Canalicular Excretion Research Articles

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

Background and AimsLipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear.Approach and ResultsWild‐type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two‐photon microscopy, and the findings in Bsep KO mice were compared to common bile duct–ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS‐induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule‐dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice.ConclusionsAbsence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Role of biopsy in Benign recurrent intrahepatic cholestasis

Benign recurrent intrahepatic cholestasis (BRIC) is an inherited self limiting cholestatic disorder. It is very rare and less common than its severe form, Progressive familial intrahepatic cholestasis (PFIC). Due to impaired canalicular biliary excretion, patients develop recurrent episodes of jaundice and intense pruritus with characteristic symptom free intervals. Most of the cases have a benign course and are managed symptomatically. Histopathological examination of liver biopsy is essential in cases of recurrent intrahepatic cholestasis to confirm the diagnosis. Here we report two cases of biopsy confirmed Benign recurrent intrahepatic cholestasis for its rarity.

Novel Mechanisms of Valproate Hepatotoxicity: Impaired Mrp2 Trafficking and Hepatocyte Depolarization.

Drug-induced liver injury (DILI) remains a major challenge in drug development. Although numerous mechanisms for DILI have been identified, few studies have focused on loss of hepatocyte polarization as a DILI mechanism. The current study investigated the effects of valproate (VPA), an antiepileptic drug with DILI risk, on the cellular mechanisms responsible for loss of hepatocyte polarization. Fully polarized collagen sandwich-cultured rat hepatocytes were treated with VPA (1-20 mM) for specified times (3-24 h). Hepatocyte viability was significantly decreased by 10 and 20 mM VPA. Valproate depolarized hepatocytes, even at noncytotoxic concentrations (≤5 mM). Depolarization was associated with significantly decreased canalicular levels of multidrug resistance-associated protein 2 (Mrp2) resulting in reduced canalicular excretion of the Mrp2 substrate carboxydichlorofluorescein. The decreased canalicular Mrp2 was associated with intracellular accumulation of Mrp2 in Rab11-positive recycling endosomes and early endosomes. Mechanistic studies suggested that VPA inhibited canalicular trafficking of Mrp2. This effect of VPA on Mrp2 appeared to be selective in that VPA had less impact on canalicular levels of the bile salt export pump (Bsep) and no detectable effect on P-glycoprotein (P-gp) canalicular levels. Treatment with VPA for 24 h also significantly downregulated levels of tight junction (TJ)-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of TJ proteins claudin 1, claudin 2, occludin, ZO1, and ZO3. These findings reveal that two novel mechanisms may contribute to VPA hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.

Inside Back Cover: Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo (J. Biophotonics 9/2018)

In vivo multiphoton imaging was used to map changes in hepatobiliary metabolism in liver fibrosis (left column) and hepatocellular carcinoma (right column). The top row shows the maps of kinetic rate constant of the uptake and esterase processing while the bottom row shows that of bile canalicular excretion of xenobiotics.Further details can be found in the article by Chih‐Ju Lin, Sheng‐Lin Lee, Wei‐Hsiang Wang, et al. (e201700338). image

Bile canalicular dynamics in hepatocyte sandwich cultures.

Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.

A physiologically based model of hepatic ICG clearance: Interplay between sinusoidal uptake and biliary excretion

Although indocyanine green (ICG) has long been used for the assessment of liver function, the respective roles of sinusoidal uptake and canalicular excretion in determining hepatic ICG clearance remain unclear. Here this issue was addressed by incorporating a liver model into a minimal physiological model of ICG disposition that accounts of the early distribution phase after bolus injection. Arterial ICG concentration–time data from awake dogs under control conditions and from the same dogs while anesthetized with 3.5% isoflurane were subjected to population analysis. The results suggest that ICG elimination in dogs is uptake limited since it depends on hepatocellular uptake capacity and on biliary excretion but not on hepatic blood flow. Isoflurane caused a 63% reduction in cardiac output and a 33% decrease in the ICG biliary excretion rate constant (resulting in a 26% reduction in elimination clearance) while leaving unchanged the sinusoidal uptake rate. The terminal slope of the concentration–time curve, K, correlated significantly with elimination clearance. The model could be useful for assessing the functions of sinusoidal and canalicular ICG transporters.

