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Background: Recurrence determines the postoperative prognosis with hepatocellular carcinoma (HCC). It is unknown how the liver dysfunction involving organic anion transporter failure causes the occurrence of HCCs. This study was designed to elucidate the link between liver dysfunction and multicentric occurrence (MO) after radical hepatectomy. Methods: Forty-nine samples of noncancerous liver tissue from HCC patients within the Milan criteria who were treated at our institution between January 2004 and August 2008 were examined as a training set by using genome-wide gene expression analysis. Using the independent 2-institutional cohort of 134 patients between September 2008 and December 2009, we performed a validation study using tissue microarray analysis. Cox proportional hazard regression analyses for MFS were performed to estimate the risk factors. Results: In the Gene Ontology database (GO:0015711), SLC22A7 expression was the best predictor of MO-free survival [MFS] (Fold, 0.726; P = 0.001). High SLC22A7 gene expression prevented the occurrence of HCC after hepatectomy (odds ratio [OR], 0.2; P = 0.004). Multivariate analyses identified SLC22A7 expression as an independent risk factor (OR, 0.3; P = 0.043). In the validation study, multivariate analyses of MFS identified SLC22A7 expression as an independent risk factor (OR, 0.5; P = 0.012). As judged by gene set enrichment analysis, SLC22A7 down regulation was associated with mitochondrion ( P = 0.008) and oxidoreductase activity ( P = 0.006). Sirtuin 3 as a regulator of mitochondrial metabolism also determined MFS ( P = 0.018). Conclusions: The mitochondrial pathways may affect SLC 22A7 function to promote the occurrence of HCC. (Word count: 246). [ABSTRACT FROM AUTHOR]

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3302-3308, 2013 [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Renal uptake of Tc-99m-MG3 involves organic anion transporter (OAT). Treatment with drugs showing OAT affinity might interfere with renal uptake of Tc-99m-MAG3, leading to misinterpretation in Tc-99m-MAG3. This study was conducted to discuss a possible drug interference with Tc-99m-MAG3 diagnosis on OAT sites. Methods: Renal uptake and plasma clearance of Tc-99m-MAG3 were analyzed in healthy volunteers under control and OAT1 and OAT3 related drug treatment conditions. An in vitro uptake study using OAT1 or OAT3 expressing cells was also conducted. Results: Both PAH and probenecid treatment induced delays in Tc-99m-MAG3 clearance from blood, and reductions in the renal uptake clearance. As a result, the normalized effective renal plasma flow estimated from Tc-99m-MAG3 clearance was significantly underestimated, whereas the glomerular filtration rate estimated from plasma creatinine levels was unchanged. The transport activity of Tc-99m-MAG3 was higher in OAT1-expressing cells than in OAT3-expressing cells. Conclusion: Drugs with OAT1 affinity affect the renal uptake of Tc-99m-MAG3 and blood clearance. This might cause misinterpretation of functional diagnosis of the kidney using Tc-99m-MAG3. [Copyright &y& Elsevier]

The thiazide diuretic chlorothiazide is poorly metabolized, and is predominantly excreted via the kidneys. We have previously shown that chlorothiazide is transported by ATP-binding cassette transporter G2, suggesting a potential role for this transporter in apical efflux of chlorothiazide in the kidney. However, because of the poor passive permeability of the drug, it is likely that uptake transporters on the basolateral membrane are also involved to facilitate vectorial transport in the renal proximal tubule. Two suggested candidate transporters for this role are the human organic anion transporters, OAT1 and OAT3. By using mammalian cells stably expressing these transporters, we have demonstrated OAT1- and OAT3-dependent uptake of chlorothiazide with Michaelis constant values of 14.5 and 37.6 µM, respectively. Furthermore, we have found that probenecid, furosemide, and diclofenac inhibit chlorothiazide transport by OAT1 and OAT3, of which the probenecide-mediated inhibition may be of clinical importance. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1683-1687, 2013 [ABSTRACT FROM AUTHOR]

Objectives Using rabbit corneal epithelial cells ( RCECs), the transport of a nonsteroidal anti-inflammatory drug ( NSAID) [3H]ketoprofen across the cornea was investigated with the aim of revealing the mechanism of uptake. Methods [3 H]Ketoprofen transport was evaluated by measuring the permeability across the RCECs layers. Key findings [3 H]Ketoprofen uptake was time, temperature and pH dependent. Maximal uptake occurred from a solution with a pH of 5.25. Uptake was also reduced by metabolic inhibitors (sodium azide and dinitrophenol ( DNP)) and proton-linked monocarboxylate transporter ( MCT) inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone ( FCCP) and α-cyano-4-hydroxycinnamic acid ( CHC)). [3 H]Ketoprofen uptake was significantly inhibited by various monocarboxylates and other NSAIDs and by MCT and/or organic anion transporter ( OAT) inhibitors probenecid and p-aminohippurate, but was unaffected by organic anion-transporting polypeptide ( OATP) inhibitors bromosulfophthalein and taurocholate. The specific uptake of [3 H]ketoprofen was saturable. Eadie-Hofstee plots indicated the involvement of high- and low-affinity components. The Km and Vmax values for the high- and low-affinity components of [3H]ketoprofen uptake were 0.56 and 24 m m, and 0.37 and 61 nmol/min/mg of protein, respectively. Benzoic acid, a substrate and inhibitor of MCTs, selectively inhibited low-affinity [3 H]ketoprofen uptake. Conversely, indometacin inhibited high-affinity [3 H]ketoprofen uptake. Conclusion The results of this study suggest that the monocarboxylate transport system partly accounts for the low-affinity component of [3 H]ketoprofen uptake, and that the carrier-mediated transport systems such as the OAT family, shared by NSAIDs account for the high-affinity component. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways. Materials and methods: Localization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer. Results: In rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes'' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts. Conclusions: These data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 and 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Rats were injected intraperitoneally with 0.64nmol/kg/day 1,25(OH)2D3 in 1ml/kg corn oil for 5days. The plasma and urine concentrations of JBP485 after intravenous administration and the uptake of JBP485 in kidney slices in vitro were determined by liquid chromatography/tandem mass spectrometry. Quantitative polymerase chain reaction, western blotting, immunohistochemical analysis and immunofluorescence were used to determine the changes in the expression of organic anion transporter (Oat)1 and Oat3 in rat kidney in response to 1,25(OH)2D3 treatment. The plasma concentrations and AUCs of JBP485 were significantly increased, while the renal clearance of JBP485 and uptake of JBP485 in kidney slices were significantly decreased after 1,25(OH)2D3 treatment. These results confirmed that 1,25(OH)2D3 inhibited renal excretion of JBP485. Moreover, 1,25(OH)2D3 decreased expression of Oat1 and Oat3 in rat kidney. Our results are novel in demonstrating an interaction between JBP485 and 1,25(OH)2D3 when they are co-administered. The mechanism of interaction between JBP485 and 1,25(OH)2D3 could be explained at least in part by inhibitory effect of 1,25(OH)2D3 on expression of Oats in rat kidney. [Copyright &y& Elsevier]

