Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn²⁺ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.     

Effects of JBP485 on the expression and function of PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells

To investigate the effect of JBP485 (an anti-inflammatory dipeptide) on PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells, the activity and expression of PEPT1 were examined. The effects of treatment with indomethacin and co-treatment with JBP485 were examined in terms of intestinal histological changes, MDA and MPO levels in rats; as well as LDH-release and oxidative stress in Caco-2 cells. Uptake of glycylsarcosine (Gly-Sar) by PEPT1 was determined by in vivo, in vitro and in situ studies. RT-PCR and Western blot were used to assess the expression of PEPT1 in rat intestine and Caco-2 cells. JBP485 caused a significant decrease in MDA and MPO levels, and improved the pathological condition of rat intestine, while attenuating Caco-2 cells damage induced by indomethacin. Uptake of Gly-Sar by PEPT1 was decreased by indomethacin treatment, whereas the Gly-Sar plasma concentration was markedly increased in JBP485 co-treated rats. Indomethacin down-regulated the expression of PEPT1 mRNA and protein in rat intestine and Caco-2 cells, and the effects were reversed after administration of JBP485. These results indicated that JBP485 not only improved intestinal injury and cell damage but also partially blocked the down-regulation of PEPT1 expression and function induced by indomethacin.     

Oat5 and NaDC1 Protein Abundance in Kidney and Urine After Renal Ischemic Reperfusion Injury

The aim of this study was to evaluate the abundance of the organic anion transporter 5 (Oat5) and the sodium-dicarboxylate cotransporter 1 (NaDC1) in kidney and urine after renal ischemic reperfusion injury. Renal injury was induced in male Wistar rats by occlusion of both renal pedicles for 0 (Group Sham), 5 (Group I5R60), or 60 (Group I60R60) min. The studies were performed after 60 min of reperfusion. The expression of Oat5 and NaDC1 was evaluated by IHC and Western blotting. Oat5 and NaDC1 abundance and alkaline phosphatase activity (AP) were assayed in urine. A decreased expression in renal homogenates and apical membranes and an increase in urinary excretion of Oat5 and NaDC1 were observed in I60R60 rats, as well as alterations of other widely used parameters for renal dysfunction and injury (plasma creatinine, urinary AP activity, kidney weight, histological lesions). In contrast, in the I5R60 group, only an increase in urinary excretion of Oat5 and mild histopathological damage was detected. This is the first study on Oat5 and NaDC1 detection in urine. These results suggest that urinary excretion of Oat5 might be an early indicator of renal dysfunction, which is useful for detection of even minor alterations in renal structural and functional integrity. (J Histochem Cytochem 57:17–27, 2009)     

Organic anion transporters involved in the excretion of bestatin in the kidney

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 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.     

Protective effect of heme oxygenase induction in ethinylestradiol‐induced cholestasis

Estrogen‐induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase‐1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol‐induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down‐regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up‐regulated Mrp3 (348%, P ≤ 0.05). Heme pre‐treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up‐regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol‐treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme‐induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up‐regulation (Mrp2/Mrp4) or down‐regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2‐dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol‐induced cholestasis.     

Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.     

Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis

It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1-5 μmol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1-5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1-5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues.     

Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy

The expression of hepatic multidrug resistance-associated protein (Mrp)1, 2, 3, and 6 and organic anion transporting polypeptides (Oatp)1 and 2 were examined in control and 20- to 21-day pregnant rats. Western analysis showed that expression of Oatp2 was decreased 50% in pregnancy, whereas expression of Oatp1 did not change. Expression of Mrp2 protein determined by Western analysis of total liver homogenate decreased to 50% of control levels in pregnant rats, consistent with studies using plasma membranes. Confocal immunohistochemistry showed that Mrp2 expression was confined to the canalicular membrane in both control and pregnant rats and was not detectable in intracellular compartments. In isolated perfused liver, the biliary excretion of 2,4-dintrophenyl-glutathione was significantly decreased in pregnancy, consistent with decreased expression of Mrp2. The expression of the basolateral transporter Mrp1 was not altered in pregnancy, whereas expression of Mrp6 mRNA was decreased by 60%. Expression of Mrp3 was also decreased by 50% in pregnant rat liver, indicating differential regulation of Mrp isoforms in pregnancy. These data also demonstrate that decreased Mrp2 expression is not necessarily accompanied by increased Mrp3 expression.     