Prediction of Hepatic Clearance in Human From In Vitro Data for Successful Drug Development

The in vivo metabolic clearance in human has been successfully predicted by using in vitro data of metabolic stability in cryopreserved preparations of human hepatocytes. In the predictions by human hepatocytes, the systematic underpredictions of in vivo clearance have been commonly observed among different datasets. The regression-based scaling factor for the in vitro-to-in vivo extrapolation has mitigated discrepancy between in vitro prediction and in vivo observation. In addition to the elimination by metabolic degradation, the important roles of transporter-mediated hepatic uptake and canalicular excretion have been increasingly recognized as a rate-determining step in the hepatic clearance. It has been, therefore, proposed that the in vitro assessment should allow the evaluation of clearances for both transporter(s)-mediated uptake/excretion and metabolic degradation. This review first outlines the limited ability of subcellular fractions such as liver microsomes to predict hepatic clearance in vivo. It highlights the advantages of cryopreserved human hepatocytes as one of the versatile in vitro systems for the prediction of in vivo metabolic clearance in human at the early development stage. The following section discusses the mechanisms underlying the systematic underprediction of in vivo intrinsic clearance by hepatocytes. It leads to the proposal for the assessment of hepatic uptake clearance as one of the kinetically important determinants for accurate predictions of hepatic clearance in human. The judicious combination of advanced technologies and understandings for the drug disposition allows us to rationally optimize new chemical entities to the drug candidate with higher probability of success during the clinical development.

263 PATERNAL ISODISOMY FOR CHROMOSOME 2 AS CAUSE OF BILE SALT EXPORT PUMP (BSEP) DEFICIENCY

Background: Bile salt export pump (BSEP) deficiency, a defect in a canalicular bile acid excretion, falls into the progressive familial intrahepatic cholestasis. It is an autosomal recessive disorder with several mutations being reported in the ABCB11 gene on chromosome 2q24−31. We describe a case of BSEP deficiency with a new mutation, where the patient received both mutated alleles from the father, through the rare phenomenon of uniparental disomy (UPD). Methods: A 4-year-old infant, underwent to orthotopic liver transplantation (OLT) at the age of 9 months because of persistent cholestasis and liver failure. Immunohistochemistry was performed with BSEP antibody. Genomic DNA was extracted from the baby and his parents. Molecular studies of ABCB11 were performed using denaturing high-pressure liquid chromatography (DHPLC) and direct sequencing. Gene copy number determination, segregation of the mutation and reconstruction of the flanking haplotype employed quantitative real time PCR (qPCR), single nucleotide polymorphisms (SNPs) genotyping, and polymorphic microsatellites. Results: Liver histology showed cirrhosis. Immunohistochemistal stain using anti-BSEP antibodies revealed wholly absent marking at canalicular margin of hepatocytes, consistent with the diagnosis of BSEP deficiency. DHPLC and sequencing analysis showed a novel, homozygous c.2620C>T transition in exon 20 of the ABCB11 gene. The mutation replaces arginine for cysteine at residue 833 (p. R833C). This change was absent in a large panel of normal chromosomes, and it was heterozygous in peripheral blood from the father, but not in several tissues obtained from the child’s mother. qPCR analyses did not disclose abnormal gene copy number in both the patient and his mother ruling out a microdeletion of ABCB11. Segregation analysis of seven intragenic SNPs and six flanking microsatellite markers showed data suggestive of UID of the region on chromosome 2q24−31 inherited from the father. Conclusion: In severe forms, infants with BSEP deficiency developed cholestasis and fibrosis, and end-stage liver disease requires OLT. Molecular studies are needed to offer measures as to avoid recurrence of the disease. The present case highlights the importance of full gene analyses to offer a more accurate genetic counselling.

258 HIGH FREE AND GLYCOCONJUGATED AND LOW TAUROCONJUGATED BA LEVELS IN SERUM ARE THE MIRROR OF INTESTINAL EVENTS AND HEPATIC UPTAKE

The endogenous estradiol metabolite, estradiol 17b-D-glucuronide (E17G), induces an acute cholestasis in rat liver due in part to retrieval of canalicular transporters such as the bile salt export pump (Bsep, Abcc11) in a process that involves PKC alpha (Hepatology 2008, 48: 1885−95). Recently, estrogen receptors (ER) have been implicated in the chronic estrogen cholestasis model induced by ethynylestradiol (JBC 2006, 281:16625−31). The aim of this study was to evaluate the involvement of ER in E17Ginduced changes in bile salt canalicular excretion. Methods: Isolated rat hepatocytes couplets were cultured for 5 h, exposed to the estrogen receptor inhibitor ICI-182,780 (ICI, 1mM) for 15min and then incubated with E17G (50mM) for 20min. Other couplet preparations were additionally incubated with PKC inhibitor Go 6976 (Go, 1 mM, 15min) before E17G incorporation. Finally, all preparations were incubated with cholyl-lysylfluorescein (CLF) a fluorescent bile salt substrate of Bsep. Couplets accumulating CLF in their vacuole were counted in a fluorescent microscope and informed as a percentage. Results were compared by ANOVA followed by Student-Newman-Keuls test. Results: See the table.

