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One of the characteristics of chronic kidney disease (CKD) is the accumulation of uremic solutes in the plasma. Less is known about the effects of uremic solutes on transporters that may play critical roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes were identified as inhibitors of OAT1 and OAT3, respectively. Several of them inhibited OAT1 or OAT3 at clinically relevant concentrations and reduced the transport of other OAT1/3 substrates in vitro. Review of clinical studies showed that the active secretion of most drugs that are known substrates of OAT1/3 deteriorated faster than the renal filtration in CKD. Collectively, these data suggest that through inhibition of OAT1 and OAT3, uremic solutes contribute to the decline in renal drug clearance in patients with CKD.

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity.

1 alpha,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR-rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill-defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)(2)D-3 treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)(2)D-3 treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2- to 20-fold) in 1,25(OH)(2)D-3-treated rats, and a 28-fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal-specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ost alpha, and Mrp4, and protein levels of PepT1 and Oatl were decreased in a dose-dependent manner, and the similar to 50% concomitant reduction in FXR, SHP, HNF-1 alpha and HNF-4 alpha mRNA expression suggests the possibility of cross-talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)(2)D-3 changes are tissue-specific, differing between the intestine and kidney which are VDR-rich organs. Copyright (C) 2009 John Wiley & Sons, Ltd.

To investigate the hypothesis that during the development of biliary atresia, early changes in hepatobiliary transport are mainly related to the inflammatory process and lead to intrahepatic cholestasis and subsequent liver injury, livers from mice with rhesus rotavirus-induced biliary atresia were analyzed for mRNA expression of hepatobiliary transporters, nuclear receptors, and inflammatory cytokines. Seven days after inoculation, despite high bile acid concentrations in the liver, gene expression of canalicular and basolateral hepatobiliary transporters and their regulatory nuclear receptors was down-regulated with concomitant increase in gene expression of inflammatory cytokines and rise in serum unconjugated bilirubin. At 14 d, hepatobiliary transporters and nuclear receptors remained down-regulated although the inflammatory response subsided. The percentage of conjugated bilirubin started to increase as extrahepatic biliary obstruction occurred. At 18 d, expression of hepatobiliary transporters remained low, expression of nuclear receptors returned to normal, while expression of inflammatory cytokines decreased further. Moreover, histology demonstrated progressive inflammation, bile duct damage, ductular proliferation, and hepatocyte necrosis. In conclusion, intrahepatic cholestasis due to inflammation-related down-regulation of basolateral and canalicular hepatobiliary transporters is an early event in the development of biliary atresia. Intrahepatic cholestasis contributes to the development of jaundice and liver injury. (Pediatr Res 66: 380-385, 2009)

To investigate the hypothesis that during the development of biliary atresia, early changes in hepatobiliary transport are mainly related to the inflammatory process and lead to intrahepatic cholestasis and subsequent liver injury, livers from mice with rhesus rotavirus-induced biliary atresia were analyzed for mRNA expression of hepatobiliary transporters, nuclear receptors, and inflammatory cytokines. Seven days after inoculation, despite high bile acid concentrations in the liver, gene expression of canalicular and basolateral hepatobiliary transporters and their regulatory nuclear receptors was down-regulated with concomitant increase in gene expression of inflammatory cytokines and rise in serum unconjugated bilirubin. At 14 d, hepatobiliary transporters and nuclear receptors remained down-regulated although the inflammatory response subsided. The percentage of conjugated bilirubin started to increase as extrahepatic biliary obstruction occurred. At 18 d, expression of hepatobiliary transporters remained low, expression of nuclear receptors returned to normal, while expression of inflammatory cytokines decreased further. Moreover, histology demonstrated progressive inflammation, bile duct damage, ductular proliferation, and hepatocyte necrosis. In conclusion, intrahepatic cholestasis due to inflammation-related down-regulation of basolateral and canalicular hepatobiliary transporters is an early event in the development of biliary atresia. Intrahepatic cholestasis contributes to the development of jaundice and liver injury. (Pediatr Res 66: 380-385, 2009)

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension. Copyright � 2008 by the American Society of Nephrology.

