Kousei Ito

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

Dysfunctional genotype combinations of polymorphic adenosine 5′-triphosphate–binding cassette transporter gene ABCG2/BCRP, which encodes a high-capacity urate secretion transporter in human gut and kidney, are major causes of gout. Gout, the “Disease of Kings” as it is often known, is a painful medical condition characterized by sharp acute pain in bone joints, due to the high deposition of uric acid crystals from the blood serum into the surrounding cartilage. It affects approximately 1% of the U.S. population and remains a significant public health concern. The prevalence of gout is much higher in certain Asian ethnic groups, and is also reportedly rising in African Americans. Current medical treatments are aimed at ameliorating pain severity, but as the underlying genetic etiology of the disease unfolds, new targets for future therapies are likely to be found. Although genome-wide association studies (GWAS) have enabled the calculation of risk predispositions for a wide variety of complex diseases, the relation of gene function to the causality of disease-related mutations has remained largely unclear. A recent U.S. population–based study supported an association between urate levels and gout in individuals carrying variants in a multifunctional transporter gene, ABCG2. This study identified Q141K as a high-risk variant in nearly 10% of gout cases in Caucasians. Now, a team led by Hirotaka Matsuo report that in a Japanese population, another risk variant in ABCG2, namely the Q126X nonfunctional mutation, confers an even higher risk associated with an increase in uric acid deposition in the blood and may cause gout in Asians. Because this gene is responsible for giving rise to a protein that transports harmful waste products and metabolites out of the kidney and gut, they extensively validate the biological activity of ABCG2 using functional assays in vitro that effectively recapitulate human data obtained from Japanese individuals afflicted with the disease. These findings lend weight to previously reported GWAS; moreover, these newly identified specific high-risk variants that block urate secretion may serve as potential intervention points for quelling the disease. 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.

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump.

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

Apical/Basolateral Surface Expression of Drug Transporters and its Role in Vectorial Drug Transport

It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug–drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood–tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin–Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.

Functional Analysis of a Canalicular Multispecific Organic Anion Transporter Cloned from Rat Liver

Transport of many organic anions across the bile canalicular membrane is mediated by the canalicular multispecific organic anion transporter (cMOAT). Previously, we cloned cDNA that may encode cMOAT from Sprague-Dawley rat liver (Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1997)Am. J. Physiol. 272, G16–G22). In the present study, the function of this cloned cDNA was investigated by examining the ATP-dependent uptake ofS-(2,4-dinitrophenyl)-glutathione (DNP-SG) into membrane vesicles isolated from an NIH/3T3 cell line transfected with an expression vector containing the cloned cDNA. Although the membrane vesicles from the control NIH/3T3 cells exhibited endogenous activity in transporting DNP-SG and leukotriene C4 in an ATP-dependent manner, the transfection of cMOAT cDNA resulted in a significant increase in the transport activity for these ligands. The uptake of DNP-SG into membrane vesicles was osmotically sensitive and was stimulated to some extent by other nucleotide triphosphates (GTP, UTP, and CTP) but not by AMP or ADP. TheK m and V max values for the uptake of DNP-SG by the membrane vesicles were 0.175 ± 0.031 μm and 11.0 ± 0.73 pmol/min/mg protein, respectively, for the transfected rat cMOAT and 0.141 ± 0.036 μm and 3.51 ± 0.39 pmol/min/mg protein, respectively, for the endogenous transporter expressed on control NIH/3T3 cells. These results suggest that the product of the previously cloned cDNA has cMOAT activity being able to transport organic anions in an ATP-dependent manner. Alternatively, it is possible that the cDNA product encodes an activator of endogenous transporter since the K m value for DNP-SG was comparable between the vector- and cMOAT-transfected cells. The transport activity found in the control NIH/3T3 cells may be ascribed to mouse cMOAT since Northern blot analysis indicated the presence of a transcript that hybridyzed to the carboxyl-terminal ATP-binding cassette sequence of the murine protein.

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver

Inchin‐ko‐to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a “bile acid‐independent” mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance‐associated protein 2 (Mrp2; Abcc2)‐mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)‐dependent uptake of Mrp2‐specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP‐dependent uptake of estradiol 17‐β‐D‐glucuronide (E217βG; by 265%), leukotriene C4 (LTC4; by 161%), taurolithocholate‐3‐sulfate (TLC‐3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2‐deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin‐treated liver tissue sections when compared with the vehicle‐treated liver tissue sections. In conclusion, genipin may enhance the bile acid‐independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases. (HEPATOLOGY 2004;39:167–178.)