Abstract: Flavonoids modulate cell signaling and inhibit oxidative enzymes. After oral consumption, they circulate in human plasma as amphiphilic glucuronide or sulfate conjugates, but it is unknown how these physiological metabolites permeate into cells. We examined the mechanisms of uptake of these conjugates into hepatocellular carcinoma (HepG2) cells, and found that uptake of quercetin-3′-O-sulfate was saturable and temperature-dependent, indicating the involvement of carrier-mediated transport. Quercetin-3-O-glucuronide was taken up predominantly via passive diffusion in these cells. Quantitative real-time PCR analysis showed high expression of OATP4C1, followed by OAT2, OAT4 and low expression of OATP1B1 in HepG2 cells, and addition of inhibitors of OATs and OATPs resulted in a significant reduction in quercetin-3′-O-sulfate uptake. The accumulation of quercetin-3′-O-sulfate was further evaluated in HEK293 cells expressing OAT2, OAT4 and OATP4C1. Uptake of quercetin-3′-O-sulfate was 2.3- and 1.4-fold higher in cells expressing OAT4 and OATP4C1 at pH 6.0, respectively, than in control HEK293 cells. siRNA knockdown of OATP4C1 expression in HepG2 cells reduced uptake of quercetin-3′-O-sulfate by ∼40%. This study highlights a role for OATs and OATPs in the cellular uptake of biologically active flavonoid conjugates. [Copyright &y& Elsevier]

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (∙ψ)- and Cl+- dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na+-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl+-dependent manner at the expense of an electrochemical gradient of protons, especially ∙ψ, across the membrane. The ∙ψ- and Cl+- dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'- disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct. [ABSTRACT FROM AUTHOR]

Abstract: Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and NO synthase. Supplementation of BH4 potentially targets cardiovascular dysfunction as well as inherited BH4 deficiencies and BH4-responsive phenylketonuria. However, the high cost/effect ratio of the recommended daily dose of BH4 supplementation acts against further popularization of this therapy. The aim of this study was to attenuate urinary excretion with the intention of improving efficacy of BH4 supplementation. The rapid excretion of BH4 in the urine was confirmed to be the major route of supplemented BH4 loss. In addition to glomerular filtration into the urine, a dominant rapid exclusion by renal secretion was observed in rats (T(1/2) =16min) when the plasma BH4 was higher than about 1nmol/mL (more than10 times higher than normal), due to BH4 supplementation. The rapidity of the process was slowed by prior administration of cyclosporin A, a representative anti-excretory drug, and the excretion decelerated to a moderate rate (T(1/2) =53min). By the combined administration of BH4 plus cyclosporin A, the blood BH4 levels were dramatically elevated. It was hypothesized that the drug interfered with kidney excretion of BH4 rather than by attenuating organ tissue distribution by inhibiting biopterin uptake from the plasma. Consistent with this hypothesis, biopterin levels after BH4 administration were elevated in major organs in the presence of anti-excretory drugs without notable change in their BH4 fraction which was consistently 95% or higher regardless of combined administration with the drugs. Targeting these putative transporters would be a promising approach for improving the efficiency of BH4 supplementation therapy. [Copyright &y& Elsevier]

Abstract: Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K m and V max were 0.679±0.007mM and 0.807±0.006nmol/mgprotein/30s for OAT1, 0.632±0.014mM and 1.303±0.015nmol/mgprotein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K i values of 92±9μM and 197±21μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K i values of 88±12μM and 160±16μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin. [Copyright &y& Elsevier]