Purpose The purpose of this study was to examine the regulation of renal organic ion transporters in cisplatin-induced acute kidney injury (AKI) and its relation with indoxyl sulfate (IS), a uremic toxin. Methods The IS concentrations in the serum and kidney were monitored by high-performance liquid chromatography. Uptake of p-aminohippuric acid, estrone-3-sulfate and tetraethylammonium were examined using renal slices. Real-time PCR and immunoblotting were performed to examine the mRNA and protein expression of rOATs, rOCTs and rMATE1 in the kidney, respectively. Results The serum and renal IS levels were markedly elevated in cisplatin-treated rats. However, this effect was largely reversed by administration of AST-120, an oral charcoal adsorbent. The functions of renal basolateral organic anion and cation transporters were reduced in cisplatin-treated rats. The levels of mRNA and protein corresponding to rOAT1, rOAT3, rOCT2 and rMATE1, but not rOCT1, were depressed in the kidney of cisplatin-treated rats. Administration of AST-120 to cisplatin-treated rats partially restored the function and expression level of these transporters. Conclusions Cisplatin-induced AKI causes down-regulation of renal organic ion transporters accompanied by accumulation of serum and renal IS. IS could be involved in the mechanism of down-regulation of rOAT1, rOAT3 and rMATE1 under cisplatin-induced AKI.

Prostaglandin E2 (PGE2) is produced in the brain during infectious/inflammatory diseases, and it mediates acute-phase responses including fever. In the recovery phase of such diseases, PGE2 disappears from the brain through yet unidentified mechanisms. Rat prostaglandin transporter (PGT), which facilitates transmembrane transport of PGE2, might be involved in the clearance of PGE2 from the brain. Here, we examined the cellular localization of PGT mRNA and its protein in the brains of untreated rats and those injected intraperitoneally with a pyrogen lipopolysaccharide (LPS) or saline. PGT mRNA was weakly expressed in the arachnoid membrane of untreated rats and saline-injected ones, but was induced in blood vessels of the subarachnoidal space and choroid plexus and in arachnoid membrane at 5 h and 12 h after LPS injection. In the same type of cells, PGT-like immunoreactivity was found in the cytosol and cell membrane even under nonstimulated conditions, and its level was also elevated after LPS injection. PGT-positive cells in blood vessels were identified as endothelial cells. In most cases, PGT was not colocalized with cyclooxygenase-2, a marker of prostaglandin-producing cells. The PGE2 level in the cerebrospinal fluid reached its peak at 3 h after LPS, and then dropped over 50% by 5 h, which time point coincides with the maximum PGT mRNA expression and enhanced level of PGT protein. These results suggest that PGT is involved in the clearance of PGE2 from the brain during the recovery phase of LPS-induced acute-phase responses.

1-{7-[(1-(3,5-Diethoxyphenyl)-3-{[(3,5-difluorophenyl)(ethyl)amino]carbo nyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide, R-146224, is a potent, specific ileum apical sodium-dependent bile acid transporter (ASBT) inhibitor; concentrations required for 50% inhibition of [3H]taurocholate uptake in human ASBT-expressing HEK-293 cells and hamster ileum tissues were 0.023 and 0.73 μM, respectively. In bile-fistula rats, biliary and urinary excretion 48 h after 10 mg/kg [14C]R-146224, were 1.49 ± 1.75% and 0.14 ± 0.05%, respectively, demonstrating extremely low absorption. In hamsters, R-146224 dose-dependently reduced gallbladder bile [3H]taurocholate uptake (ED50: 2.8 mg/kg). In basal diet-fed hamsters, 14-day 30-100 mg/kg R-146224 dose-dependently reduced serum total cholesterol (∼ 40%), high density lipoprotein (HDL) cholesterol (∼ 37%), non-HDL cholesterols (∼ 20%), and phospholipids (∼ 20%), without affecting serum triglycerides, associated with reduced free and esterified liver cholesterol contents. In normocholesterolemic cynomolgus monkeys, R-146224 specifically reduced non-HDL cholesterol. In human ileum specimens, R-146224 dose-dependently inhibited [3H]taurocholate uptake. Potent non-systemic ASBT inhibitor R-146224 decreases bile acid reabsorption by inhibiting the ileal bile acid active transport system, resulting in hypolipidemic activity. © 2006 Elsevier B.V. All rights reserved. Chemicals / CAS: carbon 14, 14762-75-5; cholesterol, 57-88-5; taurocholic acid, 145-42-6, 59005-70-8, 81-24-3; tritium, 10028-17-8; carrier protein, 80700-39-6; sodium, 7440-23-5; 1-(7-((1-(3,5-diethoxyphenyl)-3-(((3,5-difluorophenyl)(ethyl)amino)carbonyl)-4-oxo-1,4-dihydroquinolin-7-yl)oxy)heptyl)-1-methylpiperidinium bromide; Anticholesteremic Agents; Bile Acids and Salts; Cholesterol, 57-88-5; Membrane Transport Proteins; Organic Anion Transporters, Sodium-Dependent; Piperidines; Quinolines; Sodium, 7440-23-5; sodium-bile acid cotransporter, 145420-23-1; Symporters; Taurocholic Acid, 81-24-3