Itraconazole-Induced Cholestasis: Involvement of the Inhibition of Bile Canalicular Phospholipid Translocator MDR3/ABCB4

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [14C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [3H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal.

Glutathione (GSH) conjugates of 4-hydroxy-trans-2,3-nonenal (HNE) are the final products of lipid peroxidation. In the present study, the role of multidrug resistance-associated protein 2 (MRP2) in biliary excretion of GSH conjugates of HNE (HNE-SG) was studied in vitro by using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2 and in vivo using a mutant rat strain whose MRP2 expression is defective (Eisai-hyperbilirubinemic rats [EHBR]). A high-performance liquid chromatography method was developed to assay HNE-SG conjugates. Four diastereomeric HNE-SG conjugates could be separated with this method. Three of four HNE-SG conjugates were detectable after incubation of the cell monolayers with HNE. Expression of human MRP2 resulted in a 10-fold increase in HNE-SG conjugates excretion across the apical membrane of MDCK II cells. The four HNE-SG conjugates appeared swiftly in bile from Sprague Dawley rats after intravenous administration of HNE, whereas no detectable HNE-SG conjugates were observed in the bile of EHBR. These results demonstrate the role of MRP2 in the biliary excretion of HNE-SG conjugates.

Canalicular Mrp2 localization is reversibly regulated by the intracellular redox status

Oxidative stress is known to be a common feature of cholestatic syndrome. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile salt-independent bile flow, under acute oxidative stress, and a series of signaling pathways finally leading to the activation of novel protein kinase C were involved in this mechanism; however, it has been unclear whether the internalized Mrp2 localization was relocalized to the canalicular membrane when the intracellular redox status was recovered from oxidative stress. In this study, we demonstrated that decreased canalicular expression of Mrp2 induced by tertiary-butyl hydroperoxide (t-BHP) was recovered to the canalicular membrane by the replenishment of GSH by GSH-ethyl ester, a cell-permeable form of GSH. Moreover, pretreatment of isolated rat hepatocytes with colchicine and PKA inhibitor did not affect the t-BHP-induced Mrp2 internalization process but did prevent the Mrp2 recycling process induced by GSH replenishment. Moreover, intracellular cAMP concentration similarly changed with the change of intracellular GSH content. Taken together, our data clearly indicate that the redox-sensitive balance of PKA/PKC activation regulates the reversible Mrp2 localization in two different pathways, the microtubule-independent internalization pathway and -dependent recycling pathway of Mrp2.

Expression of a putative ATP-binding cassette region, homologous to that in multidrug resistance-associated protein (MRP), is hereditarily defective in Eisai hyperbilirubinemic rats (EHBR)

Abstract It is well established that several organic anions such as leukotriene C4 and S-(2,4-dinitrophenyl)-glutathione are excreted into the bile via an ATP-dependent primary active transporter located on the bile canalicular membrane. Although the molecular features of this transporter still remain to be clarified, this transporter might be a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily which has a common ABC region. In the present study, a cDNA fragment was amplified from Sprague-Dawley (SD) rat liver by PCR using degenerate primers prepared from the conserved sequence in the COOH-terminal ABC region of human multidrug resistance-associated protein (MRP), a primary active transporter. The amplified 421 bp fragment exhibited homology with a human MRP and the human MRP-like fragment (yp75a11) with homology score of 66.3% and 83.0% at the cDNA level, and 73.3% and 84.7% deduced from the amino acid level, respectively. Northern blot analysis of poly(A)+ RNA prepared from SD rat liver revealed the presence of ~5 kb and 8.5 kb mRNA species which hybridized to this fragment. In contrast, poly(A)+ RNA from Eisai hyperbilirubinemic rats (EHBR), whose primary active transporter on the bile canalicular membrane is hereditarily defective, did not hybridize to this fragment. These results suggest (1) that the impaired expression of this particular region might be related to the pathogenesis of hyperbilirubinemia in EHBR and (2) that this region might encode part of the primary active transporter on the bile canalicular membrane.

ABCG2 is a High-Capacity Urate Transporter and its Genetic Impairment Increases Serum Uric Acid Levels in Humans

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3′-azido-3′-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10−5). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.