Mrp2 is involved in benzylpenicillin-induced choleresis

Benzylpenicillin (PCG; 180 micromol/kg), a classic beta-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [(3)H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow ( approximately 2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r(2) = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r(2) = 0.968). No ATP-dependent uptake of [(3)H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.     

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [³H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(−) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(−/−) knockout mice, and Bcrp1(−/−) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(−/−) mice or in Bcrp1(−/−) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(−) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(−) rats and functional DMP uptake into isolated TR(−) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(−) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(−) rats expelled phalloidin back into blood circulation.     

Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis

It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1-5 mumol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1-5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1-5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues.     

Impaired Insulin Signaling Affects Renal Organic Anion Transporter 3 (Oat3) Function in Streptozotocin-Induced Diabetic Rats

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway.     

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.     

Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12h following lipopolysaccharide (LPS) injection (4mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2+/-0.2 microl/g liver wet weight/min vs 3.3+/-0.1 microl/g liver wet weight in controls), biliary BSP excretion (10.8+/-2.2 nmol/g/min vs 21.0+/-3.8 nmol/g/min), and biliary TC excretion (114+/-23 nmol/g/min vs 228+/-8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor alpha and interleukin-1beta release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.     

Lithospermic acid B isolated from Salvia miltiorrhiza ameliorates ischemia/reperfusion-induced renal injury in rats

The present study was designed to examine whether lithospermic acid B (LSB) isolated from Salvia miltiorrhiza has an ameliorative effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2) and Na,K-ATPase in the ischemia-reperfusion induced acute renal failure (ARF) rats. LSB showed strong antioxidant activity against production of reactive oxygen species (ROS), ROS-induced hemolysis, and production of lipid peroxide in a dose-dependent manner. Polyuria caused by down-regulation of renal AQP 2 in the ischemia-reperfusion induced ARF rats was partially restored by administration of LSB (40 mg/kg, i.p.), restoring expression of AQP 2, in renal inner and outer medulla. The expression of Na,K-ATPase alpha1 subunit in outer medulla of the ARF rats was also restored in the ARF rats by administration of LSB, while beta1 subunit level was not altered. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also partially restored in ischemia-ARF rats by administration of LSB. Histological study also showed that renal damages in the ARF rats were abrogated by administration of LSB. Taken together, these data indicate that LSB ameliorates renal defects in rats with ischemia-reperfusion induced ARF, most likely via scavenging of ROS.     

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.     

Endogenous expression of liver-specific drug transporters for organic anions in the rat hepatocytoma fusion cell line HPCT-1E3

HPCT-1E3 cells, a fusion cell line between primary rat hepatocytes and Fao Reuber hepatoma cells H35, are immortalized hybrid cells with many phenotypic properties of liver parenchyma including phase I and II metabolism and bile acid secretion. Selective elimination of endogenous compounds and drugs by the liver involves transport proteins that complementarily mediate uptake and efflux in co-operation with metabolism, but the study of this function is limited by the unavailability of an integrated in vitro model. Therefore, we investigated the expression of some important liver-specific import and export carrier proteins for organic anions in this cell line. RT-PCR analysis indicated gene expression of Oat2, Oatplal, Oatpla4, Oatplb2, Rfc-1/MTX-1, FOLR, Mrp1-6, mdr1, and Lrp. Uptake and efflux as well as inhibition studies confirmed the functional activity of Oat, Oatp, Rfc-1, Mrp, and Mdr carriers. In conclusion, the hepatocyte-like HPCT-1E3 cell line shows endogenous expression of all liver-specific carrier proteins for organic anions and may hence represent a valuable in vitro model for the study of transport phenomena and their regulation in hepatocytes.