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To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of α,β-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. α,β-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition. © 2005 Elsevier Inc. All rights reserved.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC50 values, ranging between 2.7 and 14.3 μM) followed by robinetin, myricetin and quercetin (IC50 values ranging between 13.6 and 21.8 μM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC 50 values of 15.0 and 22.2 μM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent Km for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 μM for MRP1 and 8.5 μM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1. © 2004 Elsevier Inc. All rights reserved.

The hepatobiliary system and the kidneys are the main routes by which drugs and their metabolites leave the body. Compounds that are mainly excreted into bile in general have relatively high molecular weights, arc amphipathic and highly bound to plasma proteins. In contrast, compounds that are predominantly excreted into urine have relatively low molecular weights, are more hydrophilic and generally less protein bound. The first step in drug elimination in liver and kidney is uptake into hepatocytes or into proximal tubular cells. The substrate specificity and affinity of the uptake carriers expressed at the basolateral membranes of hepatocytes and proximal tubular cells could therefore play an important role for the determination of the main elimination route or a compound. This review discusses the tissue distribution. substrate specificity, transport mechanism, and regulation of the members of the organic anion transporting polypeptide (Oatp/OATP) superfamily (Solute carrier family SLC21A) and the SLC22A family containing transporters for organic cations (OCTs) and organic anions (OATs). The Oatps/OATPs are mainly important for the hepatic uptake of large amphipathic organic anions, organic cations and uncharged substrates, whereas OCTs and OATs mediate uptake of predominantly small organic cations and anions in liver and kidney.

Together with drug metabolising enzymes, transmembrane transporters are important determinants of drug metabolism and drug clearance by the liver. Hepatic uptake of organic anions, cations, prostaglandins and bile salts is supported by dedicated transporter proteins in the basolateral (sinusoidal) membrane of hepatocytes: OATPs, OATs, OCTs, PGTs and NTCP, respectively. ATP-binding cassette (ABC) transporter proteins in the canalicular membrane of hepatocytes mediate the hepatic efflux of drugs, bile salts and metabolites against a steep concentration gradient from liver to bile. This transport is driven by ATP hydrolysis. Drugs, endogenous metabolites, bile salts and cytokines affect the expression levels of these transporters. They act through a family of ligand-activated transcription factors, the nuclear hormone receptors. Consequently, the levels of the various transporter proteins are subject to genetic polymorphism in the encoding genes as well as in these transcription factors. Adverse drug reactions may be caused by genetic or disease-induced variations of transporter expression or drug-drug interactions at the level of these transporters. (C) 2002 Elsevier Science B.V. All rights reserved.

Together with drug metabolising enzymes, transmembrane transporters are important determinants of drug metabolism and drug clearance by the liver. Hepatic uptake of organic anions, cations, prostaglandins and bile salts is supported by dedicated transporter proteins in the basolateral (sinusoidal) membrane of hepatocytes: OATPs, OATs, OCTs, PGTs and NTCP, respectively. ATP-binding cassette (ABC) transporter proteins in the canalicular membrane of hepatocytes mediate the hepatic efflux of drugs, bile salts and metabolites against a steep concentration gradient from liver to bile. This transport is driven by ATP hydrolysis. Drugs, endogenous metabolites, bile salts and cytokines affect the expression levels of these transporters. They act through a family of ligand-activated transcription factors, the nuclear hormone receptors. Consequently, the levels of the various transporter proteins are subject to genetic polymorphism in the encoding genes as well as in these transcription factors. Adverse drug reactions may be caused by genetic or disease-induced variations of transporter expression or drug-drug interactions at the level of these transporters. (C) 2002 Elsevier Science B.V. All rights reserved.

