Characterization of Urate Transport System in JAR and JEG-3 Cells, Human Trophoblast-derived Cell Lines

Urate (uric acid) is the major inert end product of purine metabolism in humans. Since it is water soluble, it requires a membranous protein called transporter for its permeation across the plasma membrane. Increased blood urate level is often seen in preeclampsia, but its precise mechanism remains unknown. Syncytiotrophoblasts function as a barrier between maternal blood and fetal one so called “blood-placental barrier”. So far, the expression of several urate transporters was identified in these cells, but it is still unclear about their contribution to urate handling in blood-placental barrier. In this study, we investigated the expression of urate transporters and the properties of ［14C］urate transport in both JAR and JEG-3, human choriocarcinoma cells as a model of human placenta. Conventional PCR analysis revealed that both JAR and JEG-3 cells express strongly breast cancer resistance protein (BCRP/ABCG2) mRNA. Uptake of ［14C］urate by these cells is time-dependent with Na＋- and Cl－-independent and voltage-insensitive manner and is not inhibited by benzbromarone, a representative renal urate transport inhibitor. Then, we focused on BCRP which shows strong mRNA expression and found that these cells have urate efflux property that is sensitive to fumitremorgin C (FMC), a BCRP inhibitor. These results suggest that BCRP is one of the important components for urate handling in human placenta in pathophysiological condition such as preeclampsia

Organic Anion Transporter 5 (Oat5) Urinary Excretion Is a Specific Biomarker of Kidney Injury: Evaluation of Urinary Excretion of Exosomal Oat5 after N-Acetylcysteine Prevention of Cisplatin Induced Nephrotoxicity

Cisplatin is a commonly used chemotherapeutic agent. Its main side-effect is nephrotoxicity. It was reported that the organic anion transporter 5 (Oat5) urinary excretion is elevated, implying renal perturbation, when no modifications of traditional markers of renal damage are still observed in cisplatin-induced acute kidney injury (AKI). It was also demonstrated that Oat5 is excreted in urine by the exosomal pathway. This study was designated to demonstrate the specific response of the urinary excretion of exosomal Oat5 to kidney injury independently of other cisplatin toxic effects, in order to strengthen Oat5 urinary levels as a specific biomarker of AKI. To accomplish that aim, we evaluated if urinary excretion of exosomal Oat5 returns to its basal levels when cisplatin renal damage is prevented by the coadministration of the renoprotective compound N-acetylcysteine. Four days after cisplatin administration, AKI was induced in cisplatin-treated male Wistar rats (Cis group), as it was corroborated by increased urea and creatinine plasma levels. Tubular damage was also observed. In cotreated animals (Cis + NAC group), plasma urea and creatinine concentrations tended to return to their basal values, and tubular damage was improved. Urinary excretion of exosomal Oat5 was notably increased in the Cis group, but when renal injury was ameliorated by N-acetylcysteine coadministration, that increase was undetected. So, in this work we observed that urinary excretion of exosomal Oat5 was only increased if renal insult is produced, demonstrating its specificity as a renal injury biomarker.Fil: Bulacio, Romina Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Anzai, Naohiko. Dokkyo Medical University; JapónFil: Ouchi, Motoshi. Dokkyo Medical University; JapónFil: Torres, Adriana Monica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular Mechanism of Ochratoxin A Transport in the Kidney

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity

Analysis of different complexes of type IIa sodium-dependent phosphate transporter in rat renal cortex using blue-native polyacrylamide gel electrophoresis

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) can be localized in the apical plasma membrane of renal proximal tubule to carry out a rate-limiting step of phosphate reabsorption. For the apical localization, NaPi-IIa is required to form a macromolecular complex with some adaptor proteins such as Na+/H+ exchanger regulatory factor 1 (NHERF-1) and ezrin. However, the detail of macromolecular complex containing NaPi-IIa in the apical membrane of the renal proximal tubular cells has not been clarified. In this study, we identified at least four different complexes (220, 480, 920, 1,100 kDa) containing NaPi-IIa by using blue-native polyacrylamide gel electrophoresis. Interestingly, LC-MS/MS analysis and immunoprecipitation analysis reveal that megalin is a component of larger complexs (920 and 1,100 kDa). In addition, NaPi-IIa can be heterogeneously co-localized with ezrin and megalin on the apical membrane of renal proximal tubuler cells by fluorescence microscopy analysis. These results suggest that NaPi-IIa can form some different complexes on the apical plasma membrane of renal proximal tubular cells

Clinical and Functional Characterization of URAT1 Variants

Idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. There is excessive urinary wasting of uric acid resulting in hypouricaemia. Patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. Here we have identified Macedonian and British patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. We have identified heterozygous missense mutations in SLC22A12 encoding the urate transporter protein URAT1 and correlate these genetic findings with functional characterization. Urate handling was determined using uptake experiments in HEK293 cells. This data highlights the importance of the URAT1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia

Identification of Carnitine Transporter CT1 Binding Protein Lin-7 in Nervous System

