Promsuk Jutabha

Molecular identification of a renal urate–anion exchanger that regulates blood urate levels

Urate, a naturally occurring product of purine metabolism, is a scavenger of biological oxidants implicated in numerous disease processes, as demonstrated by its capacity of neuroprotection. It is present at higher levels in human blood (200–500 µM) than in other mammals, because humans have an effective renal urate reabsorption system, despite their evolutionary loss of hepatic uricase by mutational silencing. The molecular basis for urate handling in the human kidney remains unclear because of difficulties in understanding diverse urate transport systems and species differences. Here we identify the long-hypothesized urate transporter in the human kidney (URAT1, encoded by SLC22A12), a urate–anion exchanger regulating blood urate levels and targeted by uricosuric and antiuricosuric agents (which affect excretion of uric acid). Moreover, we provide evidence that patients with idiopathic renal hypouricaemia (lack of blood uric acid) have defects in SLC22A12. Identification of URAT1 should provide insights into the nature of urate homeostasis, as well as lead to the development of better agents against hyperuricaemia, a disadvantage concomitant with human evolution.

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 (Km: 365 ± 42 μm). 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; Remunurate 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).

Mutations in SLC6A19 , encoding B 0 AT1, cause Hartnup disorder

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B0AT1 (ref. 4). We isolated the human homolog of B0AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

Modulation of Renal Apical Organic Anion Transporter 4 Function by Two PDZ Domain–Containing Proteins

Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K(D): 36 nM, 1.2 microM, and 41.7 microM, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E1S transport activities in HEK293 cells (1.2- to 1.4-fold), and the deletion of the OAT4 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by alteration in V(max) of E(1)S transport via OAT4 and was associated with the increased surface expression level of OAT4 protein. This study indicates that the functional activity of OAT4 is modulated through the PDZ interaction with the network of PDZK1 and NHERF1 and suggests that OAT4 is involved in the regulated apical organic anion handling in the renal proximal tubules, provided by the PDZ scaffold.

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.

Recent advances in renal urate transport: characterization of candidate transporters indicated by genome-wide association studies

Humans have higher serum uric acid levels than other mammalian species owing to the genetic silencing of the hepatic enzyme uricase that metabolizes uric acid into allantoin. Urate (the ionized form of uric acid) is generated from purine metabolism and it may provide antioxidant defense in the human body. Despite its potential advantage, sustained hyperuricemia has pathogenetic causes in gout and renal diseases, and putative roles in hypertension and cardiovascular diseases. Since the kidney plays a dominant role in maintaining plasma urate levels through the excretion process, it is important to understand the molecular mechanism of renal urate handling. Although the molecular identification of a kidney-specific urate/anion exchanger URAT1 in 2002 paved the way for successive identification of several urate transport-related proteins, the entire picture of effective renal urate handling in humans has not yet been clarified. Recently, several genome-wide association studies identified a substantial association between uric acid concentration and single nucleotide polymorphisms in at least ten genetic loci including eight transporter-coding genes. In 2008, we functionally characterized the facilitatory glucose transporter family member SLC2A9 (GLUT9), one of the candidate genes for urate handling, as a voltage-driven urate transporter URATv1 at the basolateral side of renal proximal tubules that comprises the main route of the urate reabsorption pathway, in tandem with URAT1 at the apical side. In this review, recent findings concerning these candidate molecules are presented.

LAT1 Is a Critical Transporter of Essential Amino Acids for Immune Reactions in Activated Human T Cells

Activation of T cells accompanies remarkable enhancement of metabolism. Sufficient and continuous nutrient supply is therefore important to support immune reaction in T cells. However, the mechanism of the promotion of nutrient incorporation in activated T cells has not been elucidated. In this study, we show that L-type amino acid transporter 1 (LAT1) is a major transporter for essential amino acids into activated human T cells. CD3/CD28 stimulation in primary human T cells triggered dramatic induction of LAT1 expression mediated by NF-κB and AP-1. Functional disturbance of LAT1 by a specific inhibitor and by small interfering RNA in human T cells suppressed essential amino acid uptake and induced a stress response mediated by DNA damage–inducible transcript 3 to attenuate cytokine production via inhibition of NF-κB and NFAT activities. These results uncover the previously unknown mechanism by which T cells accelerate essential amino acid uptake upon activation and adapt to essential amino acid starvation. Our results also raise the possibility for application of an LAT1 inhibitor as a new drug for therapy of disease caused by exaggerated immune response.