The Interactions between the Chronic Exposure to Aluminum and Liver Regeneration on Bile Flow and Organic Anion Transport in Rats

The chronic exposure to Aluminum (Al) may compromise different liver functions, mainly during the hepatic regeneration. The aim of this study is to investigate the interactions between the chronic i.p. exposure to Al and hepatic regeneration (HR) on bile flow and organic anion transport in experimental animals. For this purpose, we studied bile flow and fractional transfer rates for the transport of hepatic organic anions (hepatic uptake, sinusoidal efflux, and canalicular excretion), as well as parameters related with the oxidative stress (OS), on rats chronically treated with Al at 0 and 2 days of HR. The Al treatment and time of HR caused a decrease in the biliary flow and in the hepatic uptake and canalicular excretion constants. In addition, Al and HR increased the lipoperoxidation associated with a reduction of the glutathione content and glutathione peroxidase and catalase enzyme's activities. Since the effects of Al and HR on biliary flow and transport systems were additive, but not on the oxidative status, different mechanisms might be involved on these alterations. Even though the OS may play a key role on the hepatic deleterious effects, there is no unique cause-effect relationship between OS and liver dysfunction in this experimental animal model.

In Vitro Biliary Clearance of Angiotensin II Receptor Blockers and 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Inhibitors in Sandwich-Cultured Rat Hepatocytes: Comparison with in Vivo Biliary Clearance

Previous reports have indicated that in vitro biliary clearance (Cl(biliary)) determined in sandwich-cultured hepatocytes correlates well with in vivo Cl(biliary) for limited sets of compounds. This study was designed to estimate the in vitro Cl(biliary) in sandwich-cultured rat hepatocytes (SCRHs) of angiotensin II receptor blockers and HMG-CoA reductase inhibitors that undergo limited metabolism, to compare the estimated Cl(biliary) values with published in vivo Cl(biliary) data in rats, and to characterize the mechanism(s) of basolateral uptake and canalicular excretion of these drugs in rats. The average biliary excretion index (BEI) and in vitro Cl(biliary) values of olmesartan, valsartan, pravastatin, rosuvastatin, and pitavastatin were 15, 19, 43, 45, and 20%, respectively, and 1.7, 3.2, 4.4, 46.1, and 34.6 ml/min/kg, respectively. Cl(biliary) predicted from SCRHs, accounting for plasma unbound fraction, correlated with reported in vivo Cl(biliary) for these drugs. The rank order of Cl(biliary) values predicted from SCRHs was consistent with in vivo Cl(biliary) values. Bromosulfophthalein inhibited the uptake of all drugs. BEI and Cl(biliary) values of olmesartan, valsartan, pravastatin, and rosuvastatin, known multidrug resistance-associated protein (Mrp) 2 substrates, were reduced in SCRHs from Mrp2-deficient (TR(-)) compared with wild-type (WT) rats. Although Mrp2 plays a minor role in pitavastatin biliary excretion, pitavastatin BEI and Cl(biliary) were reduced in TR(-) compared with WT SCRHs; Bcrp expression in SCRHs from TR(-) rats was decreased. In conclusion, in vitro Cl(biliary) determined in SCRHs can be used to estimate and compare in vivo Cl(biliary) of compounds in rats and to characterize transport proteins responsible for their hepatic uptake and excretion.

Update on Progressive Familial Intrahepatic Cholestasis

Three distinct forms of familial intrahepatic cholestasis are the result of mutations in the ATP8B1, ABCB11, and ABCB4 genes. The pathophysiologies of the latter 2 of these diseases are well characterized and are the result of abnormalities in canalicular excretion of bile acids and phospholipids, respectively. The molecular pathophysiology of the systemic disease associated with mutations in ATP8B1 remains unclear. In all of these diseases, wide variations in clinical phenotypes have been observed. The variability can be ascribed at least in part to predicted genotype:phenotype correlations. Disease- and genotype-specific prognoses and therapeutic approaches may exist, although much more information needs to be ascertained before clinicians can confidently make decisions based on genetic information.

