Organic Cation Transporters OCT1 Research Articles

The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin.

Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines.

OCT1 mediates hepatic uptake of sumatriptan and loss-of-function OCT1 polymorphisms affect sumatriptan pharmacokinetics.

The low bioavailability of the anti-migraine drug sumatriptan is partially caused by first-pass hepatic metabolism. In this study, we analyzed the impact of the hepatic organic cation transporter OCT1 on sumatriptan cellular uptake, and of OCT1 polymorphisms on sumatriptan pharmacokinetics. OCT1 transported sumatriptan with high capacity and sumatriptan uptake into human hepatocytes was strongly inhibited by the OCT1 inhibitor MPP(+) . Sumatriptan uptake was not affected by the Met420del polymorphism, but was strongly reduced by Arg61Cys and Gly401Ser, and completely abolished by Gly465Arg and Cys88Arg. Plasma concentrations in humans with two deficient OCT1 alleles were 215% of those with fully active OCT1 (P = 0.0003). OCT1 also transported naratriptan, rizatriptan, and zolmitriptan, suggesting a possible impact of OCT1 polymorphisms on the pharmacokinetics of other triptans as well. In conclusion, OCT1 is a high-capacity transporter of sumatriptan and polymorphisms causing OCT1 deficiency have similar effects on sumatriptan pharmacokinetics as those observed in subjects with liver impairment.

Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes.

BackgroundSLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na+/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein.ResultsWe detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK293 cells. But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis.ConclusionsAlthough different kinds of assays were used to screen for transport function, SLC10A4 failed to show transport activity for a series of neurotransmitters and neuromodulators, indicating that SLC10A4 does not seem to represent a typical neurotransmitter transporter such as DAT, SERT, CHT1 or VMAT2.

Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1.

BackgroundThe organic cation transporter OCT1 (SLC22A1) mediates the uptake of vitamin B1, cationic drugs, and xenobiotics into hepatocytes. Nine percent of Caucasians lack or have very low OCT1 activity due to loss-of-function polymorphisms in OCT1 gene. Here we analyzed the global genetic variability in OCT1 to estimate the therapeutic relevance of OCT1 polymorphisms in populations beyond Caucasians and to identify evolutionary patterns of the common loss of OCT1 activity in humans.MethodsWe applied massively parallel sequencing to screen for coding polymorphisms in 1,079 unrelated individuals from 53 populations worldwide. The obtained data was combined with the existing 1000 Genomes data comprising an additional 1,092 individuals from 14 populations. The identified OCT1 variants were characterized in vitro regarding their cellular localization and their ability to transport 10 known OCT1 substrates. Both the population genetics data and transport data were used in tandem to generate a world map of loss of OCT1 activity.ResultsWe identified 16 amino acid substitutions potentially causing loss of OCT1 function and analyzed them together with five amino acid substitutions that were not expected to affect OCT1 function. The variants constituted 16 major alleles and 14 sub-alleles. Six major alleles showed improper subcellular localization leading to substrate-wide loss in activity. Five major alleles showed correct subcellular localization, but substrate-specific loss of activity. Striking differences were observed in the frequency of loss of OCT1 activity worldwide. While most East Asian and Oceanian individuals had completely functional OCT1, 80 % of native South American Indians lacked functional OCT1 alleles. In East Asia and Oceania the average nucleotide diversity of the loss-of-function variants was much lower than that of the variants that do not affect OCT1 function (ratio of 0.03) and was significantly lower than the theoretically expected heterozygosity (Tajima’s D = −1.64, P < 0.01).ConclusionsComprehensive genetic analyses showed strong global variations in the frequency of loss of OCT1 activity with selection pressure for maintaining OCT1 activity in East Asia and Oceania. These results not only enable pharmacogenetically-based optimization of drug treatment worldwide, but may help elucidate the functional role of human OCT1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0172-0) contains supplementary material, which is available to authorized users.

