OATP1B1 mRNA Research Articles

Use of Traditional and Proteomic Methods in the Assessment of a Preclinical Model of Preeclampsia.

Recent studies have demonstrated downregulation of breast cancer resistance protein (BCRP/ABCG2) in placenta obtained from women with preeclampsia (PE). BCRP is highly expressed in placenta and plays an important role in preventing xenobiotics from entering the fetal compartment. While PE is often therapeutically managed with drugs that are substrates of BCRP, there are limited studies on the impact of PE on fetal drug exposure. Due to ethical concerns, use of preclinical models is an important approach. Thus, by using proteomic and traditional methods, we characterized transporter changes in an immunological rat model of PE to determine its utility and predictive value for future drug disposition studies. PE was induced by daily administration of low-dose endotoxin (0.01-0.04 mg/kg) to rats on gestational days (GD) 13 to 16, urine was collected, and rats were sacrificed on GD17 or GD18. PE rats shared similar phenotype to PE patients, including proteinuria, and increased levels of TNF-α and IL-6. Transcript and protein levels of Bcrp were significantly downregulated in placenta of PE rats on GD18. Mdr1a, Mdr1b and Oatp2b1 mRNA were also decreased in PE. Proteomics revealed activation of various hallmarks of PE including immune activation, oxidative stress, endoplasmic reticulum stress and apoptosis. Overall, our results demonstrated that the immunological PE rat model exhibits numerous similarities to human PE along with dysregulation of placental transporters. Therefore, this model may be useful in examining the impact of PE on the maternal and fetal disposition of BCRP substrates. Significance Statement Fully characterizing preclinical models of disease is necessary in order to determine their validity to human conditions. Combining traditional and proteomic methods of model characterization, we identified numerous phenotypic similarities between our model of PE and human disease. The alignment with human pathophysiological changes allows for more confident use of this preclinical model.

6-Hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor elevated in renal failure patients

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [3H]estrone-3-sulfate (E1S), although some directly inhibited [3H]E1S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [3H]E1S uptake, even after the inhibitor was washed out before [3H]E1S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [3H]E1S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [3H]E1S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.

Post-transcriptional regulation of OATP2B1 transporter by a microRNA, miR-24

Human OATP2B1, a member of organic anion transporting polypeptide family, is expressed in several tissues, including small intestine and liver, and contributes to cellular uptake of endogenous compounds and various drugs. Altered expression of OATP2B1 affects pharmacokinetics of substrate drugs; however, limited information is available on the regulation of OATP2B1 expression. This study aimed to explore microRNAs (miRNAs) that regulate OATP2B1 expression using HEK293 cells transfected with an expression plasmid of OATP2B1 including 3′-UTR (HEK/OATP2B1) and Caco-2 as a model of human intestine. Computational analysis predicted that three miRNAs, miR-143, miR-125b and miR-24, may bind to the 3′-UTR of OATP2B1 mRNA. A luciferase assay using a plasmid containing the 3′-UTR of OATP2B1 gene demonstrated that only miR-24 significantly reduced its expression. The overexpression of miR-24 decreased the expression of OATP2B1 mRNA and protein in HEK/OATP2B1 and Caco-2 cells and uptake of [3H]estrone-3-sulfate by HEK/OATP2B1 cells. However, a statistically significant increase of endogenous OATP2B1 expression was not observed by miR-24 inhibitor in Caco-2 cells. In conclusion, it was found that miR-24 negatively regulates OATP2B1 expression, resulting in suppression of OATP2B1 activity, while its contribution to regulation of apparent expression of OATP2B1 is considered to depend on tissues and cell types.

Absence of OATP1B (Organic Anion-Transporting Polypeptide) Induction by Rifampin in Cynomolgus Monkeys: Determination Using the Endogenous OATP1B Marker Coproporphyrin and Tissue Gene Expression.

