause renal hypouricemia type 2 (RHUC2) by their decreased urate reabsorption on both sides of the renal proximal tubule cells. However, a previously reported GLUT9 mutation, P412R, was unlikely to be pathogenic. These findings also enable us to propose a physiological model of the renal urate reabsorption via GLUT9 and URAT1 and can lead to a promising therapeutic target for gout and related cardiovascular diseases.