Background. p-Cresyl sulfate (PCS), a recently identified anionic uremic toxin, is the main circulating metabolite of p-cresol. In cases of chronic kidney disease (CKD), it might be associated with cardiovascular outcomes and the progression of CKD. However, the renal excretion pathway of PCS is currently unknown. The objective of the present study was to determine whether organic anion transporters (OATs), which are renal tubular basolateral membrane transporters, play an important role in this process.Methods. The uptake of PCS was investigated using rat renal cortical slices and human proximal tubular cells (HK-2). The active uptake velocity was calculated by subtracting the uptake velocity at 4°C (nonspecific uptake) from that at 37°C.Results. As evidenced by renal cortical slice experiments, the uptake of PCS was saturable with a mean Km of 231.6 μM, indicating that the active transport is involved in the basolateral uptake of PCS. Similar results were also observed in HK-2 cells. The active transport of PCS was significantly suppressed by inhibitors of OATs, such as probenecid, benzylpenicillin, p-aminohippuric acid and estrone sulfate. Similar inhibitions were observed in the presence of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropionate, OATs substrates among uremic toxins. In contrast, digoxin and tetraethylammonium that did not interact with OATs had little inhibitory effect.Conclusions. The findings of the present study strongly suggest that PCS serves as a substrate for OATs, is preferentially recognized by OAT3 and plays a key role in the renal tubular secretion process. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) play key roles in the influx and efflux transport of endogenous substrates in the brain and cerebrospinal fluid. The organic anion transporter (OAT) 3 and organic cation transporter (OCT) 3, which belong to the solute carrier (SLC) 22A family, are expressed at the BBB and BCSFB, and regulate the excretion of endogenous and exogenous organic anions and cations. Our recent research provides novel molecular and functional evidence that indoxyl sulfate, an anionic uremic toxin, undergoes efflux transport at the BBB via OAT3 and creatinine, a uremic guanidino compound, undergoes efflux transport at the BCSFB via OCT3. Renal impairment is associated with the accumulation of uremic toxins in blood and uremic encephalopathy. It is conceivable that uremic encephalopathy is related to inhibition or dysfunction of efflux transport systems for uremic toxins in the brain. Here, we review the function of OAT3 and OCT3 at the BBB and BCSFB in the context of their roles in the progression of renal failure. [ABSTRACT FROM AUTHOR]

Abstract: Organic anion transporter 1 (Oat1), first identified as NKT, is a multispecific transporter responsible for the handling of drugs and toxins in the kidney and choroid plexus, but its normal physiological role appears to be in small molecule metabolite regulation. Metabolites transported by Oat1 and which are altered in the blood and urine of the murine Oat1 knockout, may serve as templates for further drug design. This may lead to better tissue targeting of drugs or design of Oat1 inhibitors that prolong the half-life of current drugs. Due to the multispecificity of the transporter, 19 of known targeted metabolites have different chemical structures and properties that make constructing a common pharmacophore model difficult. Here we propose an approach that clustered the metabolites into four distinct groups which allowed for the construction of a consensus pharmacophore for each cluster. The screening of commercial molecular databases determined the top candidates whose interaction with Oat1 was confirmed in an experimental model of organic anion transport. Thus, these candidate selections represent potential molecules for further drug design. [Copyright &y& Elsevier]

The response to chemotherapy by tumor cells depends on the concentration of cytostatics accumulated inside the cells. The accumulation of anticancer drugs in tumor cells is mainly dependent on functional expression of efflux and influx transporters and to a minor extent on passive diffusion through the membrane. Efflux transporters of the ABC family are partially responsible for the chemoresistance of cancer cells by secreting these cytostatics. Over the past decades, the role of ABC transporters in the chemoresistance of various malignant tumors has been very well documented. By contrast, very little is known about the impact on tumor therapy of influx transporters belonging to the solute carrier transporters (SLC family). In this review, we focus on the interaction of SLC22 transporters with cytostatics, the expression of these transporters in tumor cells as well as their impact on the chemosensitivity of cancer cells. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 200-208. The choroid plexuses (CP) are responsible for transport of micronutrients into brain and clearance of toxic compounds, in addition to its barrier function and production of CSF. Multidrug resistance-associated protein (Mrp) 4 is one transport protein highly expressed in CP tissue and is characterized as a versatile pump for toxicants and signalling molecules. Aim of the study was to determine transport characteristics of a fluorescent cAMP analog in rat CP and to define whether fluo-cAMP can be used for analyses of function, substrate/inhibitor specificity and regulation of Mrp4. Confocal imaging was used to analyze transport mechanisms in absence and presence of various modulators of organic anion transport in freshly isolated and functionally intact CP. Fluo-cAMP transport was saturable, selective, concentrative and metabolism-dependent, following an active two-step mechanism composed of apical uptake into epithelial cells and basolateral efflux. Uptake included a Na+-dependent and a Na+-independent component and was inhibited by estrone sulfate, taurocholate and sildenafil indicating involvement of organic anion transporting polypeptide Oatp1a5. Efflux was composed of an indirect Na+-dependent component and a component inhibitable by, for example, the MRP4 substrates/inhibitors, sulindac sulfide and 4-(2-aminoethyl) benzenesulfonyl fluoride. Therefore, fluo-cAMP can be used as fluorescent model compound for studying involvement of Mrp4 in signalling pathways and neuroprotection in CP. [ABSTRACT FROM AUTHOR]

Abstract: Methotrexate has a clinically important pharmacokinetic interaction with nonsteroidal anti-inflammatory drugs (NSAIDs) mainly through its competition for tubular secretion via the renal organic anion transporter 3 (OAT3). We have previously reported the usefulness of OAT3-transfected renal tubular cells for screening of the drugs which interfere with the pharmacokinetics of methotrexate. Celecoxib, a cyclooxygenase (COX) 2 inhibitor, has not been reported to interact with methotrexate, but the mechanisms are unclear why the interaction did not occur. The purpose of this study was to evaluate the effect of celecoxib on methotrexate tubular secretion using a renal cell line stably expressing human OAT3 (S2-hOAT3), and to evaluate the pharmacokinetic interaction of the two drugs in rats. [3H]methotrexate uptake into S2-hOAT3 cells was significantly inhibited by celecoxib in a concentration-dependent manner and the Ki value was 35.3µM. However, methotrexate serum concentrations and urinary excretion of methotrexate over 24h in rats were not affected by celecoxib (50, 200mg/kg). Celecoxib serum concentrations were increased by the increase in celecoxib dosage and the maximum drug concentration (Cmax) was 20.6µM (celecoxib 200mg/kg), which did not reach the Ki value obtained in the in vitro study. These results indicated that celecoxib inhibited the secretion of methotrexate via hOAT3, which suggested that celecoxib was a substrate of hOAT3. However, co-administration of the two drugs at clinical dosage did not affect the pharmacokinetics of methotrexate, because the serum concentrations did not reach the Ki value. Although the accumulation study using S2-hOAT3 cells was useful to predict the interaction between the new drug and methotrexate in vivo, a comparison of the Ki value with the Cmax in clinical dosage was necessary to evaluate the degree of this interaction. [Copyright &y& Elsevier]