Aim: Organic anion-transporting polypeptides OATP1B1 and OATP1B3 are sinusoidal membrane transporters mediating liver uptake of a wide range of substrates including conjugated and unconjugated bilirubin, xenobiotics and drugs. Absence of OATP1Bs in the liver causes Rotor syndrome. Our aim was to correlate OATP1B expression with hyperbilirubinemia in common liver diseases. Methods: Immunoreactivity of five antibodies against human OATP1Bs was tested on frozen and formalin-fixed paraffin-embedded liver tissue of mouse strains transgenic for SLCO1B1 or SLCO1B3 and on human specimens. The proportion of hepatocytes expressing OATP1Bs was then assessed immunohistologically in formalin-fixed paraffin-embedded liver samples obtained from patients with hepatocellular and primary biliary liver diseases. UGT1A1 promoter TATA-box and SLCO1B1 rs4149056 genotyping was performed to rule out individuals predisposed to hyperbilirubinemia. Results: The most specific detection of OATP1B3 was achieved with the H-52 (sc-98981) antibody. OATP1B1 was specifically recognized with the ESL (ab15441) anti-OATP1B1 antibody, but only in frozen sections. The MDQ (ab15442) anti-OATP1B1 antibody cross-reacted with both OATP1B proteins in liver tissue of the transgenic mouse strains. Expression of the OATP1B proteins was decreased in advanced liver diseases and inversely correlated with serum bilirubin levels. The reduction was more pronounced in advanced primary biliary diseases (1.9±1.1 vs. 2.7±0.6; P=0.009). Conclusions: Down-regulation of OATP1B proteins may contribute to pathogenesis of jaundice accompanying advanced cholestatic liver diseases.

The function of the blood-testis barrier is to protect germ cells from harmful influences; thus, it also impedes the delivery of chemotherapeutic drugs to the testis. The barrier has three components: first, a physicochemical barrier consisting of continuous capillaries, Sertoli cells in the tubular wall, connected together with narrow tight junctions, and a myoid-cell layer around the seminiferous tubule. Second, an efflux-pump barrier that contains P-glycoprotein in the luminal capillary endothelium and on the myoid-cell layer; and multidrug-resistance associated protein 1 located basolaterally on Sertoli cells. Third, an immunological barrier, consisting of Fas ligand on Sertoli cells. Inhibition of P-glycoprotein function offers the opportunity to increase the delivery of cytotoxic drugs to the testis. In the future, visualisation of function in the blood-testis barrier may also be helpful to identify groups of patients in whom testis conservation is safe or to select drugs that are less harmful to fertility.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S 2 hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S 2 hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S 2 hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K m value of 22.9±2.44 μM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner ( K i =44.4–336.4 μM), with the following order of potency: probenecid>piroxicam>octanoate>citrinin. The efflux of OTA by S 2 hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S 2 hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.

A major problem in the treatment of patients with acute myeloid leukemia (AXIL) is the occurrence of resistance to structurally and functionally unrelated chemotherapeutic agents, called multidrug resistance (MDR). One of the known MDR mechanisms is the overexpression of adenosine triphosphate, (ATP)-dependent efflux pumps. Permeability-glycoprotein (P-gp), the best characterized of the human drug efflux pumps, has been shown to be associated with poor treatment outcome in AML patients. Besides P-gp, in addition the multidrug resistance protein 1 (MRP1) appeared to contribute to the observed resistance in AML. Alternative transporter proteins, such as the MRP1 homologues MRP2, MRP3, MRP5 and MRP6, and the breast cancer resistance protein (BCRP), have been shown to be expressed at variable levels in AML patient cells. The latter proteins have been described to confer resistance to chemotherapeutic agents, such as daunorubicin, mitoxantrone, etoposide and 6-mercaptopurine, which are generally used in the treatment of AML patients; however, they have not yet proven to play a role in drug resistance in AML.The present review gives an overview of the current knowledge concerning these drug transporters, with a focus on the role of the transporter proteins in AML.