Integrated physiology of proximal tubular organic anion transport

Purpose of reviewRenal organic anion transport proteins play important roles in the reabsorption and the secretion of endogenous and exogenous compounds. This review focuses on the interpretation of the physiological integration of identified transport molecules in the renal proximal tubules. Recent findingsTo date, molecular identification of organic anion transport proteins is still continuing: rodent organic anion transporter 5, organic anion-transporting polypeptide 4C1, voltage-driven organic anion transporter 1, multidrug resistance-associated protein 4, and sodium-coupled monocarboxylate transporter have yielded additional information in this field. In addition, particularly at the apical membrane of the proximal tubules, the importance of the PDZ (PSD-95, DglA, and ZO-1) binding domain proteins has emerged in the formation of the multimolecular complex as a functional unit of membrane transport. Finally, discovery of dicarboxylate receptors in the renal tubular cells raises the possibility that dicarboxylate anions function as intrarenal signaling molecules. This novel aspect of renal organic anion transport, the potential modulation of signaling via dicarboxylate receptors, may be of significant relevance to renovascular hypertension and other renal diseases. SummaryComprehensive understanding of the multimolecular complex, which is composed of transporters and their related signaling elements and is supported by the scaffold proteins underneath the plasma membrane, may be useful in clarifying complex transport phenomena such as renal apical organic anion handling. In addition to the recent proteomics approaches and conventional molecular physiology, it is necessary to develop novel methods to analyze the overall function of the multimolecular complex for the post-genomic era.

Roles of Organic Anion Transporters in the Renal Excretion of Perfluorooctanoic Acid

Perfluorooctanoic acid, an environmental contaminant, is found in both wild animals and human beings. There are large species and sex differences in the renal excretion of perfluorooctanoic acid. In the present study, we aimed to characterize organic anion transporters 1-3 (OAT1-3) in human beings and rats to investigate whether the species differences in the elimination kinetics of perfluorooctanoic acid from the kidneys can be attributed to differences in the affinities of these transporters for perfluorooctanoic acid. We used human (h) and rat (r) OAT transient expression cell systems and measured the [(14)C] perfluorooctanoic acid transport activities. Both human and rat OAT1 and OAT3 mediated perfluorooctanoic acid transport to similar degrees. Specifically, the kinetic parameters, K(m), were 48.0 +/- 6.4 microM for h OAT1; 51.0 +/- 12.0 microM for rOAT1; 49.1 +/- 21.4 microM for hOAT3 and 80.2 +/- 17.8 microM for rOAT3, respectively. These data indicate that both human and rat OAT1 and OAT3 have high affinities for perfluorooctanoic acid and that the species differences in its renal elimination are not attributable to affinity differences in these OATs between human beings and rats. In contrast, neither hOAT2 nor rOAT2 transported perfluorooctanoic acid. In conclusion, OAT1 and OAT3 mediated perfluorooctanoic acid transport in vitro, suggesting that these transporters also transport perfluorooctanoic acid through the basolateral membrane of proximal tubular cells in vivo in both human beings and rats. Neither human nor rat OAT2 mediated perfluorooctanoic acid transport. Collectively, the difference between the perfluorooctanoic acid half-lives in human beings and rats is not likely to be attributable to differences in the affinities of these transporters for perfluorooctanoic acid.

A novel transporter of SLC22 family specifically transports prostaglandins and co-localizes with 15-hydroxyprostaglandin dehydrogenase in renal proximal tubules

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na+-independent and saturable transport of PGE2 when expressed in a proximal tubule cell line (S2). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE2, PGF2α, and PGD2. Similar to PGE2 receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an α-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE2 receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the ω-tail tip forming a “hydrophobic core” accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE2, the metabolite of PGE2 produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE2 transport more efficient. By removing extracellular PGE2, OAT-PG is proposed to be involved in the local PGE2 clearance and metabolism for the inactivation of PG signals in the kidney cortex.