Organic anion-transporting polypeptide (OATP) 1B induction is an evolving mechanism of drug disposition and interaction. However, there are contradictory reports describing OATP1B expression in hepatocytes and liver biopsies after administration of an inducer. This study investigated the in vivo effects of the common inducer rifampin (RIF) on the activity and expression of cynomolgus monkey OATP1B1 and OATP1B3 transporters, which are structurally and functionally similar their human OATP1B counterparts. Multiple doses of oral RIF (15 mg/kg) resulted in a steady 3.9-fold increase of CYP3A biomarker, 4β-hydroxycholesterol (4βHC), in the plasma samples collected before each RIF dose during the treatment period (i.e., predose). In contrast, the predose plasma levels of OATP1B biomarkers coproporphyrin (CP) I and CPIII did not change when compared with RIF treatment. The trough concentration, area under plasma concentration-time curve (AUC), and half-life of RIF decreased markedly during RIF treatment, suggesting that RIF induced its own clearance. Consequently, RIF treatment increased CPI and CPIII AUCs substantially after a single administration and, to a lesser extent, after multiple administrations compared with preadministration AUCs. In addition, OATP1B1 and OATP1B3 mRNA expressions were not modulated by RIF treatment (0.85-1.3-fold), whereas CYP3A8 expression was increased 3.7-5.0-fold, which correlated well with the predose levels of CP and 4βHC. Rifampin treatment showed 2.0-3.3-fold increases in P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) expression in the small intestine. Collectively, these findings indicate that monkey OATP1B and OATP1B3 are not induced by RIF, and further investigation of OATP1B induction by RIF and other nuclear receptor activators in humans is warranted. SIGNIFICANCE STATEMENT: In this study, combined endogenous biomarker and gene expression data suggested that RIF did not induce OATP1B in cynomolgus monkeys. For the first time, the study determines transporter gene expression in the nonhuman primate liver, gut, and kidney tissues after administration of RIF for 7 days, leading to a better understanding of the induction of OATP1B and other major drug transporters. Finally, it provides evidence to strengthen the claim that coproporphyrin is a suitable endogenous probe of OATP1B activity.

Regulation of Organic Anion Transporting Polypeptide 2B1 Expression by MicroRNA in the Human Liver.

Organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is an uptake transporter expressed in several tissues, including the liver, intestine, brain, kidney, and skeletal muscle. Hepatocyte nuclear factor 4 alpha (HNF4α) is known as an important transcriptional factor of OATP2B1 in the liver. It has been reported that there are large interindividual differences in OATP2B1 mRNA and protein expressions in human livers. The mechanism causing the interindividual differences in OATP2B1 expression is still unclear. MicroRNAs (miRNAs) control gene expression by leading translational repression and/or degradation of the target mRNA. There is no significant correlation between OATP2B1 mRNA and protein expression, suggesting that post-transcriptional regulating mechanisms, such as miRNAs, play an important role in the interindividual differences in OATP2B1 expression. In this study, we hypothesized that certain miRNAs cause the interindividual differences in OATP2B1 expression in the human liver. In silico analysis showed that miR-24 was a candidate miRNA regulating OATP2B1 expression. It has been reported that miR-24 degrades HNF4α mRNA expression. We revealed that the miR-24 expression level was negatively correlated with OATP2B1 mRNA, protein, and HNF4α mRNA expression levels in human livers. Transfection by the miR-24 precursor decreased the luciferase activity in the transfected cells with the vector containing the OATP2B1 3' untranslated region (3'UTR) or SLCO2B1 promoter region. In HepaRG cells, miR-24 decreased the OATP2B1 and HNF4α expression levels. These results suggest that miR-24 represses not only the translation of OATP2B1 but also the transcription of OATP2B1 by HNF4α mRNA degradation.

LncRNA HOTAIR modulates the expression of OATP1B1 in HepG2 cells by sponging miR-206/miR-613

OATP1B1 is an important drug transporter with a complex regulatory mechanism. In this study, we wanted to investigate how LncRNA HOTAIR regulates the expression of OATP1B1 through its action on miR-206/miR-613 in HepG2 cells. The expression level of LncRNA HOTAIR, miR-206/miR-613, and OATP1B1 mRNA was detected by RT-qPCR, and the OATP1B1 protein level was detected by Western blot. The competitive endogenous RNA mechanism was validated by bioinformatics analysis and a dual-luciferase reporter gene assay. Our results showed that over- or under-expression of LncRNA HOTAIR correspondingly significantly increased or decreased the protein level of OATP1B1 in HepG2 cells, while no significant change in OATP1B1 mRNA level was observed. In addition, the stimulatory or inhibitory effect of LncRNA HOTAIR on OATP1B1 protein expression was correspondingly reversed by miR-206/miR-613 mimic or miR-206/miR-613 inhibitor. Finally, the reporter gene assay revealed that LncRNA HOTAIR can sponge miR-206/miR-613, which breaks the binding site of miR-206/miR-613 and OATP1B1 mRNA 3’-UTR, eliminating the stimulatory effect of LncRNA HOTAIR on OATP1B1 protein. Thus, we conclude that LncRNA HOTAIR can affect the expression of OATP1B1 in HepG2 cells by sponging miR-206/miR-613, which, in turn, prevents the binding of miR-206/miR-613 and OATP1B1 mRNA 3’-UTR.