P0222 : Relevance of organic cation transporter OCT1 (SLC22A1) in diethylnitrosamine-initiated and phenobarbital-promoted hepatocellular carcinoma in OCT3-knockout mice

P0222 : Relevance of organic cation transporter OCT1 (SLC22A1) in diethylnitrosamine-initiated and phenobarbital-promoted hepatocellular carcinoma in OCT3-knockout mice

Relevance of organic cation transporter OCT1 (SLC22A1) in Diethylnitrosamine-initiated and Phenobarbital-promoted hepatocellular carcinoma in OCT3- knockout mice

Background: Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates. OCTs became pharmaceutically interesting, because they are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. Recent data have shown a down-regulation of OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma (HCC) and human cholangiocarcinoma (CCC), associated with tumor progression and a worse patient survival. The OCT1 down-regulation in HCC may affect the ability of sorafenib and platin derivates to reach active intracellular concentrations in these tumors.

Cisplatin-induced renal injury is independently mediated by OCT2 and p53.

Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2(-/-)mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2(-/-)mice, we hypothesized that alternate pathways are involved in the observed injury. Studies were done in wild-type, Oct1/2(-/-), or p53-deficient animals, all on an FVB background, receiving cisplatin intraperitoneally at 15 mg/kg. Cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays. KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that the most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wild-type (P = 2.40 × 10(-11)) and Oct1/2(-/-)mice (P = 1.92 × 10(-8)). This was confirmed by demonstrating that homozygosity for a p53-null allele partially reduced renal tubular damage, whereas loss of p53 in Oct1/2(-/-)mice (p53(-/-)/Oct1/2(-/-)) completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(-/-)/Oct1/2(-/-)mice. These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Von Hippel-Lindau Exonic Methylation Analysis Using MALDI-TOF Mass Spectrometry

Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation. Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated. To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.

Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat

Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 μM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

PP177—Inhibitory Interaction of 125 Drugs with the Renally Expressed Organic Cation Transporter OCT2: Development of a Chemoinformatics-Based Model to Predict Transporter Inhibition in Silico

The pivotal role of the organic cation transporter OCT2 for the renal excretion of cationic drugs raises the risk for drug–drug interactions in which 1 drug reduces OCT2-mediated elimination of a second drug. In vivo testing of drug–drug interactions is time-consuming and costly. Therefore, additional strategies are needed to predict drug–drug interactions (DDIs). We used a large set of experimental data to develop a chemoinformatics-based model to predict OCT2-inhibition in silico.

Morphine is a substrate of the organic cation transporter OCT1 and polymorphisms in OCT1 gene affect morphine pharmacokinetics after codeine administration

We investigated whether morphine and its pro-drug codeine are substrates of the highly genetically polymorphic organic cation transporter OCT1 and whether OCT1 polymorphisms may affect morphine and codeine pharmacokinetics in humans.Morphine showed low transporter-independent membrane permeability (0.5×10−6cm/s). Morphine uptake was increased up to 4-fold in HEK293 cells overexpressing human OCT1. The increase was concentration-dependent and followed Michaelis-Menten kinetics (KM=3.4μM, VMAX=27pmol/min/mg protein). OCT1-mediated morphine uptake was abolished by common loss-of-function polymorphisms in the OCT1 gene and was strongly inhibited by drug-drug interactions with irinotecan, verapamil and ondansetron. Morphine uptake in primary human hepatocytes was strongly reduced by MPP+, an inhibitor of organic cation transporters, and morphine was not a substrate of OCT3, the other organic cation transporter expressed in human hepatocytes. In concordance with the in vitro data, morphine plasma concentrations in healthy volunteers were significantly dependent on OCT1 polymorphisms. After codeine administration, the mean AUC of morphine was 56% higher in carriers of loss-of-function OCT1 polymorphisms compared to non-carriers (P=0.005). The difference remained significant after adjustment for CYP2D6 genotype (P=0.03). Codeine itself had high transporter-independent membrane permeability (8.2×10−6cm/s). Codeine uptake in HEK293 cells was not affected by OCT1 overexpression and OCT1 polymorphisms did not affect codeine AUCs.In conclusion, OCT1 plays an important role in the hepatocellular uptake of morphine. Carriers of loss-of-function OCT1 polymorphisms may be at higher risk of adverse effects after codeine administration, especially if they are also ultra-rapid CYP2D6 metabolizers.