A major problem in the treatment of patients with acute myeloid leukemia (AXIL) is the occurrence of resistance to structurally and functionally unrelated chemotherapeutic agents, called multidrug resistance (MDR). One of the known MDR mechanisms is the overexpression of adenosine triphosphate, (ATP)-dependent efflux pumps. Permeability-glycoprotein (P-gp), the best characterized of the human drug efflux pumps, has been shown to be associated with poor treatment outcome in AML patients. Besides P-gp, in addition the multidrug resistance protein 1 (MRP1) appeared to contribute to the observed resistance in AML. Alternative transporter proteins, such as the MRP1 homologues MRP2, MRP3, MRP5 and MRP6, and the breast cancer resistance protein (BCRP), have been shown to be expressed at variable levels in AML patient cells. The latter proteins have been described to confer resistance to chemotherapeutic agents, such as daunorubicin, mitoxantrone, etoposide and 6-mercaptopurine, which are generally used in the treatment of AML patients; however, they have not yet proven to play a role in drug resistance in AML. The present review gives an overview of the current knowledge concerning these drug transporters, with a focus on the role of the transporter proteins in AML.

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

BackgroundHerb-drug interactions (HDIs) resulting from concomitant use of herbal products with clinical drugs may cause adverse reactions. Organic anion transporter 1 (OAT1) and 3 (OAT3) are highly expressed in the kidney and play a key role in the renal elimination of substrate drugs. So far, little is known about the herbal extracts that could modulate OAT1 and OAT3 activities.MethodsHEK293 cells stably expressing human OAT1 (HEK-OAT1) and OAT3 (HEK-OAT3) were established and characterized. One hundred seventy-two extracts from 37 medicinal and economic plants were prepared. An initial concentration of 5 µg/ml for each extract was used to evaluate their effects on 6-carboxylfluorescein (6-CF) uptake in HEK-OAT1 and HEK-OAT3 cells. Concentration-dependent inhibition studies were conducted for those extracts with more than 50% inhibition to OAT1 and OAT3. The extract ofJuncus effusus, a well-known traditional Chinese medicine, was assessed for its effect on thein vivopharmacokinetic parameters of furosemide, a diuretic drug which is a known substrate of both OAT1 and OAT3.ResultsMore than 30% of the plant extracts at the concentration of 5 µg/ml showed strong inhibitory effect on the 6-CF uptake mediated by OAT1 (61 extracts) and OAT3 (55 extracts). Among them, three extracts for OAT1 and fourteen extracts for OAT3 were identified as strong inhibitors with IC50values being Juncus effususshowed a strong inhibition to OAT3in vitro, and markedly altered thein vivopharmacokinetic parameters of furosemide in rats.ConclusionThe present study identified the potential interactions of medicinal and economic plants with human OAT1 and OAT3, which is helpful to predict and to avoid potential OAT1- and OAT3-mediated HDIs.

Introduction: For the elimination of environmental chemicals and metabolic waste products, the body is equipped with a range of broad specificity transporters that are present in excretory organs as well as in several epithelial blood-tissue barriers. Areas covered: ABCC2 and ABCC3 (also known as MRP2 and MRP3) mediate the transport of various conjugated organic anions, including many drugs, toxicants and endogenous compounds. This review focuses on the physiology of these transporters, their roles in drug disposition and how they affect drug sensitivity and toxicity. It also examines how ABCC2 and ABCC3 are coordinately regulated at the transcriptional level by members of the nuclear receptor (NR) family of ligand-modulated transcription factors and how this can be therapeutically exploited. Expert opinion: Mutations in both ABCC2 and ABCC3 have been associated with changes in drug disposition, sensitivity and toxicity. A defect in ABCC2 is associated with Dubin-Johnson syndrome, a recessively inherited disorder characterized by conjugated hyperbilirubinemia. Pharmacological manipulation of the activity of these transporters can potentially improve the pharmacokinetics and thus therapeutic activity of substrate drugs but also affect the physiological function of these transporters and consequently ameliorate associated disease states.