Abstract: Objective: Our goal was to use genetic variants to identify factors contributing to the muscular side effects of statins. Background: Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are usually well tolerated medications, but muscle symptoms, ranging from mild myalgia to clinically important rhabdomyolysis are an important side effect of these drugs and a leading cause of noncompliance. Recent results suggest that genetic factors increase the risk of statin-related muscle complaints. We performed a systematic review of the medical literature to determine genetic factors associated with statin myopathy. Methods: We identified English language articles relating statin myopathy and genetic diseases and gene variants via a PubMed search. Articles pertinent to the topic were reviewed in detail. Results/Conclusions: Our review suggests that some patients are susceptible to statin myopathy because of pre-existing subclinical inherited muscular disorders, or genetic variation in statin uptake proteins encoded by SLCO1B1 or the cytochrome P enzyme system. Variations in genes affecting pain perception and polymorphism in vascular receptors may also contribute to statin myopathy. None of the variants identified in this review suggested novel metabolic mechanisms leading to statin myopathy. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Many transport proteins, including the clinically important organic anion transporters (OATs), appear to function via an "alternating access" mechanism. In analyzing the kinetics of these transporters, the terms K(m) and V(max) are often treated in the field as denoting, respectively, the affinity of the substrate for the transporter and the turnover (conformational switch) rate of the substrate-transporter complex. In fact, the expressions for both these parameters have very complex forms comprising multiple rate constants from conformational switch as well as association/dissociation steps in the cycling of the transporter and, therefore, do not have straightforward physical meanings. However, if the rapid equilibrium assumption is made (namely, that the association/dissociation steps occur far more rapidly than the conformational switch steps), these expressions become greatly simplified and their physical meaning clear, though still distinct from the conventional interpretations. V(max) will be a function of not just the rate of substrate-transporter complex turnover but also the rate of the "return" conformational switch and will vary largely with the slower of these two steps (the rate-limiting step). K(m) will be seen to be related to substrate affinity by a term that varies inversely with the substrate-transporter complex turnover rate, essentially because the greater this rate, the greater the extent to which transporters will be distributed in a conformation inaccessible to substrate. Here, an intuitive approach is presented to demonstrate these conclusions. The phenomena of trans-stimulation and trans-inhibition are discussed in the context of this analysis. [ABSTRACT FROM AUTHOR]

Abstract: Hypertension is an exceedingly common disease with potentially devastating complications. Unfortunately, existing treatments are often only partially effective, indicating the utility of the development of novel therapeutics. It has recently been discovered that loss of renal organic anion transporter 3 (Oat3) results in decreased blood pressure. This suggests (regardless of the underlying mechanisms, which are, as yet, unclear) that inhibition of Oat3 function might also decrease blood pressure, and, therefore, that inhibitors of Oat3 might serve as novel drugs in the treatment of hypertension. In support of this hypothesis, it has been found that at least some potent Oat3 inhibitors can lower blood pressure. These recent findings are discussed in the context of the alternating access model that has been proposed for organic anion transporter function, with particular attention to the little-appreciated phenomenon of trans-inhibition. [Copyright &y& Elsevier]

Abstract: Coadministration of methotrexate and nonsteroidal anti-inflammatory drugs (NSAIDs) can cause a pharmacokinetic interaction and a subsequent increase in blood methotrexate concentrations. methotrexate and most NSAIDs are excreted into urine via organic anion transporter 3 (OAT3). The purpose of this study was to evaluate NSAIDs that compete less with methotrexate by using the renal cell line stably expressing human OAT3 (S2-hOAT3) in vitro. We also confirmed the pharmacokinetic interaction of methotrexate with NSAIDs in vivo. [3H]methotrexate uptake into S2-hOAT3 cells was inhibited by most NSAIDs in a concentration-dependent manner, but aspirin, salicylate, tiaramide, and acetaminophen did not inhibit uptake. Inhibition by sulindac and pranoprofen was weaker at therapeutic drug concentrations. Furthermore, methotrexate concentrations in rat serum were significantly increased in a NSAID concentration-dependent manner when concentrations of coadministered NSAIDs increased above the Ki values obtained in the in vitro study. On the other hand, drugs that were not substrates of hOAT3, such as acetaminophen, did not interact with methotrexate. The magnitude of the pharmacokinetic interaction between methotrexate and NSAIDs was significantly correlated with results of the accumulation study in vitro and was not significantly correlated with a reduction of urinary creatinine excretion. In conclusion, methotrexate and most NSAIDs are substrates of hOAT3, and those drugs compete via hOAT3 in tubular secretion, the major mechanism of the interaction between methotrexate and NSAIDs. The accumulation study using S2-hOAT3 cells might be useful for screening of potential interactions between methotrexate and new NSAIDs in vivo. [Copyright &y& Elsevier]

