Makoto Hosoyamada

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.

Expression Cloning and Characterization of a Novel Multispecific Organic Anion Transporter

Numerous drugs and endogenous compounds are efficiently excreted from the renal proximal tubule via carrier-mediated pathways. Transepithelial excretion of organic anions occurs via their accumulative transport from the blood into the proximal tubule cells across the basolateral membrane and subsequent secretion into the urine through the apical membrane. Here we report on the isolation of a novel complementary DNA from rat kidney that encodes a 551-amino acid residue protein (OAT1) with 12 putative membrane-spanning domains. When expressed in Xenopus laevisoocytes, OAT1 mediated sodium-independent para-aminohippurate (PAH) uptake (K m = 14.3 ± 2.9 μm). The uptake rate of PAH was increased by the outwardly directed dicarboxylate gradient, consisting with the idea that OAT1 is an organic anion/dicarboxylate exchanger. OAT1 displayed remarkably wide substrate selectivity, covering endogenous substrates such as cyclic nucleotides, a prostaglandin and uric acid, and a variety of drugs with different structures (e.g. antibiotics, a nonsteroidal anti-inflammatory drug, diuretics, an antineoplastic drug, and a uricosuric drug). The Northern blot analysis and in situhybridization revealed that OAT1 is exclusively expressed in the particular segment of the proximal tubule in the kidney. These data suggest that OAT1 is a multispecific organic anion transporter at the basolateral membrane of the proximal tubule. Isolation of OAT1 will facilitate elucidation of the molecular basis of drug kinetics and the development of new drugs lacking unwanted side effects.

Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney

Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1-1 cDNA and hOAT1-2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) ( K m = 9.3 ± 1.0 μM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and α-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1-1 cDNA and hOAT1-2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) (Km = 9. 3 +/- 1.0 microM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and alpha-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.

Clinical and Molecular Analysis of Patients with Renal Hypouricemia in Japan-Influence of URAT1 Gene on Urinary Urate Excretion

Renal hypouricemia is an inherited and heterogeneous disorder characterized by increased urate clearance (CUA). The authors recently established that urate was reabsorbed via URAT1 on the tubular apical membrane and that mutations in SLC22A12 encoding URAT1 cause renal hypouricemia. This study was undertaken to elucidate and correlate clinical and genetic features of renal hypouricemia. The SLC22A12 gene was sequenced in 32 unrelated idiopathic renal hypouricemia patients, and the relationships of serum urate levels, and CUA/creatinine clearance (Ccr) to SLC22A12 genotype were examined. Uricosuric (probenecid and benzbromarone) and anti-uricosuric drug (pyrazinamide) loading tests were also performed in some patients. Three patients had exercise-induced acute renal failure (9.4%), and four patients had urolithiasis (12.5%). The authors identified eight new mutations and two previously reported mutations that result in loss of function. Thirty patients had SLC22A12 mutations; 24 homozygotes and compound heterozygotes, and 6 heterozygotes. Mutation G774A dominated SLC22A12 mutations (74.1% in 54 alleles). Serum urate levels were significantly lower and CUA/Ccr was significantly higher in heterozygotes compared with healthy subjects; these changes were even more significant in homozygotes and compound heterozygotes. These CUA/Ccr relations demonstrated a gene dosage effect that corresponds with the difference in serum urate levels. In contrast to healthy subjects, the CUA/Ccr of patients with homozygous and compound heterozygous SLC22A12 mutations was unaffected by pyrazinamide, benzbromarone, and probenecid. The findings indicate that SLC22A12 was responsible for most renal hypouricemia and that URAT1 is the primary reabsorptive urate transporter, targeted by pyrazinamide, benzbromarone, and probenecid in vivo.

Uric Acid Causes Vascular Smooth Muscle Cell Proliferation by Entering Cells via a Functional Urate Transporter

Background: Soluble uric acid stimulates vascular smooth muscle cell (VSMC) proliferation by activating mitogen-activated protein kinases, and stimulating COX-2 and PDGF synthesis. The mechanism by which uric acid enters the VSMC is not known. We hypothesized that uric acid enters via transporters similar to that observed in the kidney. Methods: We studied the uptake of uric acid into rat VSMC under polarized and depolarized conditions and in the presence of organic anion transport (OAT) inhibitors (probenecid and benzbromarone) or p-aminohippurate (PAH). We also examined the ability of probenecid to inhibit uric acid-induced VSMC proliferation and monocyte chemoattractant protein-1 (MCP-1) synthesis. Results:14C-Urate uptake was shown in VSMC and was enhanced under depolarized conditions. 14C-Uric acid uptake was inhibited by probenecid and benzbromarone, as well as by unlabelled urate and PAH. Probenecid blocked VSMC proliferation and MCP-1 expression in response to uric acid. VSMC did not express rOAT1-3, rOAT-5 or URAT-1 mRNA by PCR, but did express the voltage-sensitive transporter (UAT) by both PCR and RNase protection assay. Conclusions: Urate enters VSMC by both voltage-sensitive and OAT pathways, and the uptake, cell proliferation and MCP-1 expression can be blocked by OAT inhibitors. The specific transporter(s) responsible for the urate uptake remains to be determined.