Berberine Promotes OATP1B1 Expression and Rosuvastatin Uptake by Inducing Nuclear Translocation of FXR and LXRα.

Berberine, a quinoline alkaloid, can be used in combination with statins to enhance hypolipidemic effects and reduce the dose and side effects of statins. The hypolipidemic effects of statins in the liver are mainly regulated by organic anion transporting polypeptides (OATPs), and the expression of OATPs is regulated by nuclear receptors. Berberine has been reported to affect nuclear receptors. However, whether berberine affects the uptake of statins by regulating nuclear receptor-mediated expression of OATPs remains to be determined. The aim of this study was to investigate the effects of berberine on the expression of OATP1B1 in HepG2 and explore the underlying mechanism. In HepG2 cells, 10–50 μM berberine significantly increased the uptake of rosuvastatin by inducing the expression of OATP1B1 mRNA and protein. Dual-Luciferase reporter assay showed that luciferase activity of hFXR and hLXRα activated OATP1B1 promoter was increased by 2.5–50 μM berberine in a concentration-dependent manner, with half-maximal effective concentration (EC50) of 12.19 ± 0.86 and 32.15 ± 2.32 μM, respectively. In addition, after silencing FXR or LXRα by small interfering RNA (siRNA), berberine-induced OATP1B1 expression was significantly attenuated. Western blot analysis of FXR and LXRα protein levels in the cytoplasm and nucleus of HepG2 cells after treatment with berberine showed that berberine induced nuclear translocation and activation of FXR and LXRα. In conclusion, berberine-induced nuclear translocation of FXR and LXRα could activate OATP1B1 promoter, resulting in enhanced expression of OATP1B1 and increased uptake of rosuvastatin.

Characterization of Liver- and Cancer-type-Organic Anion Transporting Polypeptide (OATP) 1B3 Messenger RNA Expression in Normal and Cancerous Human Tissues.

Membrane transport protein organic anion transporting polypeptide (OATP) 1B3 mediates the cellular uptake of many clinically important drugs including anti-cancer drugs (e.g., paclitaxel). In addition to the well-recognized hepatic expression and function of OATP1B3 [herein named liver-type (Lt) OATP1B3], OATP1B3 also expresses in cancers and has been postulated to play a role in cancer therapy, presumably by facilitating the influx of anti-cancer drugs. Recently, a cancer type (Ct)-OATP1B3 mRNA variant was identified in colon and lung cancer tissues, which encodes truncated Ct-OATP1B3 with negligible transport activity. Other than in colon and lung cancers, reports on mRNA expression of OATP1B3 in other cancers cannot distinguish between the Ltand Ct-OATP1B3. The current studies were designed to characterize the expression of Lt- and Ct-OATP1B3 mRNA in ovarian, prostate, bladder, breast, and lung tissues. Lt- and Ct-OATP1B3 isoform-specific PCR primers were utilized to determine the mRNA levels of Lt- and Ct-OATP1B3, respectively. An expression vector expressing green fluorescent protein (GFP)-tagged Lt-OATP1B3 was transiently transfected into the ovarian cancer cell line SKOV3. Confocal live-cell microscopy was utilized to determine the localization of GFP-Lt-OATP1B3 in SKOV3 cells. For the first time, Lt-OATP1B3 mRNA was detected in ovarian, prostate, bladder and breast cancers. The localization of GFP-Lt-OATP1B3 on the plasma membrane of SKOV3 cells after transient transfection was readily detected by confocal microscopy. Our findings are supportive of the potential role of Lt-OATP1B3 in cancer cells.

Cancer-Type OATP1B3 mRNA in Extracellular Vesicles as a Promising Candidate for a Serum-Based Colorectal Cancer Biomarker.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.

Cancer-type OATP1B3 mRNA has the potential to become a detection and prognostic biomarker for human colorectal cancer.