Reichel V, Miller DS, Fricker G. Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus. Am J Physiol Regul Integr Comp Physiol 295: R131 1-R1319, 2008. First published July 23, 2008; doi: 10.11 52/ajpregu.90373.2008.-Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepitheliallvascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na[sup+]-replacement, but was not affected by a 10-fold increase in buffer K[sup+]. In shark CP, Na[sup+]-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K[sup+] medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the MrpI inhibitor MK571. In rat CP, TR accumulation in subepitheliall vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals. [ABSTRACT FROM AUTHOR]

Background/Aims: Renal organic anion transporters (OAT1 and OAT3) localized in the basolateral membrane mediate the uptake of organic anions from the blood into proximal tubules. This study aimed to examine the effects of renal ischemia/reperfusion injury (IRI) on the expression of cortical renal OAT1 and OAT3 and the functional impact. Methods: Male rats underwent a right nephrectomy and clamping of the left renal pedicle for 50 min or sham operation, followed by reperfusion for 1, 2, 4 and 6 days. The expression of OAT1 and OAT3 was detected by RT-PCR, immunohistochemistry and Western blot analysis. Na+-K+-ATPase activity was also estimated. Results: The renal clearance of para-aminohippurate was significantly decreased on day 1 in IRI rats compared with sham-operated rats and returned to normal when the tubular injury recovered. There were significant increases in the mRNA and protein levels of OAT1 and OAT3 in renal cortex homogenates and basolateral membranes on day 1 after IRI, while on days 2 and 4 after IRI, the renal expression of OAT1 and OAT3 decreased gradually but was still significantly higher than that of the sham-operated rats. The Na+-K+-ATPase activity in renal cortex homogenates decreased significantly on day 1 after IRI but gradually increased on days 2, 4 and 6. Conclusions: Renal para-aminohippurate clearance was depressed in response to IRI; however, the expressions of renal cortex OAT1 and OAT3 were significantly elevated in the early stage of IRI which may have substantial impact on renal excretion of some drugs and toxic compounds. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Multidrug resistance protein 1 (MRP1/ABCC1) is a 190kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr324 in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated. [Copyright &y& Elsevier]

Abstract: The immunosuppressant mycophenolate mofetil (MMF) is frequently administered with calcineurin inhibitors and corticosteroids to recipients of organ transplantations. However, the renal handling of the active metabolite mycophenolic acid (MPA) and 7-O-MPA-glucuronide (MPAG) has been unclear. The purpose of the present study was to assess the interaction of MPA and MPAG with the human renal organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), by conducting uptake experiments using HEK293 cells stably expressing these transporters. MPA and MPAG inhibited the time-dependent uptake of p-[14C]aminohippurate by hOAT1 and that of [3H]estrone sulfate by hOAT3. The apparent 50% inhibitory concentration (IC50) of MPA for hOAT1 and hOAT3 was estimated at 10.7 and 1.5μM, respectively. In the case of MPAG, the IC50 values were calculated at 512.3μM for hOAT1 and 69.1μM for hOAT3. Eadie–Hofstee plot analyses showed that they inhibited hOAT1 noncompetitively and hOAT3 competitively. No inhibitory effects of tacrolimus, cyclosporin A and azathioprine on transport of p-[14C]aminohippurate by hOAT1 and of [3H]estrone sulfate by hOAT3 were observed. No transport of MPA by these transporters was observed. On the other hand, the uptake of MPAG into cells was stimulated by the expression of hOAT3, but not hOAT1. These findings propose the possibility that the administration of MMF decreases the renal clearance of drugs which are substrates of hOAT1 and hOAT3. Present data suggest that hOAT3 contributes to the renal tubular secretion of MPAG. [Copyright &y& Elsevier]

The effect of acute renal failure (ARF) induced by ischemia/reperfusion (I/R) of rat kidney on the expression of organic anion transporters (OATs) was examined. The level of serum indoxyl sulfate (IS), a uremic toxin and substrate of OATs in renal tubules, shows a marked increase with the progression of ARF. However, this increase was significantly attenuated by ingestion of cobalt. The level of mRNA and protein of both rOAT1 and rOAT3 were markedly depressed in the ischemic kidney. The uptake of p-aminohippuric acid (PAH) and estrone sulfate (ES) by renal slices of ischemic rats was significantly reduced compared to control rats. Renal slices taken from ischemic rats treated with cobalt displayed significantly elevated levels of ES uptake. Cobalt intake did not affect PAH uptake, indicating the functional restoration of rOAT3 but not rOAT1. The expression of Na+/K+-ATPase was markedly depressed in the ischemic kidney, suggesting that the inward Na+ gradient in renal tubular cells had collapsed, thereby reducing the outward gradient of α-ketoglutarate, a driving force of both rOATs. The decreased expression of Na+/K+-ATPase was significantly restored by cobalt treatment. Our results suggest that the downregulation of renal rOAT1 and rOAT3 could be responsible for the increase in serum IS level of ischemic rats. Cobalt treatment has a significant protective effect on ischemia-induced ARF, being accompanied by the restoration of rOAT3 and/or Na+/K+-ATPase function.Kidney International (2007) 71, 539–547. doi:10.1038/sj.ki.5002104; published online 24 January 2007 [ABSTRACT FROM AUTHOR]

