Ken Ogasawara

Identification of multidrug and toxin extrusion (MATE1 and MATE2-K) variants with complete loss of transport activity

H+/organic cation antiporters (multidrug and toxin extrusion: MATE1 and MATE2-K) play important roles in the renal tubular secretion of cationic drugs. We have recently identified a regulatory single nucleotide polymorphism (SNP) of the MATE1 gene (−32G>A). There is no other information about SNPs of the MATE gene. In this study, we evaluated the functional significance of genetic polymorphisms in MATE1 and MATE2-K. We sequenced all exons of MATE1 and MATE2-K genes in 89 Japanese subjects and identified coding SNPs (cSNPs) encoding MATE1 (V10L, G64D, A310V, D328A and N474S) and MATE2-K (K64N and G211V). All the variants except for MATE1 V10L showed significant decrease in transport activity. In particular, MATE1 G64D and MATE2-K G211V variants completely lost transport activities. When membrane expression level was evaluated by cell surface biotinylation, those of MATE1 (G64D and D328A) and MATE2-K (K64N and G211V) were significantly decreased compared with that of wild type. These findings suggested that the loss of transport activities of the MATE1 G64D and MATE2-K G211V variants were due to the alteration of protein expression in cell surface membranes. This is the first demonstration of functional impairment of the MATE family induced by cSNPs.

Kidney-specific expression of human organic cation transporter 2 (OCT2/SLC22A2) is regulated by DNA methylation.

Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfering with the transactivation by USF1.

Analysis of regulatory polymorphisms in organic ion transporter genes (SLC22A) in the kidney.

AbstractOrganic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1–4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (−578_−576delAAG) was identified; −578_−576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of −578_−576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.

Pharmacokinetic Significance of Renal OAT3 (SLC22A8) for Anionic Drug Elimination in Patients with Mesangial Proliferative Glomerulonephritis

PurposeOur previous studies showed that the mRNA level of human organic anion transporter (hOAT) 3 in the kidney was correlated with the rate of elimination of an anionic antibiotic cefazolin. However, the correlation coefficient was not so high. In the present study, therefore, we enrolled more patients to examine whether additional factors were responsible for the correlation.MethodshOAT mRNA levels in renal biopsy specimens were quantified using the real-time polymerase chain reaction method. The elimination rates for the free fraction of cefazolin were determined in patients with various renal diseases.ResultsIn the present study, the coefficient of correlation between the hOAT3 mRNA level and the elimination rates for the free fraction of cefazolin was not so high in the patients overall as in our previous study (r = 0.536). However, following the classification of renal diseases, a better correlation was obtained in patients with mesangial proliferative glomerulonephritis (r = 0.723). In contrast, multiple regression analyses including gender, age, and liver function did not result in any improvements in the correlation coefficients.ConclusionsThese results suggest that the hOAT3 mRNA level is a significant marker of pharmacokinetics with which to predict the rate of elimination of cefazolin in patients with mesangial proliferative glomerulonephritis.

Human Organic Anion Transporter 3 Gene Is Regulated Constitutively and Inducibly via a cAMP-Response Element

Human organic anion transporter (OAT) 3 (SLC22A8) is localized to the basolateral membranes of renal tubular epithelial cells and plays a critical role in the excretion of anionic compounds. We previously reported that interindividual variation in the OAT3 mRNA level corresponded to interindividual differences in the rate of renal excretion of cefazolin. However, there is little information available on the molecular mechanisms regulating the gene expression of OAT3. Therefore, in the present study, we examined the transcriptional regulation of human OAT3. A deletion analysis of the OAT3 promoter suggested that the region spanning -214 to -77 base pairs was essential for basal transcriptional activity. This region contained a perfectly conserved cAMP-response element (CRE), and a mutation here led to a reduction in promoter activity. Electrophoretic mobility shift assays showed that CRE-binding protein (CREB)-1 and activating transcription factor (ATF)-1 bound to CRE. The activity of the OAT3 promoter was increased through the phosphorylation of CREB-1 and ATF-1 by treatment with 8-bromo-cAMP. This paper reports the first characterization of the human OAT3 promoter and shows that CREB-1 and ATF-1 function as constitutive and inducible transcriptional regulators of the human OAT3 gene via CRE.

Adaptive responses of renal organic anion transporter 3 (OAT3) during cholestasis.

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of beta-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis.

Impact of regulatory polymorphisms in organic anion transporter genes in the human liver

Objectives The aim of this study was to evaluate whether genetic polymorphisms are responsible for individual variation in the expression levels of hepatic organic anion transporters. Methods The mRNA levels of multidrug resistance-associated protein 2, organic anion transporting polypeptide (OATP) 1B1, OATP1B3 and OATP2B1 were determined by real-time polymerase chain reaction using liver tissue samples from 102 Japanese. The association between the mRNA levels of these transporters and the disappearance rate of indocyanine green was examined. The promoter region of each transporter gene was sequenced and the effects of single nucleotide polymorphisms (SNPs) in the promoter region [regulatory SNPs (rSNPs)] on mRNA levels were evaluated. Western blot analysis using hepatic crude plasma membrane was carried out to assess protein levels. Results Expression levels of hepatic organic anion transporters vary considerably among individuals and possible links were observed between mRNA levels of these transporters and the disappearance rate of indocyanine green. In the OATP2B1 promoter region, we identified an rSNP (−282G>A) that significantly increased mRNA expression. Western blot analysis showed a remarkable increase in the OATP2B1 protein level in the homozygotes for the rSNP with a high mRNA level. In contrast, none of the rSNPs of multidrug resistance-associated protein 2, OATP1B1 and OATP1B3 affected the mRNA expression. Conclusion OATP2B1 −282G>A is a major factor affecting expression, suggesting a contribution to inter-individual differences in the expression level of OATP2B1.

Characterization of the Basal Promoter Element of Human Organic Cation Transporter 2 Gene

Human organic cation transporter 2 (hOCT2; SLC22A2) is abundantly expressed in the kidney, and it plays important roles in the renal tubular secretion of cationic drugs. Although the transport characteristics of hOCT2 have been studied extensively, there is no information available for the transcriptional regulation of hOCT2. The present study was undertaken to identify the cis-element and trans-factor for basal expression of hOCT2. The transcription start site was located 385 nucleotides above the translation start site by using 5′-rapid amplification of cDNA ends. An approximately 4-kilobase fragment of the hOCT2 promoter region was isolated and the promoter activities were measured in the renal epithelial cell line LLC-PK1. A deletion analysis suggested that the region spanning –91 to –58 base pairs was essential for basal transcriptional activity. This region lacked a TATA-box but contained a CCAAT box and an E-box. Electrophoretic mobility shift assays showed that specific DNA/protein complexes were present in the E-box but not in the CCAAT box, and supershift assays revealed that upstream stimulatory factor 1 (USF-1), which belongs to the basic helix-loop-helix-leucine zipper family of transcription factors, bound to the E-box. Mutation of the E-box resulted in a decrease in hOCT2 promoter activity, and overexpression of USF-1 enhanced the hOCT2 promoter activity in a dose-dependent manner. This article reports the first characterization of the hOCT2 promoter and shows that USF-1 functions as a basal transcriptional regulator of the hOCT2 gene via the E-box.