Obstructive cholestasis induces TNF-alpha- and IL-1 -mediated periportal downregulation of Bsep and zonal regulation of Ntcp, Oatp1a4, and Oatp1b2

Inverse acinar regulation of Mrp2 and 3 represents an adaptive response to hepatocellular cholestatic injury. We studied whether obstructive cholestasis (bile duct ligation) and LPS treatment affect the zonal expression of Bsep (Abcb11), Mrp4 (Abcc4), Ntcp (Slc10a1), and Oatp isoforms (Slco1a1, Slco1a4, and slco1b2) in rat liver, as analyzed by semiquantitative immunofluorescence. Contribution of TNF-alpha and IL-1beta to transporter zonation in obstructive cholestasis was studied by cytokine inactivation. In normal liver Bsep, Mrp4, Ntcp, and Oatp1a1 were homogeneously distributed in the acinus, whereas Oatp1a4 and Oatp1b2 expression increased from zone 1 to 3. Glutamine synthetase-positive pericentral hepatocytes exhibited markedly lower Oatp1a4 expression than the remaining zone 3 hepatocytes. In cholestatic liver Bsep and Ntcp immunofluorescence in periportal hepatocytes significantly decreased to 66 +/- 4% (P < 0.01) and 67 +/- 7% (P < 0.05), whereas it was not altered in pericentral hepatocytes. Oatp1a4 was significantly induced in hepatocytes with a primarily low expression, i.e., in periportal hepatocytes and in glutamine synthetase-positive pericentral hepatocytes. Likewise, Oatp1b2 was upregulated in periportal hepatocytes. Mrp4 zonal induction was homogeneous. Inactivation of TNF-alpha and IL-1beta prevented periportal downregulation of Bsep. Recruitment of neutrophils and polymorphonuclear cells mainly occurred in the periportal zone. Likewise, IL-1beta induction was largely found periportally. No significant transporter zonation was seen following LPS treatment. In conclusion, zonal downregulation of Bsep in obstructive cholestasis is associated with portal inflammation and is mediated by TNF-alpha and IL-1beta. Periportal downregulation of Ntcp and induction of Oatp1a4 and Oatp1b2 may represent adaptive mechanisms to reduce cholestatic injury in hepatocytes with profound downregulation of Bsep and Mrp2.     

Interleukin-6 regulates hepatic transporters during acute-phase response

Cholestasis develops during inflammatory conditions characterized by the release of cytokines like interleukin-6 (IL-6), which is the major player in the hepatic acute-phase response. However, the exact contribution of IL-6 to transporter down-regulation is unclear. Therefore, we compared wild-type and IL-6-deficient mice after IL-6-injection and induction of an aseptic (turpentine-injection) or septic (LPS-injection) acute-phase response. Down-regulation of basolateral (Ntcp, Oatp1, and Mrp3) and canalicular (Mrp2, Bsep) transporter mRNA occurred after treatment with IL-6, turpentine, and LPS. In IL-6-deficient mice, turpentine failed to decrease mRNA-levels of basolateral and canalicular transporters, whereas LPS-mediated down-regulation of Ntcp, Mrp3, and Mrp2 was abolished at later time points (24 h). In conclusion, induction of an aseptic and septic acute-phase response leads to the down-regulation of basolateral and canalicular organic anion transporters. IL-6 is required for transporter down-regulation during aseptic inflammation. Furthermore, IL-6 also contributes to transporter regulation during LPS-induced cholestasis at more delayed time points.     

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.     