Abstract: Investigations of the blood–brain barrier (BBB) in diabetes have yielded contradictory results. It is possible that diabetes differentially affects paracellular and transcellular permeabilities via modulation of tight junction and transport proteins, respectively. Fluorescein (FL), a marker for paracellular permeability, is a substrate for the transport proteins organic anion transporter (OAT)-3 and multidrug resistance protein (MRP)-2 at the BBB. Furthermore, MRP-2-mediated efflux of FL can be upregulated by glucose. In this study, streptozotocin-induced diabetes led to decreased brain distribution of FL measured by in situ brain perfusion, consistent with activation of an efflux transport system for FL at the BBB. This change was paralleled by increased protein expression of MRP-2, but not OAT-3, in cerebral microvessels. These data indicate that diabetes may lead to changes in efflux transporters at the BBB and have implications for delivery of therapeutics to the central nervous system. [Copyright &y& Elsevier]

Abstract: We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss. [Copyright &y& Elsevier]

The choroid plexuses (CPs) form a protective interface between the blood and the ventricular cerebrospinal fluid (CSF). To probe into the pathways by which CPs provide brain protection, we sought to evaluate the efficiency of glutathione conjugation in this barrier as a mechanism to prevent the entry of blood-borne electrophilic, potentially toxic compounds into the CSF, and we investigated the fate of the resulting metabolites. Rat CPs, as well as human CPs from both fetal and adult brains, displayed high glutathione-S-transferase activities. Using an in vitro model of the blood–CSF barrier consisting of choroidal epithelial cells cultured in a two-chambered device, we showed that glutathione conjugation can efficiently prevent the entry of 1-chloro-2,4-dinitrobenzene (CDNB) into the CSF, a model for electrophilic compounds. The duration of this enzymatic protection was set by the concentration of CDNB to which the epithelium was exposed, and this barrier effect was impaired only on severe epithelial intracellular glutathione and cysteine depletion. The conjugate was excreted from the choroidal cells in a polarized manner, mostly at the blood-facing membrane, via a high-capacity transport process, which is not a rate-limiting step in this detoxification pathway, and which may involve transporters of the ATP-binding cassette c(Abcc) and/or solute carrier 21 (Slc21) families. Supplying the choroidal epithelium at the blood-facing membrane with a therapeutically relevant concentration of N-acetylcysteine sustained this neuroprotective effect. Thus, glutathione conjugation at the CP epithelium coupled with the basolateral efflux of the resulting metabolites form an efficient blood–CSF enzymatic barrier, which can be enhanced by pharmacologically increasing glutathione synthesis within the epithelial cells.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 1165–1175. doi:10.1038/sj.jcbfm.9600267; published online 4 January 2006 [ABSTRACT FROM AUTHOR]

Aims To investigate the association between torsemide renal clearance and genetic variation in the basolaterally expressed renal organic anion transporters OAT1 and OAT3 and in the luminally situated OAT4. Methods We analysed 22 polymorphisms in the OAT coding genes SLC22A6, SLC22A8 and SLC22A11 and their haplotypes and measured torsemide renal clearance in 95 healthy men. In addition, the effect of torsemide on the OAT-mediated transport was studied in vitro. Results In stably transfected HEK293 cells torsemide (100 µm) inhibited the uptake by human OAT1, OAT3 and OAT4 by 63.1, 58.1 and 68.0%, respectively. Torsemide renal clearance ranged from 6.5 to 43.1 ml min−1 with a log-normal distribution and a geometric mean of 15.6 ml min−1 (15.0–16.1 ± SEM). No clear outlier group was observed. AA carriers of the polymorphism rs11231809 in SLC22A11 had a torsemide renal clearance of 13.3 ml min−1 (12.7–13.9) compared with 15.1 ml min−1 (14.5–15.8) in AT and 18.0 ml min−1 (16.7–19.5) in TT carriers ( P = 0.002). The two most frequent haplotypes at SLC22A11 showed an association with torsemide renal clearance. Homozygous carriage of these two haplotypes resulted in renal clearances of 21.2 ml min−1 (19.0–23.7) and 11.8 ml min−1 (10.5–13.5), respectively. No association between reanl clearance and genetic variation in SLC22A6 or SLC22A8 was observed. Conclusions Genetic variation in the gene encoding the luminally expressed OAT4 rather than in the basolaterally expressed OATs may affect the renal clearance of torsemide. [ABSTRACT FROM AUTHOR]

Abstract: Adefovir is transported by the organic anion transporter (OAT1) and the multidrug resistant protein (MRP2, 4 and 5). We studied adefovir clearance in rat after inhibition of transporters by probenecid and in mutant transport-deficient (TR−) rats, in which MRP2 is lacking. After treatment by probenecid or placebo, pharmacokinetics of adefovir 10mg/kg was studied via population nonlinear mixed effect modeling. The fraction of drug excreted in the urine was low. Renal clearance of adefovir was significantly lower (P <0.05) in probenecid TR− rats (0.03±0.02l/h) than in normal control (0.09±0.05l/h), in normal probenecid (0.10±0.07l/h) and in TR− control rats (0.13±0.07l/h). In vivo in rats MRP2 mutation alone did not affect adefovir clearance suggesting that MRP2 does not play a critical role in the secretion of adefovir. Additional pharmacological inhibition of transporters decreased renal clearance, which may reflect inhibition of compensating transport mechanisms activated when MRP2 is lacking. [Copyright &y& Elsevier]