Function and Localization of Urate Transporter 1 in Mouse Kidney

Mouse renal-specific transporter (RST) cDNA, the amino acid sequence of which has 74% identity with that of human urate transporter 1 (hURAT1), is potentially the mouse homologue of hURAT1, the gene responsible for hereditary renal hypouricemia. The aim of this study is to determine the location and characteristics of RST molecule in mouse kidney and investigate urate transport by RST using the Xenopus oocyte expression system. RST transported (14)C-urate in a Michaelis-Menten manner. The K(m) and the V(max) values of RST-dependent urate transport were 1213 +/- 222 micro M and 268.8 +/- 38.0 pmol/oocyte per hr, respectively (n = 3). RST-dependent urate transport was cis-inhibited significantly by 1 mM probenecid (68.7 +/- 9.4%), 50 micro M benzbromarone (67.9 +/- 6.4%), and 10 mM lactate (50.9 +/- 9.5%). However, 1 mM p-aminohippurate (PAH), 1 mM xanthine, and 1 mM oxonate did not inhibit RST-dependent urate transport. Substitution of Cl anion with gluconate in the external solution enhanced RST-dependent urate transport. Pre-injected pyrazinoic acid (PZA) or L-lactate trans-stimulated RST-dependent urate transport. Using immunohistochemistry for mouse kidney, the brush border or intracellular membrane of proximal tubules was stained by an affinity-purified antibody that recognized mouse URAT1 (mURAT1) expressed on Xenopus oocyte. Using Western blotting, anti-mURAT1 antibody detected 70-kD and 62-kD protein bands. The 70-kD protein was N-glycosylated and was identified as a Triton X-100 insoluble brush border membrane protein. RST mRNA and protein levels were higher in male kidneys than female. RST transported urate similar to hURAT1 and, therefore, appears to be mURAT1-the mouse homologue of hURAT1.

Uricosuric Action of Losartan via the Inhibition of Urate Transporter 1 (URAT 1) in Hypertensive Patients

BACKGROUND The angiotensin receptor blocker losartan inhibited urate transporter 1 (URAT1) according to in vitro experiments. However, it is still unknown whether the inhibitory effect of losartan on URAT1 contributes to its uricosuric action in humans. METHODS Thirty-two patients with hypertension and nine patients with idiopathic renal hypouricemia (five with and four without hypertension) were enrolled for this study. Hypertensive patients were prescribed oral losartan (50 mg/day, n = 16) or candesartan (8 mg/day, n = 16). Before and after 1-month treatment, the serum concentration of urate (Sur) and creatinine (Scr), and the clearance value of urate (Cur) and creatinine (Ccr) were determined. Clearance studies using the URAT1 inhibitor benzbromarone (100 mg/day) or losartan (50 mg/day) loading test were also performed in these patients. RESULTS Blood pressure (BP) significantly decreased in the patients treated with either losartan or candesartan. Losartan significantly reduced Sur, which was associated with a concomitant increase in the Cur/Ccr ratio, whereas candesartan did not alter these parameters. In hypertensive patients with loss-of-function mutation of URAT1, losartan did not alter either Sur or Cur/Ccr, nor did benzbromarone. The lack of effect of URAT1 inhibitors on renal excretion of urate was independent of the renal function of hypouricemic patients. On the other hand, both losartan and benzbromarone increased Cur/Ccr ratio in hypertensive patients harboring the wild URAT1 gene, regardless of the presence of hypouricemia. CONCLUSIONS These findings suggested that losartan inhibited URAT1 and thereby it lowered Sur levels in hypertensive patients.

Characterization of organic anion transport inhibitors using cells stably expressing human organic anion transporters

The organic anion transport system is involved in the tubular excretion of various clinically important drugs. The purpose of this study was to characterize the effects of various organic anion transport inhibitors on organic anion transport using proximal tubule cells stably expressing human organic anion transporter 1 (human-OAT1) and human-OAT3, which are localized to the basolateral membrane of the proximal tubule. Organic anion transport inhibitors including betamipron, cilastatin, KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) and probenecid significantly inhibited human-OAT1- and human-OAT3-mediated organic anion uptake in a dose-dependent manner. Kinetic analyses revealed that these inhibitions were competitive. The Ki values of betamipron, cilastatin, KW-3902 and probencid for human-OAT1 were 23.6, 1470, 7.82 and 12.1 microM, whereas those for human-OAT3 were 48.3, 231, 3.70 and 9.0 microM. These results suggest that betamipron and probenecid could inhibit both human-OAT1- and human-OAT3-mediated organic anion transport in vivo, whereas cilastatin could inhibit only human-OAT3-mediated one. In contrast, KW-3902 did not exert the effects of significance, whereas KW-3902 was the most potent.

Characterization of ochratoxin A transport by human organic anion transporters

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.

Cloning, functional characterization, and localization of a rat renal Na^+ -dicarboxylate transporter

We report here the isolation, functional characterization, tissue distribution, and membrane localization of rat renal Na+-dicarboxylate transporter (rNaDC-1). rNaDC-1 consists of 2,245 nucleotides, and the deduced amino acid sequence showed 73% and 75% identity to rabbit and human NaDC-1, respectively. When expressed in Xenopus laevis oocytes, rNaDC-1 mediated sodium-dependent uptake of di- and tricarboxylates. Substrates of rNaDC-1 evoked inward currents in oocytes expressed with rNaDC-1; succinate, α-ketoglutarate, and glutarate were relatively high-affinity substrates, and citrate was a low-affinity substrate of rNaDC-1. The coupling ratio of citrate to charge was determined to be 1:1 at pH 7.4; influx of one positive charge per citrate molecule suggests a symport of three Na+with a divalent citrate. Expression of rNaDC-1 mRNA was detected in the kidney and the small and large intestines. Immunohistochemistry using polyclonal antibodies raised against the 14 amino acids at the COOH terminus of rNaDC-1 revealed that rNaDC-1 is localized exclusively in the luminal membrane of S2 and S3.