Functional expression of organic anion transporters in hepatic organoids reconstructed by rat small hepatocytes

Small hepatocytes (SHs) are hepatic progenitor cells with hepatic characteristics. They can proliferate to form colonies in culture and change their morphology from flat to rising/piled-up with bile canaliculi (BC), which results in maturation. In this study, we examined whether SHs could express hepatic transporters with polarity, whether the transporters could transport organic anion substrates into BC, and whether the secreted substances could be recovered from BC. Immunocytochemistry and RT-PCR were carried out. [(3)H]-labeled estrogen derivatives were used to measure the functions of the transporters in SHs isolated from normal and multidrug resistance-associated protein (Mrp) 2-deficient rats. The results showed that organic anion-transporting proteins (Oatps) 1 and 2, Na(+)-dependent taurocholate co-transporting polypeptide (Ntcp), Mrp2, and bile-salt export pump (Bsep) were well expressed in rising/piled-up cells and that their expression was correlated to that of hepatocyte nuclear factor 4alpha. Although small SHs expressed not Oatps and Mrp2 but Mrp3, rising/piled-up SHs expressed Oatp1 and 2 and Mrp2 proteins in the sinusoidal and BC membranes, respectively. On the other hand, breast cancer resistant protein (Bcrp) and Mrp3 expression decreased as SHs matured. The substrate transported via Oatps and Mrp2 was secreted into BC and it accumulated in both BC and cyst-like structures. The secreted substrate could be efficiently recovered from BC reconstructed by SHs derived from a normal rat, but not from an Mrp2-deficient rat. In conclusion, SHs can reconstitute hepatic organoids expressing functional organic anion transporters in culture. This culture system may be useful to analyze the metabolism and excretion mechanisms of drugs.     

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 Organic Anion Transporting Polypeptide 1c1 and Monocarboxylate Transporter 8 in the Rat Placental Barrier and the Compensatory Response to Thyroid Dysfunction

Thyroid hormones (THs) must pass from mother to fetus for normal fetal development and require the expression of placental TH transporters. We investigate the compensatory effect of placental organic anion transporting polypeptide 1c1 (Oatp1c1) and monocarboxylate transporter 8 (Mct8) on maternal thyroid dysfunction. We describe the expressions of these two transporters in placental barriers and trophoblastic cell populations in euthyroidism and thyroid dysfunction resulting from differential iodine nutrition at gestation day (GD) 16 and 20, that is, before and after the onset of fetal thyroid function. Immunohistochemistry revealed that in the blood-placenta barrier, these two TH transporters were strongly expressed in the villous interstitial substance and were weakly expressed in trophoblast cells. Levels of Oatp1c1 protein obviously increased in the placental fetal portion during maternal thyroid deficiency at GD16. Under maternal thyroid deficiency after the production of endogenous fetal TH, quantitative PCR analysis revealed down-regulation of Oatp1c1 occurred along with up-regulation of Mct8 in trophoblast cell populations isolated by laser capture microdissection (LCM); this was consistent with the protein levels in the fetal portion of the placenta. In addition, decreased D3 mRNA at GD16 and increased D2 mRNA on two gestational days were observed in trophoblast cells with thyroid dysfunction. However, levels of Oatp1c1 mRNA at GD16 and D3 mRNA at GD20 were too low to be detectable in trophoblast cells. In conclusion, placental Oatp1c1 plays an essential compensatory role when the transplacental passage of maternal THs is insufficient at the stage before the fetal TH production. In addition, the coordinated effects of Oatp1c1, Mct8, D2 and D3 in the placental barrier may regulate both transplacental TH passage and the development of trophoblast cells during thyroid dysfunction throughout the pregnancy.     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na+/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [3H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [3H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [3H]demethylphalloin concentrations was saturable with apparent Km values of 5.7 microM (Oatp1b2), 17 microM (OATP1B1) and 7.5 microM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [3H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3−1 and Can 10