We aimed to clarify diagnostic and prognostic biomarker potentials of cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in colorectal cancer (CRC) patients. Ct-OATP1B3 mRNA levels in 97 CRC and adjacent normal colon tissues were measured by real-time PCR. The receiver operating characteristic curve analysis and the Kaplan-Meier curve analysis were performed to characterize its biomarker potentials. Ct-OATP1B3 mRNA showed noticeable diagnostic power (the area under the receiver operating characteristic = 0.91) in the CRC patients. Additionally, the higher/lower mRNA expression was clearly associated with better/poorer overall survival in the CRC patients (p < 0.05). Ct-OATP1B3 mRNA has the potential to be a tissue-based biomarker for definitive diagnosis and prognostic stratification in CRC.

Effects of Cangfudaotan Tang on Expression of Organic Anion Transporting Polypeptide (oatp2b1) in Liver and Kidney Tissues of Rats with Phlegm Dampness Type Polycystic Ovary Syndrome (PCOS)

Objective: To explore the effect of Cangfudaotan Tang on phlegm dampness type of PCOS and the role of oatp2b1 in transportation and transformation of phlegm dampness. Methods: 36 SD female rats were randomly divided into three groups: blank control group, model group and Cangfudaotan Tang group, 12 cases in each one. After PCOS rat models were made, rats of Cangfudaotan Tang group were treated with Cangfudaotan Tang (1.42 g/kg/d) by intragastric administration for 14 days; blank control and model group were given with isodose saline. The expression of oatp2b1 mRNA/Protein in liver and kidney tissues was measured and the level of testosterone (T), follicle stimulating hormone(FSH), estradiol (E2), luteinizing hormone(LH), Serum total cholesterol (TG), Triacylglycerols (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were detected at the same time. Results: Compared with blank control group, the expression of oatp2b1 mRNA and the level of TC, TG, LDL, LH, FSH, T in model group were significantly increased (P < 0. 05), while the level of HDL was significantly decreased (P < 0. 05); compared with model group, the expression of oatp2b1 mRNA and the level of TC, TG, LDL in Cangfudaotan Tang group were significantly lowered (P < 0.05); the level of HDL was significantly higher; the oatp2b1 protein in kidney and liver tissues had different degrees of expression, while there was no statistical significance among the three groups. Conclusions: Oatp2b1 might be one of the material bases participating in transportation and transformation of phlegmy dampness. The mechanism of Cangfudaotan Tang treating phlegm dampness type of PCOS may be achieved by regulating the expression of oatp2b1.

Unique expression features of cancer-type organic anion transporting polypeptide 1B3 mRNA expression in human colon and lung cancers.

BackgroundWe have previously identified the cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in several human colon and lung cancer tissues. Ct-OATP1B3 is a variant of the liver-type OATP1B3 (Lt-OATP1B3) mRNA, which is a hepatocyte plasma membrane transporter with broad substrate specificity. However, in cancer tissues, both the detailed characteristics of Ct-OATP1B3 mRNA expression and its biological functions remain unclear. With this point in mind, we sought to characterize Ct-OATP1B3 mRNA expression in colon and lung cancer tissues. In addition, we attempted to obtain functional implication of Ct-OATP1B3 in cancer cells.MethodsMatched pairs of cancer and normal tissues were collected from 39 colon cancer and 28 lung cancer patients. The OATP1B3 mRNA expression levels in each of these tissues were separately determined by quantitative real-time polymerase chain reaction. Mann–Whitney U test and Fisher’s exact test were used in statistical analysis. The Ct-OATP1B3 functional expression in colon cancer cells was then examined by Western blotting and transport analyses.ResultsCt-OATP1B3 mRNA, but not Lt-OATP1B3 mRNA, was abundantly expressed in colon cancer tissues at a higher detection frequency (87.2%) than that of the adjacent normal tissues (2.6%). Furthermore, it was found that Ct-OATP1B3 mRNA expression was often detected in early colon cancer stages (88.9%, n = 18), and that its expression was associated with well-differentiated colon cancer statuses. On the other hand, Ct-OATP1B3 mRNA also showed a predominant and cancer-associated expression profile in lung tissues, although at frequencies and expression levels that were lower than those obtained from colon cancer. As for attempts to clarify the Ct-OATP1B3 functions, neither protein expression nor transport activity could be observed in any of the cell lines examined.ConclusionsBased on the unique characteristics of the Ct-OATP1B3 mRNA expression profile identified in this study, Ct-OATP1B3 mRNA can be expected to become a biomarker candidate for use in colon (and lung) cancer diagnosis. Simultaneously, our results advance the possibility that Ct-OATP1B3 might play yet unidentified roles, in addition to transporter function, in cancer cell biology.