Use of Sandwich-Cultured Hepatocytes To Evaluate Impaired Bile Acid Transport as a Mechanism of Drug-Induced Hepatotoxicity

Drug-induced liver toxicity is a significant problem in drug development and clinical practice, yet its mechanisms are not well understood. Growing evidence suggests that inhibition of bile acid transport may be one mechanism of hepatotoxicity. A number of hepatic transporters work in concert to transport bile acids and xenobiotics from blood to bile, and many drugs have been shown to perturb this process with detrimental consequences. Hepatocytes cultured in a sandwich configuration maintain transporter activity and liver-specific metabolic functions; thus, the sandwich-cultured hepatocyte model represents a useful tool for evaluating hepatotoxicity caused by interference with hepatic transporters. As an example, the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone is one such drug that has been shown to inhibit bile acid transport in vitro. Data presented in this manuscript indicate that troglitazone inhibits both basolateral uptake and canalicular excretion of taurocholate in a concentration-dependent manner in both sandwich-cultured and suspended human and rat hepatocytes. These data confirm both the interaction of troglitazone with bile acid transporters in hepatocytes and the utility of the sandwich-cultured hepatocyte model to study such interactions.

Altered Pharmacokinetics of Daunorubicin in Rats with CCl4-Induced Hepatic Injury

The effect of CCl4-induced experimental hepatic injury (CCl4-EHI) on the pharmacokinetics of daunorubicin was investigated systemically in rats, in an attempt to elucidate the major determinants of the effect of CCl4-EHI on the pharmacokinetics of the drug. CCl4-EHI was induced in rats by a single intraperitoneal injection of CCl4 (1 mL/kg rat), and a 24 h fasting period. Daunorubicin was administered intravenously to control and EHI rats at a dose of 11.3 mg/mL/kg and the in vivo pharmacokinetics was studied. The in vitro uptake of the drug into isolated hepatocytes and canalicular liver plasma membrane (cLPM) vesicles, as well as the liver microsomal degradation of the drug, were also determined. The area under the plasma concentration-time curve (AUC) of daunorubicin was increased by 1.6 times, resulting in a 34% decrease in the systemic clearance (CL) in rats with CCl4-EHI. The apparent biliary (CLbile) and urinary (CLurine) clearance of the drug were unchanged, whereas the AUC of daunorubicinol, the major metabolite of daunorubicin, was decreased by 66% in rats with CCl4-EHI. EHI seemed to affect the hepatobiliary elimination of the drug in several ways: the in vitro intrinsic sinusoidal uptake clearance was decreased by 20%; the in vitro intrinsic canalicular excretion clearance of the drug was increased by 1.7 times; and the in vitro liver microsomal degradation of daunorubicin was significantly retarded. CCl4-EHI appears to impair the hepatic metabolism of daunorubicin, thereby decreasing the CL and increasing the AUC of daunorubicin.

Differential Regulation of Hepatic Transporters in the Absence of Tumor Necrosis Factor-α, Interleukin-1β, Interleukin-6, and Nuclear Factor-κB in Two Models of Cholestasis

Hepatic transporters are responsible for uptake and efflux of bile acids and xenobiotics as an essential aspect of liver function. When normal vectorial transport of bile acids by the apical uptake and canalicular excretion transporters is disrupted, cholestasis ensues, leading to accumulation of toxic bile constituents and considerable hepatocellular damage. The purpose of this study was to assess the role of cytokines and nuclear factor-kappaB (NF-kappaB) in the transcriptional regulation of transporters in two models of cholestasis, lipopolysaccharide (LPS) administration and bile duct ligation (BDL). In wild-type (WT) and knockout mouse strains lacking tumor necrosis factor (TNF) receptor-1, interleukin (IL)-1 receptor I, IL-6, or inhibitor of kappaB(IkappaB) kinase beta, transporter mRNA levels in liver were determined using branched DNA signal amplification 16 h after LPS administration or 3 days after BDL. In WT mice, LPS administration tended to decrease mRNA levels of organic anion-transporting polypeptide (Oatp) 2, Na(+)-taurocholate cotransporting polypeptide (Ntcp), Oatp1, Oatp4, bile salt excretory protein (Bsep), multidrug resistance-associated protein (Mrp) 2, and Mrp6 compared with saline treatment, whereas it increased Mrp1, 3, and 5 levels. Similar changes were observed in each knockout strain after LPS administration. Conversely, BDL decreased only Oatp1 expression in WT mice, meanwhile increasing expression of Mrp1, 3, and 5 and Oatp2 expression in both WT and knockout strains. Because the transcriptional effects of BDL- and LPS-induced cholestasis reflect dissimilarity in hepatic transporter regulation, we conclude that these disparities are not due to the individual activity of TNF-alpha, IL-1, IL-6, or NF-kappaB but to the differences in the mechanism of cholestasis.