Sinusoidal and apical transporters are responsible for the uptake and biliary elimination of many compounds by hepatocytes. Few in vitro models are however available for analyzing such functions. The expression and bile-acid inducibility of 13 transporters and two nuclear receptors were investigated in the new rat polarized lines, Can 3−1 and Can 10, and in their unpolarized parent, Fao. The relative abundance of mRNA, the protein level, and their localization were examined by real-time quantitative PCR, Western blotting, immunofluorescence, and confocal microscopy. Compared with rat liver, mRNA levels of Fao cells were: negligible for Bsep/Abcb11; lower for the uptake transporters Ntcp and Oatps; similar for SHP, FXR, and Bcrp/Abcg2; and higher (four–fold to 160-fold) for the efflux pumps Mdr1b/Abcb1b, Mdr2/Abcb4, Mrp1/Abcc1, Mrp2/Abcc2, Mrp3/Abcc3, Abcg5, and Abcg8. This profile was mostly maintained (and improved for Bsep) in Can 10. Some transporters were less well expressed in Can 3−1. In both lines, sinusoidal (Ntcp, Mrp3) and canalicular transporters (Mdr-P-glycoproteins detected with C219 antibody, Mrp2) were localized at their correct poles. Bile-acid effects on polarity and mRNA levels of transporters were analyzed after a 6-day treatment with 50 μM taurocholic, chenodeoxycholic (CDCA), or ursodeoxycholic acid (UDCA). No polarization of Fao cells was induced; Can 10 and Can 3−1 polarity was maintained. CDCA and UDCA induced marked enhancement of the volume of Can 10 bile canaliculi. CDCA upregulated Bsep, Mdr2, SHP, Mdr1b, and Oatp2/1a4 in Can 10 (two- to seven-fold) and in Fao cells. Thus, Can 10 constitutes an attractive polarized model for studying vectorial hepatobiliary transport of endogenous and xenobiotic cholephilic compounds. This work was supported by a grant from Egide (PAI Picasso) and the Acción Integrada Hispano-Francesa (HF2003-0089). This research group is part of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF; grant BFU2005-24983-E/BFI) and belongs to the “Centro de Investigación Biomédica en Red” for Hepatology and Gastroenterology Research (CIBERehd), Instituto de Salud Carlos III, Spain.     

Gender-divergent profile of bile acid homeostasis during aging of mice

Aging is a physiological process with a progressive decline of adaptation and functional capacity of the body. Bile acids (BAs) have been recognized as signaling molecules regulating the homeostasis of glucose, lipid, and energy. The current study characterizes the age-related changes of individual BA concentrations by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in serum and liver of male and female C57BL/6 mice from 3 to 27 months of age. Total BA concentrations in serum increased 340% from 3 to 27 months in female mice, whereas they remained relatively constant with age in male mice. During aging, male and female mice shared the following changes: (1) BA concentrations in liver remained relatively constant; (2) the proportions of beta-muricholic acid (βMCA) increased and deoxycholic acid (DCA) decreased between 3 and 27 months in serum and liver; and (3) total BAs in serum and liver became more hydrophilic between 3 and 27 months. In female mice, (1) the mRNAs of hepatic BA uptake transporters, the Na(+)/taurocholate cotransporting polypeptide (Ntcp) and the organic anion transporting polypeptide 1b2 (Oatp1b2), decreased after 12 months, and similar trends were observed for their proteins; (2) the mRNA of the rate-limiting enzyme for BA synthesis, cholesterol 7α-hydroxylase (Cyp7a1), increased from 3 to 9 months and remained high thereafter. However, in male mice, Ntcp, Oatp1b2, and Cyp7a1 mRNAs remained relatively constant with age. In summary, the current study shows gender-divergent profiles of BA concentrations and composition in serum and liver of mice during aging, which is likely due to the gender-divergent expression of BA transporters Ntcp and Oatp1b2 as well as the synthetic enzyme Cyp7a1.     

Effect of maternal cholestasis and treatment with ursodeoxycholic acid on the expression of genes involved in the secretion of biliary lipids by the neonatal rat liver