Novel Mechanism of Impaired Function of Organic Anion-Transporting Polypeptide 1B3 in Human Hepatocytes: Post-Translational Regulation of OATP1B3 by Protein Kinase C Activation

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.

Intestinal Ischemia-Reperfusion Suppresses Biliary Excretion of Hepatic Organic Anion Transporting Polypeptides Substrate

Intestinal ischemia-reperfusion (I/R) causes gut dysfunction and promotes multi-organ failure. The liver and kidney can be affected by multi-organ failure after intestinal I/R. Organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs) are recognized in a broad spectrum from endogenous compounds to xenobiotics, including clinically important drugs. Therefore, it is important for understanding the pharmacokinetics to obtain evidence of alterations in OATPs and OATs expression and transport activities. In the present study, we investigated the expression of rat Oatps and Oats after intestinal I/R. We used intestinal ischemia-reperfusion (I/R) model rats. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Plasma concentration and biliary excretion of sulfobromophthalein (BSP), which is used as a model compound of organic anion drugs, were measured after intravenous administration in intestinal I/R rats. Although Oat1 and Oat3 mRNA levels were not altered in the kidney, Oatp1a1, Oatp1b2 and Oatp2b1 mRNA levels in the liver were significantly decreased at 1-6 h after intestinal I/R. Moreover, Oatp1a1 and Oatp2b1 protein expression levels were decreased at 1 h after intestinal I/R. Plasma concentration of BSP, which is a typical substrate of Oatps, in intestinal I/R rats reperfused 1 h was increased than that in sham-operated rats. Moreover, the area under the concentration-time curve (AUC₀₋₉₀) in intestinal I/R rats reperfused 1 h was significantly increased than that in sham-operated rats. The total clearance (CL(tot)) and the biliary clearance (CL(bile)) in intestinal I/R rats reperfused 1 h were significantly decreased than those in sham-operated rats. Oatp1a1 and Oatp2b1 expression levels are decreased by intestinal I/R. The decreases in these transporters cause alteration of pharmacokinetics of organic anion compound. The newly found influence of intestinal I/R on the expression and function of Oatps may be a key to perform appropriate drug therapy.

Influence of Serum in Hemodialysis Patients on the Expression of Intestinal and Hepatic Transporters for the Excretion of Pravastatin

It is known that the lipid-lowering agent pravastatin, which is not metabolized by cytochrome P450, is eliminated as an unchanged drug in bile and urine. It is interesting to note that the non-renal clearance of pravastatin in end-stage renal failure patients is decreased compared with that of healthy volunteers. This study investigated the influence of uremic serum and toxins on the transport mechanisms of pravastatin to elucidate the cause of decreased non-renal clearance in end-stage renal failure patients. Caco-2 and Hep3B cells were used as models of intestinal epithelial cells and hepatocytes respectively. Normal and uremic serum were deproteinized by treatment with methanol. 3-Carboxy-4-methyl-5propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, 3-indoxyl sulfate, and p-cresol were chosen as uremic toxins. Uremic serum-treated Caco-2 cells exhibited significantly increased accumulation of pravastatin and significantly decreased expression of MRP2 mRNA compared with normal serum-treated Caco-2 cells. In addition, the expression of MRP2 mRNA tended to decrease in cells treated with CMPF, indole-3-acetic acid, or 3-indoxyl sulfate. Uremic serum-treated Hep3B cells showed a significantly decreased initial uptake rate of pravastatin; furthermore, the expressions of OATP1B1 and OATP2B1 mRNA were decreased compared to normal serum-treated Hep3B cells. These results suggest that the decrease in the non-renal clearance of pravastatin in end-stage renal failure patients is partly induced by the downregulation of intestinal MRP2 and hepatic OATP1B1 and/or OATP2B1 by various uremic toxins in end-stage renal failure patients.

Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β- d-glucuronide (E 2-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly ( p < 0.01) decreased the accumulation of estrone-3-sulfate and E 2-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant ( p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation ( R 2 = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Regional absorption of fexofenadine in rat intestine

Purpose Expression of Pgp along the GI-tract of rats increases towards distal segments. We assessed the in vivo relevance by determining the absorption of fexofenadine from proximal and distal parts of rat intestine. Since it has been reported that fexofenadine is also actively taken up by OATP/oatps, we quantified rat oatp1a5 and oatp2b1 mRNA in gut mucosa to elucidate a possible contribution of an oatp-mediated active uptake of fexofenadine. Methods Absorption was determined after drug administration into ligated segments of rat duodenum/upper jejunum and terminal ileum, respectively, in the absence and the presence of the Pgp blocker PSC-833s. Portal vein blood was sampled. Expression of Pgp in the mucosa of proximal and distal intestinal segments was analyzed by Western Blot. Oatp1a5 and oatp2b1 mRNA in intestinal mucosa was quantified by real-time qRT-PCR. Results Portal vein AUC 0–90 min of fexofenadine was significantly higher after absorption from proximal segments compared to distal segments. Accordingly, Pgp expression was significantly lower in proximal compared to distal segments. Inhibition of Pgp by PSC-833 affected fexofenadine absorption only in distal segments resulting in AUC values comparable to the proximal data. Both oatp1a5 and oatp2b1 mRNA expression increased along the small intestine. Conclusion The study demonstrates that Pgp is responsible for a limitation of fexofenadine absorption from distal small intestine. These findings are supported by the found pattern of expression for oatp1a5 and oatp2b1, showing that an active oatp-mediated uptake plays no role for fexofenadine absorption in rats.

DNA Methylation Profiles of Organic Anion Transporting Polypeptide 1B3 in Cancer Cell Lines

Multispecific organic anion transporter, OATP1B3/SLCO1B3, is expressed in several cancer cell lines as well as tumor tissues, and its expression sensitizes the cells to some anti-cancer agents. The present study was aimed to characterize the DNA methylation profiles around the transcriptional start site (TSS) of OATP1B3 and correlate them with the mRNA expression in cancer and immortalized cell lines. The mRNA expression and DNA methylation profiles of OATP1B3 were determined by RT-PCR and bisulfite sequencing, respectively. The expression of OATP1B3 mRNA was detected in DLD-1, TFK-1, PK-8, and PK-45P cells, but below the limit of detection in HepG2, Caco-2, and HEK293 cells. Bisulfite sequencing demonstrated that CpG dinucleotides around the TSS are differentially methylated among cell lines and partly associated with the mRNA expression profile of OATP1B3. Furthermore, treatment with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, significantly increased the mRNA expression of OATP1B3 in HepG2 and Caco-2 cells by 18- and 14-fold, respectively, but not in DLD-1 and TFK-1 cells. DNA methylation-dependent gene silencing is at least partly involved in the regulation of OATP1B3 expression in cancer/immortalized cell lines.

The Prostaglandin Transporter OATP2A1 Is Expressed in Human Ocular Tissues and Transports the Antiglaucoma Prostanoid Latanoprost

Latanoprost, a prostaglandin F(2alpha) analogue, has become one of the most widely used medications for the treatment of glaucoma. The authors hypothesized that organic anion transporting polypeptides (OATPs) are responsible for the uptake of latanoprost into ocular tissues and, hence, that they contribute to the interindividual differences in drug concentrations and effects. Expression of prostaglandin (PG) transporters (OATP2A1, OATP2B1) in human ocular tissues was determined using real-time RT-PCR and immunofluorescence. The inhibitory interactions between latanoprost and its active metabolite (the free acid) and the uptake of prototypical substrates (PGE(2) and bromosulfophthalein) were tested in stably transfected human embryonic kidney cells overexpressing either OATP2A1 or OATP2B1. These cells were also used to investigate whether latanoprost and latanoprost acid are substrates of OATP2A1 or OATP2B1. OATP2A1 and OATP2B1 mRNA expression was highest in the choroid/retinal pigment epithelium (RPE) complex and ciliary body. OATP2A1 protein expression was most prominent in the RPE and in epithelial and endothelial cell layers of anterior segment tissues, such as cornea, conjunctiva, iris, and ciliary body, whereas OATP2B1 protein was additionally expressed in trabecular meshwork, Schlemm canal, and choroidal vasculature. Latanoprost and latanoprost acid significantly inhibited both OATP2A1 and OATP2B1. Uptake experiments demonstrated that latanoprost acid is effectively transported by OATP2A1 (affinity constant [K(m)], 5.4 microM; maximum uptake rate [V(max)], 21.5 pmol/mg protein/min) and less effectively by OATP2B1. The results presented herein suggest that at least OATP2A1 plays a role in the intraocular disposition of the therapeutically used prostanoid latanoprost.

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K m and V max values were 66.8 ± 30.3 μM and 49.7 ± 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na +-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).