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of arteriosclerosis. Clinical studies show that uremic toxins deteriorate the arteriosclerosis in renal failure patients. Indoxyl sulfate (IS) is a strong protein-bound uremic toxin, but the effect of IS on VSMC proliferation has not been studied. We examined the effect of IS on rat VSMC proliferation, assessed by a cell counting kit (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] assay) and by [3H]thymidine incorporation in vitro. We further evaluated a contribution of mitogen-activated protein kinase (MAPK; p44/42 MAPK) to VSMC proliferation by IS. Immunohistochemical staining was performed for VSMCs using antirat organic anion transporter (OAT)3 antibody. The mRNA expressions of platelet-derived growth factor (PDGF)-A and -C chains, and PDGF-β receptor were evaluated by real-time PCR. IS stimulated the proliferation of VSMCs in a concentration-dependent manner and activated p44/42 MAPK. Concentration of IS needed to stimulate the proliferation of rat VSMC was about 250 μM, which is compatible with that in the serum of end-stage renal failure patients. PD98059 (10 μM), a selective inhibitor of MAPK/extracellular signal-regulated kinase, inhibited the IS-induced (250 μM) VSMC proliferation and phosphorylation of MAPK. Probenecid (0.5 mM), an inhibitor and substrate of OAT, inhibited the IS-induced (250 μM) VSMC proliferation. Rat OAT3 was detected in VSMCs. The mRNA expressions of PDGF-C chain and PDGF-β receptor were significantly increased by IS. We conclude that IS directly stimulates rat VSMC proliferation and activates MAPK in vitro. This might be one of the mechanisms underlying the progression of atherosclerotic lesions in end-stage renal disease patients.Kidney International (2006) 69, 1780–1785. doi:10.1038/sj.ki.5000340; published online 12 April 2006 [ABSTRACT FROM AUTHOR]

We isolated the human liver-specific organic anion transporter gene, LST-2 (OATP8/SLCO1B3), which is exclusively expressed in the basolateral membrane of the hepatocytes. In this study, we analyzed the transcriptional regulation of the LST-2 gene in hepatocyte-derived cells and the effect of bile acid. Methods. Transcriptional activity of the LST-2 gene was measured using a human LST-2 promoter-luciferase reporter plasmid under various concentrations of bile acids. Electrophoresis mobility shift assays of farnesoid X receptor (FXR), hepatocyte nuclear factor (HNF) 1α, and HNF3β were performed. Results. Luciferase analysis showed that the 5′-flanking region from -180 to -20bp is responsible for LST-2 transcriptional activity. By site-directed mutation analysis, it was revealed that the consensus binding sites for FXR, HNF1α and HNF3β play important roles in the transcriptional activity of the LST-2 gene. By electrophoresis mobility shift assay, we observed specific protein-DNA complexes of FXR, HNF1α, and HNF-3β. Luciferase activity was increased fivefold when chenodeoxycholate or deoxycholate were added. Northern blot analyses revealed that the expression of LST-2 was increased by addition of chenodeoxycholate or deoxycholate in a dose-dependent manner. Conclusions. This study demonstrated that the transcription of the LST-2 gene is regulated by three transcription factors, FXR, HNF1α, and HNF3β. HNF1α and HNF3β might contribute to its liver-specific expression, and FXR might play a role in its transcriptional activation by bile acids. [ABSTRACT FROM AUTHOR]

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p -aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ , resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters. [ABSTRACT FROM AUTHOR]

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood–brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [3H]CMPF, [14C]HA and [3H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [3H]CMPF, [14C]HA and [3H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [3H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain. [ABSTRACT FROM AUTHOR]

Y-700, a novel xanthine oxidase inhibitor, was recently developed for the treatment of hyperuricemia and gout. Since the major elimination route of this compound is hepatic metabolism and excretion, the aim of the present study was to characterize the uptake mechanism of Y-700 in the liver, which is also the pharmacological target of Y-700. Efficient uptake of Y-700 was observed both in the liver in vivo and in isolated rat hepatocytes. The uptake was Na+-dependent, saturable and inhibited both by ATP-depressants and various organic anions. Indomethacin competitively inhibited Y-700 uptake, whereas the inhibitory effect of organic cations and nucleosides was not so remarkable. Saturable and Na+-dependent uptake of Y-700 was also observed in freshly isolated human hepatocytes. Uptake of Y-700 by sinusoidal membrane transporters, such as organic anion transporter (Oat) 2 and organic anion transporting polypeptide (OATP)-B, OATP-C, OATP-8, and Oatp1, could not be detected although uptake of Y-700 in the oocytes expressing sodium/taurocholate cotransporting polypeptide (NTCP) was slightly observed. In conclusion, active transport system(s), which specifically recognize certain types of anionic compounds, are involved in the hepatic uptake of Y-700 and, at least partially, relevant to its elimination from the circulation as well as delivery to pharmacological target. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:336–347, 2006 [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to examine prostaglandin E1 (PGE1) transport in rat kidney. [3H]PGE1 administered intravenously was accumulated most abundantly in the renal cortex. Infusion of probenecid and bromcresol green (BCG) decreased [3H]PGE1 accumulation in the renal cortex after injection of [3H]PGE1. To further investigate PGE1 transport in the kidney, [3H]PGE1 uptake by renal cortical slices was examined. Probenecid and BCG inhibited [3H]PGE1 uptake by the slices. Unlabeled PGE1 decreased [3H]PGE1 uptake by renal cortical slices in a concentration-dependent manner. The inhibitory effect of various dicarboxylates with different carbon atoms on [3H]PGE1 uptake was maximal at 6 carbon atoms. Preloading cortical slices with glutarate significantly increased [3H]PGE1 uptake. [3H]PGE1 uptake was inhibited by various eicosanoids and compounds with other structures (p-aminohippurate, benzylpenicillin, estrone-3-sulfate, etc.). These findings suggest that PGE1 uptake by renal cortical slices may be mediated by the members of the organic anion transporter family. [Copyright &y& Elsevier]