Substrate- and Cell Contact-Dependent Inhibitor Affinity of Human Organic Cation Transporter 2: Studies with Two Classical Organic Cation Substrates and the Novel Substrate Cd2+

Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.

Polyamine Transport by the Polyspecific Organic Cation Transporters OCT1, OCT2, and OCT3

Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 μM in OCT1, ~170 μM in OCT2, and ~60 μM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.

Renal Uptake of Substrates for Organic Anion Transporters Oat1 and Oat3 and Organic Cation Transporters Oct1 and Oct2 is Altered in Rats with Adenine-Induced Chronic Renal Failure

Chronic renal failure (CRF) leads to decreased drug renal clearance and glomerular filtration rate. However, little is known about renal tubular excretion and reabsorption in CRF. We examined transport activity of renal transporters using rats with adenine-induced CRF. We examined the effect of adenine-induced CRF on mRNA level, protein expression of transporters expressed in kidney by real-time polymerase chain reaction, and western blotting. In vivo kidney uptake clearances of benzylpenicillin and metformin, which are typical substrates for renal organic anion transporters Oat1 and Oat3 and organic cation transporters Oct1 and Oct2, respectively, were evaluated. Protein and mRNA expression levels of Oat1, Oat 3, Oct1, and Oct2 were significantly decreased in adenine-induced CRF rats. On the contrary, levels of P-glycoprotein and Mdr1b mRNA were significantly increased in adenine-induced CRF rats. The mRNA expression levels of Oatp4c1, Mate1, Urat1, Octn2, and Pept1 were significantly decreased. Kidney uptake clearance of benzylpenicillin and that of metformin were significantly decreased in adenine-induced CRF rats. Also, serum from CRF rats did not affect Oat1, Oat3, Oct1, and Oct2 function. In conclusion, our results indicate that adenine-induced CRF affects renal tubular handling of drugs, especially substrates of Oat1, Oat3, Oct1, and Oct2.

Transport of biguanides by human organic cation transporter OCT2

Biguanides have the severe side effect of lactic acidosis. Although both metformin and phenformin are biguanide derivatives, there is a difference in the frequency at which they induce lactic acidosis. However, the reasons for the difference are not clear. Metformin has been reported to be mainly excreted into urine by human organic cation transporter 2 (hOCT2). The present study was designed to investigate the renal transport of metformin and phenformin, focusing on hOCT2, using hOCT2–expressing oocytes. Both biguanides were found to be good substrates for hOCT2. However, phenformin exhibited a higher affinity and transport activity than metformin. The Km values for metformin and phenformin were 235 and 37.4μM, with CLint (Vmax/Km) values of 71.9×10−3μL/min per oocyte and 209×10−3μL/min per oocyte, respectively. This is the first report that has compared the transport profiles of these biguanides in hOCT2–expressing oocytes. The results suggest that plasma concentration of phenformin in subjects carrying hOCT2 variant may be higher compared to reference subjects, as reported in metformin. In addition, the relationship between plasma concentration of these biguanides and blood lactate level as well as the possible reasons for the difference in the associated frequency of occurrence of lactic acidosis are discussed.

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human cholangiocellular carcinoma and their prognostic significance

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human cholangiocellular carcinoma and their prognostic significance

Association of genetic variation in the organic cation transporters OCT1, OCT2 and multidrug and toxin extrusion 1 transporter protein genes with the gastrointestinal side effects and lower BMI in metformin-treated type 2 diabetes patients