Obesity has been reported as an independent risk factor for chronic kidney disease, leading to glomerulosclerosis and renal insufficiency. To assess the relationship between a reduced nephron number and a particular susceptibility to obesity-induced renal damage, mice underwent uninephrectomy (UNX) followed by either normal chow or high-fat diet (HFD) and were compared with sham-operated control mice. After 20weeks of dietary intervention, HFD-fed control mice presented characteristic features of progressive nephropathy, including albuminuria, glomerulosclerosis, renal fibrosis and oxidative stress. These changes were more pronounced in HFD-fed mice that had undergone uninephrectomy. Analysis of gene expression in mouse kidney by whole genome microarrays indicated that high fat diet led to more changes in gene expression than uninephrectomy. HFD affected mainly genes involved in lipid metabolism and transport, whereas the combination of UNX and HFD additionally altered the expression of genes belonging to cytoskeleton remodeling, fibrosis and hypoxia pathways. Canonical pathway analyses identified the farnesoid X receptor (FXR) as a potential key mediator for the observed changes in gene expression associated with UNX-HFD. In conclusion, HFD-induced kidney damage is more pronounced following uninephrectomy and is associated with changes in gene expression that implicate FXR as a central regulatory pathway.

The efficacy of chemotherapy for malignant primary or metastatic brain tumours is still poor. This is at least partly due to the presence of the blood-brain barrier (BBB). The functionality of the BBB can be explained by physicochemical features and efflux pump mechanisms. An overview of the literature is presented with emphasis on oncology. The BBB consists of capillary endothelial cells that lack fenestrations and are connected together with continuous tight junctions, with a high electrical resistance. Permeability of tight junctions can be increased in vitro by contraction of the cytoskeleton, caused by bradykinin agonists. Different efflux pumps are present in the BBB. Examples are P-glycoprotein (P-gp), organic anion transporters, (OAT) and multidrug-resistance-associated proteins (MRP)(1 and 3). These pumps act as a multi-specific efflux pump for various chemotherapeutic drugs. Experiments have shown that P-gp can be inhibited by different non-chemotherapeutic substrates such as cyclosporin A. The functionality in vivo of P-gp can be measured with positron emission tomography and [C-11]-verapamil or with single photon emission computer tomography and Tc-99m-sestamibi, MRP1 and MRP3 act as organic anion transporters that in vitro act as efflux pumps for substances that are conjugated or co-transported with glutathione and glucuronide, respectively. Methotrexate has been recently demonstrated to be transported by MRP1 and MRP3. Results of studies which demonstrate the clinical relevance and applicability of BBB modulators are eagerly awaited. (C) 2000 Harcourt Publishers Ltd.

Cholestasis in primary biliary cirrhosis results from impairment of bile flow either by reduced transport at the level of the canaliculi or by disturbed bile flow through damaged intrahepatic bile ductules. Whatever its cause, the expression of hepatic transport proteins will be affected. In cholestatic rats: the expression of the multispecific organic anion transporter mrp2 is decreased; the bile salt export pump bsep and the phospholipid transporter mdr2 are less affected; the carrier protein for hepatic uptake of bile salts ntcp is sharply down-regulated; Mrp3, a basolateral ATP-dependent transporter for glucuronides and bile salts, is upregulated. Thus, bile salts that cannot exit the hepatocyte because of the cholestasis are effectively removed across the basolateral membrane. These may be adaptive responses in defence against overloading of hepatocytes with cytotoxic bile salts. These responses show that the expression of hepatic transporter proteins is highly regulated. This occurs by transcriptional and post-transcriptional mechanisms. Primary biliary cirrhosis starts as a disease of the small intrahepatic bile ducts and therefore the experimental evidence for 'cross-talk' between hepatocytes and cholangiocytes is of great interest for this disease and needs to be further investigated. New insights in bile physiology may enable the development of new therapies for cholestatic liver diseases as primary biliary cirrhosis.

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (,MDR1) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, me describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane, In a panel of human tumors, including primary turners as well as drug-treated breast cancer and acute myeloid leukemia samples. BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.