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Background Sex differences in drug pharmacokinetics have been well recognized and gender has been considered a risk factor for adverse events to medications. The aim of this study was to investigate the effect of gender on the expression of hepatocellular transport proteins involved in uptake and secretion of organic anions in rat. Materials and methods Expression of the rat liver organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps) was analysed by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis and immunofluorescence microscopy in male and female rats. Regulation of these transport proteins in response to the steroid dehydroepiandrosterone (DHEA) was investigated. Results In untreated rats, protein expression significantly differed between genders being higher (Mrp2, Mrp3), comparable [Oatp1a1 (Oatp1); Oatp1b2 (Oatp4)] or lower [Oatp1a4 (Oatp2)] in female than in male rat. DHEA treatment over 3 days (100 mg d−1) led to a further increase in Mrp3 expression only in female rats. Mrp2 expression was not influenced by DHEA treatment. Oatp1a1 and Oatp1b2 were significantly down-regulated after DHEA treatment in both male and female rats. In contrast, Oatp1a4 was down-regulated in male rats only. Conclusions In rat, liver transport proteins of the Oatp and Mrp family are expressed and regulated in a gender-specific manner according to sexual differences in the hepatic metabolism of steroids and drugs. These findings may partly explain the well-known sex differences in hepatic handling of organic anions. [ABSTRACT FROM AUTHOR]

Abstract: We examined the substrate specificity of human organic anion transporter (hOAT) 1 and hOAT3 for various cephalosporin antibiotics, cephaloridine, cefdinir, cefotiam, ceftibuten, cefaclor, ceftizoxime, cefoselis and cefazolin by using HEK293 cells stably transfected with hOAT1 or hOAT3 cDNA (HEK-hOAT1, HEK-hOAT3). Additionally, we examined the uptake of various compounds by these transfectants. The mRNA level of hOAT3 in HEK-hOAT3 was about three-fold that of hOAT1 in HEK-hOAT1. Functional expression of hOAT1 and hOAT3 was confirmed by the uptake of p-[14C]aminohippurate and [3H]estrone sulfate, respectively. p-[14C]Aminohippurate, [3H]estrone sulfate, [14C]captopril, [3H]methotrexate, [3H]ochratoxin A, [3H]leucovorin and [3H]cimetidine were shown to be substrates for hOAT1 and hOAT3, and [3H]dehydroepiandrosterone sulfate was shown to be a substrate for hOAT3. All cephalosporin anitibiotics tested were shown to inhibit the uptake of p-[14C]aminohippurate and [3H]estrone sulfate via hOAT1 and hOAT3, respectively, in a dose-dependent manner, and the IC50 values of these antibiotics, except for cefaclor, for the hOAT1-mediated uptake of p-[14C]aminohippurate were within four-fold of those for the hOAT3-mediated uptake of [3H]estrone sulfate. The uptake of cephaloridine, cefdinir and cefotiam by HEK-hOAT3 was 35–50-fold greater than that by control cells. Moreover, the accumulation of the other cephalolsporin antibiotics was significantly greater in HEK-hOAT3 than in control cells. In contrast, the uptake of these antibiotics by HEK-hOAT1 was within two-fold of that by control cells. In conclusion, hOAT3 plays a more important role than hOAT1 in the renal secretion of cephalosporin antibiotics. [Copyright &y& Elsevier]

Background. Excretion by the kidney of a variety of organic anionic drugs and metabolites is mediated by a family of multispecific organic anion transporters (OAT genes) that are part of the SLC22 family of solute carriers. Different OATs localize to the apical (OAT2, OAT4, and RST/URAT) or basolateral (OAT1/NKT and OAT3) membranes of the renal proximal tubule; the net transport of organic anions from blood to urine is believed to require both apical and basolateral OATs. These genes are also thought to mediate transport of organic anionic drugs and metabolites (e.g., urate) across choroid plexus, retina, placenta, and possibly olfactory mucosa. The extent of functional redundancy among OATs remains uncertain, but closely related OAT genes are tightly linked in the genome. Hence, a better understanding of human variation in organic anionic drug excretion may be obtained by studying OAT genes in combination rather than individually. Methods. We have analyzed single nucleotide polymorphisms (SNPs) in OAT1 (NKT), OAT2, OAT3, OAT4, and URAT1 (human homologue of RST) in an ethnically diverse sample of 96 individuals (192 haploid genomes). Ka/Ks analysis was also performed as well as haplotype reconstruction using the software program Arelquin. Results. The data indicate that ( 1) nonsynonymous SNPs in OAT1 and OAT3 may not be frequent so it will be important to consider promoter region SNPs that regulate gene expression; ( 2) certain ethnic groups may have a high prevalence of nonsynonymous SNPs in particular OATs (e.g., OAT4 in Sub-Saharan Africans); ( 3) there are individuals who have nonsynonymous SNPs in apical and basolateral OATs; ( 4) nonsynonymous OAT4 SNPs may be more frequent, raising the possibility of altered maternofetal transport of drugs and metabolites; and ( 5) combinations of synonymous SNPs in OAT1 and OAT3 also occur in certain individuals. In addition, Ka/Ks analysis of human, chimp and rodent genes suggests that OAT4 is under accelerated selection pressure, perhaps reflecting specific human environmental exposures during evolution. In contrast, Ka/Ks analysis for URAT1 suggests decelerated selection pressure. Haplotype reconstruction also supports this view. Conclusion. Together, these data suggest that, in order to understand the effect of SNPs in genes of the SLC22 family on drug handling as well as excretion of metabolites like uric acid, it is important to consider the entire set of organic anion transporters. It will be particularly interesting to determine if individuals with nonsynonymous apical and basolateral SNPs have altered handling (and toxicity) of organic anionic drugs and metabolites. Certain OAT family members appear to be under greater evolutionary selection pressure. [ABSTRACT FROM AUTHOR]