Metformin is the most widely used oral antidiabetic drug for the treatment of type 2 diabetes (T2D). So far, the number of polymorphisms in SLC22A1, SLC22A2, and SLC47A1 genes coding for organic cation transporter 1 (OCT1), OCT2, and multidrug and toxin extrusion transporter 1 (MATE1) metformin transporters have been described in association with the efficacy of metformin. However, there is no information on the influence of genetic variations within these genes on the side effects of metformin. In this study, we assessed whether five single-nucleotide polymorphisms and two indel polymorphisms are associated with the side effects of metformin in patients with T2D. Seven polymorphisms in OCT1, OCT2, and MATE1 genes were compared between 53 T2D patients with side effects of metformin and 193 metformin users without symptoms of metformin intolerance. We found a statistically significant association between the A allele of the rs628031 (P=0.012, odds ratio=0.389, confidence interval 95% [0.186-0.815]) as well as 8 bp insertion (rs36056065) in the OCT1 gene (P=0.002, odds ratio=0.405, confidence interval 95% [0.226-0.724]) and the presence of the side effects of metformin. Two genetic variations in OCT1 that are in strong linkage disequilibrium may predispose toward an increased prevalence of the side effects of metformin in patients with T2D.

Organic cation transporters OCT1 and OCT2 determine the accumulation of lamivudine in CD4 cells of HIV-infected patients

Identifying factors that determine concentrations of antiretroviral drugs in CD4 cells are important for improving therapeutic efficacy. Experimental models indicate that the nucleoside reverse transcriptase inhibitor lamivudine is transported by the organic cation transporters 1 and 2 (OCT1 and OCT2, respectively). Here, we tested whether OCT1 and OCT2 contribute to the uptake of lamivudine into native CD4 cells of human immunodeficiency virus (HIV)-infected individuals. CD4 cells obtained by non-activated cell sorting from 35 individuals with HIV-1 infection were incubated with lamivudine (10 μM, 30 min), and intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate were determined by liquid chromatography tandem mass spectrometry. The expression of OCT1 and OCT2 mRNA was measured by quantitative real-time polymerase chain reaction (PCR). A model of OCT2-transfected CD4 cells was established for mechanistic investigations. Intracellular concentrations of lamivudine and its active metabolite lamivudine triphosphate showed strong linear correlations with each other and with the CD4 mRNA expression of OCT1 and OCT2 (r > 0.80). Coincubation with protease inhibitors (ritonavir, nelfinavir) that inhibit OCT1 and OCT2 yielded decreased intracellular concentrations of lamivudine and lamivudine triphosphate. Incubation of CD4 cells from healthy donors transfected with an OCT2 expression vector yielded increased concentrations of lamivudine and lamivudine triphosphate. Our studies indicate a role of OCT1 and OCT2 for the cellular accumulation of lamivudine in HIV-infected individuals.

Organic Anion and Cation SLC22 “Drug” Transporter (Oat1, Oat3, and Oct1) Regulation during Development and Maturation of the Kidney Proximal Tubule

Proper physiological function in the pre- and post-natal proximal tubule of the kidney depends upon the acquisition of selective permeability, apical-basolateral epithelial polarity and the expression of key transporters, including those involved in metabolite, toxin and drug handling. Particularly important are the SLC22 family of transporters, including the organic anion transporters Oat1 (originally identified as NKT) and Oat3 as well as the organic cation transporter Oct1. In ex vivo cultures of metanephric mesenchyme (MM; the embryonic progenitor tissue of the nephron) Oat function was evident before completion of nephron segmentation and corresponded with the maturation of tight junctions as measured biochemically by detergent extractability of the tight junction protein, ZO-1. Examination of available time series microarray data sets in the context of development and differentiation of the proximal tubule (derived from both in vivo and in vitro/ex vivo developing nephrons) allowed for correlation of gene expression data to biochemically and functionally defined states of development. This bioinformatic analysis yielded a network of genes with connectivity biased toward Hnf4α (but including Hnf1α, hyaluronic acid-CD44, and notch pathways). Intriguingly, the Oat1 and Oat3 genes were found to have strong temporal co-expression with Hnf4α in the cultured MM supporting the notion of some connection between the transporters and this transcription factor. Taken together with the ChIP-qPCR finding that Hnf4α occupies Oat1, Oat3, and Oct1 proximal promoters in the in vivo differentiating rat kidney, the data suggest a network of genes with Hnf4α at its center plays a role in regulating the terminal differentiation and capacity for drug and toxin handling by the nascent proximal tubule of the kidney.