In juvenile rats born from mothers with obstructive cholestasis during pregnancy (OCP), transient latent cholestasis together with alterations in the secretion of biliary lipids have been reported. Here we investigated whether the expression of genes involved in this function is already modified at birth and examined the effect of treating pregnant rats with ursodeoxycholic acid (UDCA; i.g., 60 microg/100 g b.w./day). Cholanemia was markedly higher in mothers with OCP, and was further increased by UDCA. In the Control pups, cholanemia increased after birth, whereas in OCP and OCP+UDCA pups, hypercholanemia decreased after birth. Steady-state mRNA levels in neonatal liver were measured by real-time quantitative RT-PCR. The expression of basolateral bile acid transporters was not affected by OCP and was unchanged (Oatp1/1a1 and Oatp4/1b2) or moderately increased (Ntcp and Oatp2/1a4) by UDCA. In both groups, the expression of ABC proteins was either not modified (Bsep, Bcrp and Mrp2) or enhanced (Mrp1 and Mrp3), that of phospholipid flippase Mdr2 was not changed, whereas that of cholesterol transporter Abcg5/Abcg8 was impaired. The expression of the nuclear receptor FXR was not affected by OCP or UDCA, whereas that of SHP and key enzymes in bile acid synthesis (Cyp7a1, Cyp8b1 and Cyp27) was increased in both groups. In conclusion, OCP affects the expression in the neonatal liver of genes involved in hepatobiliary function, which cannot be prevented, at this stage, by treating pregnant rats with UDCA, even though this treatment has been found to partially restore normal lipid secretion later during post-natal development.     

Transporters, enzymes, and enalapril removal in a rat (CC531-induced) liver metastatic model

Temporal changes in physiological spaces, protein expression of transporters and enzymes, and enalapril removal were appraised in the metastatic liver tumor model developed from male Wag/Rij rats after the intraportal injection of CC531 colon adenocarcinoma cells; sham-operated preparations received PBS. Liver tissue spaces, investigated with multiple indicator dilution technique in liver perfusion studies, were unchanged at week 3 after tumor induction. At week 4, however, the sinusoidal blood volume and albumin Disse space in tumor-bearing livers were slightly lower compared with those of shams. Increased levels of the canalicular ATP transporters, P-glycoprotein, multidrug resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) at week 2 (P < 0.05), unchanged levels of Ntcp, Oatp1a1, Oatp1a4, and Mct2, but decreased levels of cytochrome P450 3a2 (Cyp3a2) and glutathione S-transferase (Gst4-4) at week 4 (P < 0.05) were observed in peritumor vs. sham-operated liver tissues with Western blotting. The steady-state extraction ratio of enalapril, a substrate that enters the liver rapidly via Oatp1a1 and primarily undergoes metabolism by the carboxylesterases, was unaffected by liver metastasis at week 4 regardless of its delivery via the portal vein or hepatic artery into the perfused liver preparations.     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na⁺/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [³H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [³H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [³H]demethylphalloin concentrations was saturable with apparent K_m values of 5.7 μM (Oatp1b2), 17 μM (OATP1B1) and 7.5 μM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [³H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

Bile acids via FXR initiate the expression of major transporters involved in the enterohepatic circulation of bile acids in newborn mice

The enterohepatic circulation (EHC) of bile acids (BAs) plays a pivotal role in facilitating lipid absorption. Therefore, initiation of the EHC in newborns is of crucial importance for lipid absorption from milk. The purpose of this study was to determine at what age BA transporters in liver are expressed, and the mechanism for their initiation. Serum and liver samples were collected from C57BL/6 mice at 2 days before birth and various postnatal ages. Messenger RNA assays revealed a dramatic increase at birth in the expression of the BA transporters (Ntcp, Bsep, Mrp4, Ostβ), as well as the phospholipid floppase Mdr2 in mouse liver, with the highest expression at 1 day of age. The mRNA expression of the ileal BA transporters (Ostα and Ostβ) also markedly increased at birth. Meanwhile, taurine-conjugated cholic acid markedly increased in both serum and liver of newborns, correlated with upregulation of the classic pathway of BA biosynthesis in newborn liver. The mRNA levels of the major BA sensors, FXR and PXR, were increased at 1 day of age, and their prototypical target genes were upregulated in liver. The mRNA expression of transporters involved in the EHC of BAs was similar in wild-type and PXR-null mice. In contrast, in FXR-null mice, the "day 1 surge" pattern of Ntcp, Bsep, Ostβ, and Mdr2 was blocked in newborn mouse liver, and the induction of Ostα and Ostβ was also abolished in ileums of FXR-null mice. In conclusion, at birth, BAs from the classic pathway of synthesis trigger the induction of transporters involved in EHC of BAs in mice, through activation of the nuclear receptor FXR.