_L-Carnitine is an essential component of mitochondrial fatty acid b-oxidation in the muscle and may control the acetyl moiety levels in the brain for acetylcholine synthesis. Carnitine transporter 1(CT1)is the high affinity _L-carnitine transporter whose localization was observed in the kidney, testis, liver, skeletal muscle and brain. To clarify the molecular mechanism of carnitine transport, we sought to find the interacting protein that may be related to the transport function of CT1. Using the intracellular C-terminal region of rat CT1 containing PDZ(PSD95/DLG/ZO-1)motif as bait, we performed the yeast two-hybrid screening against rat brain cDNA library. Thirty two positive clones were obtained from the 2.7×10^7 clones screened. One of them was PDZ domain-containing protein Lin-7. We found that Lin-7 interacts specifically with C-termini of CT1:deletion and mutation of the CT1 C-terminal PDZ-motif abolished the interaction with Lin-7 in the yeast two-hybrid assay. In addition, a PDZ domain within Lin-7 associates with the CT1 C-terminal. The association of CT1 with Lin-7 enhanced _L-carnitine transport activities in HEK293 cells although there is no statistical significance. Coexpression of Lin-7 and CT1 is identified in motor neurons of the spinal cord ventral horn together with Lin-2, a binding partner of Lin-7 known to assemble proteins involved in synaptic vesicle exocytosis and synaptic junctions. Therefore, Lin-7 interacts with CT1 and may regulate their subcellular distribution or function in central nervous system

ヒト ジンゾウ ニョウサン トランス ポーター URAT 1 ト スイヨウ セイ ヨード ケイ ゾウエイザイ IODIPAMIDE ノ ソウゴ サヨウ

降圧薬などいくつかの薬剤は本来の薬理作用とは別に尿酸降下作用を持つものがあり,水溶性ヨード系造影剤もその一つでiodipamideやdiatrizoateでの尿酸排泄亢進が報告されていた.長らく不明のままであった腎尿酸輸送機構の分子実体は2002年の腎尿細管尿酸トランスポーターURAT1(Urate Transporter 1)の分子同定によりその理解が飛躍的に進んだ.本研究ではURAT1と水溶性造影剤のiodipamideおよびdiatrizoateの相互作用を検討することで,その尿酸排泄促進作用の分子機序の解明を目的とする.URAT1の尿酸輸送活性の測定にはURAT1安定発現HEK293細胞(HEK-URAT1)細胞を用いた.IodipamideはHEK-URAT1細胞でのRI標識尿酸取込みを著明に阻害した(IC_:1.19±0.08?μM)のに対し,diatrizoateは1?mMまでの範囲では50%以上の阻害作用を示さなかった.1?mMまでのiodipamideはHEK-URAT1細胞の生存率に影響を与えなかった.IodipamideによるURAT1媒介尿酸輸送への阻害作用のキネティクス解析の結果,その阻害は競合阻害であり,阻害定数Ki値は11.03?μMであった.以上より,iodipamideは尿酸トランスポーターURAT1と相互作用をすることを初めて確認できた.このことからiodipamideは細胞外からURAT1の尿酸結合部位に結合し,競合して阻害を行うことで,腎尿細管の経上皮性尿酸再吸収を抑制し,ひいては血清尿酸値を低下させるものと考えられた.Drug-induced hypouricemia has been found in several drugs such as probenecid, benzbromarone and angiotensin II receptor blocker(ARB)losartan. Xray contrast agents such as iodipamide and diatrizoate, used for the intravenous cholangiography and excretory urography, were reported to have uricosuric effct beside their original action. After the molecular identification of renal apical urate transporter URAT1 as an entrance of urate into the epithelial cells of proximal tubules, this protein is thought to be major determinant for renal reabsorption of urate that affect the blood urate levels in human. The purpose of this study is to examine whether iodipamide and diatrizoate act on URAT 1 . In URAT 1 -stably expressing HEK293(HEK-URAT1)cells, iodipamide inhibited [^C] urate uptake dose-dependently(IC_ , 1.19±0.08 μM), while diatrizoate did not. Up to the concentration of 1 mM, iodipamide incubation for 24 hr did not affect the viability of HEK293-URAT1 cells. Lineweaver-Burk plot of the kinetic analysis by URAT1-mediated urate uptake with or without iodipamide indicated that its interaction occurs in a competitive manner(Ki:11.03 μM). These results suggest that uricosuric effect of iodipamide can be explained by the interaction of iodipamide with urate-binding site of URAT1, and the inhibition of urate reabsorption from extracellular side by iodipamide causes uricosuria leading to induce hypouricemia

Molecular Mechanism of the Urate-lowering Effects of Calcium Channel Blockers

Hyperuricemia has recently been recognized as one of the risk factors for cardiovascular diseases. Some calcium channel blockers(CCBs), commonly used in the treatment of hypertension, have been reported to decrease serum urate level. Here, we tried to elucidate the molecular mechanism of the urate-lowering effects of CCBs. We performed [^C]urate uptake in cells stably expressing human urate transporter 1, a major contributor of renal urate reabsorption and a major target of uricosuric drugs such as benzbromarone and losartan(HEK-URAT1), together with mock(HEK-mock)cells to analyze the uricosuric action of CCBs. We also measured the activity of human xanthine oxidase(XO)to determine whether CCBs have inhibitory effects on urate production. The CCBs tested were nifedipine, nilvadipine, nitrendipine, benidipine, nisoldipine, nicardipine, efonidipine, amlodipine, azelnidipine, verapamil and diltiazem. We found for the first time that at least seven CCBs in the dihydropyridine subgroup interacted with URAT1-mediated urate uptake in HEK-URAT1 cells. Among these CCBs, nifedipine, nilvadipine and nitrendipine strongly inhibited URAT1-mediated urate uptake. Their IC_s were 15.8, 0.018 and 0.40?μM, respectively. In contrast, urate production mediated by XO was weakly inhibited by nifedipine and nisoldipine. In summary, URAT1 interacted with various CCBs differently, whereas XO, a major enzyme for urate production in the liver, did not interact with most of CCBs. Although CCBs were not excreted from the urine basically, their urate-lowering effects may be associated with the inhibition of renal urate reabsorption mediated by renal urate transporters such as URAT1 with their metabolites, and the results for structure-activity information in this study will provide a clue for developing new uricosuric drugs targeting URAT1