ETHYNYLESTRADIOL INCREASES EXPRESSION AND ACTIVITY OF RAT LIVER MRP3

We evaluated the effect of ethynylestradiol (EE) administration (5 mg/kg b.wt. s.c., for 5 consecutive days) on the expression and activity of multidrug resistance-associated protein 3 (Mrp3) in rats. Western blotting analysis revealed decreased Mrp2 (-41%) and increased Mrp3 (+200%) expression by EE. To determine the functional impact of up-regulation of Mrp3 versus Mrp2, we measured the excretion of acetaminophen glucuronide (APAP-glu), a common substrate for both transporters, into bile and perfusate in the recirculating isolated perfused liver (IPL) model. APAP-glu was generated endogenously from acetaminophen (APAP), which was administered as a tracer dose (2 micromol/ml) into the perfusate. Biliary excretion of APAP-glu after 60 min of perfusion was reduced in EE-treated rats (-80%). In contrast, excretion into the perfusate was increased by EE (+45%). Liver content of APAP-glu at the end of the experiment was reduced by 36% in the EE group. The total amount of glucuronide remained the same in both groups. Taken together, these results indicate that up-regulation of Mrp3 led to an exacerbated basolateral versus canalicular excretion of conjugated APAP in IPL. We conclude that induced expression of basolateral Mrp3 by EE may represent a compensatory mechanism to prevent intracellular accumulation of common Mrp substrates, either endogenous or exogenous, due to reduced expression and activity of apical Mrp2.

Deglycosylation of Na+/K+-ATPase causes the basolateral protein to undergo apical targeting in polarized hepatic cells

Polarized epithelia, such as hepatocytes, target their integral membrane proteins to specific apical or basolateral membrane domains during or after biogenesis. The roles played by protein glycosylation in this sorting process remain controversial. We report here that deglycosylation treatments in well-polarized hepatic cells by deglycosylation drugs, or by site-directed mutagenesis of the N-linked-glycosylation residues, all cause the Na+/K+-ATPase beta-subunit to traffic from the native basolateral to the apical/canalicular domain. Deglycosylated beta-subunits are still able to bind and therefore transport the catalytic alpha-subunits to the aberrant apical location. Such apical targeting is mediated via the indirect transcytosis pathway. Cells containing apical Na+/K+-ATPase appear to be defective in maintaining the ionic gradient across the plasma membrane and in executing hepatic activities that are dependent upon the ionic homeostasis such as canalicular excretion.

Effect of a new hepatoprotective agent, YH-439, on the hepatobiliary transport of organic cations (OCS): Selective inhibition of sinusoidal OCs uptake without influencing glucose uptake and canalicular OCs excretion

The effect of a new hepatoprotective agent, YH-439, on the hepatobiliary transport of a model organic cation (OC), TBuMA (tributylmethylammonium), was investigated. The area under the plasma concentration-time curve (AUC) from time zero to 4 h following iv administration of TBuMA (6.6 micromol/kg) was increased significantly when YH-439 in corn oil (300 mg/kg) was orally administered to rats 24 h prior to the experiment. Nevertheless, the cumulative biliary excretion of TBuMA remained unchanged. As a consequence, the apparent biliary clearance (CLb) of TBuMA was decreased significantly as a result of YH-439 pretreatment, consistent with the fact that the in vivo excretion clearance of TBuMA across the canalicular membrane (CLexc) was not changed by the pretreatment. The in vitro uptake of TBuMA into isolated hepatocytes was decreased by one half by the pretreatment, owing to a decrease in the apparent Vmax and CLlinear, but the Km for the process remained constant. Most interestingly, however, the sinusoidal uptake of glucose, a nutrient, into hepatocytes was not influenced by the pretreatment, suggesting the YH-439 pretreatment specifically impaired the sinusoidal uptake of OCs. Thus, the OC-specific inhibition of hepatic uptake, without influencing the uptake of glucose, a nutrient, appeared to be associated with the hepatoprotective activity of YH-439.

Mechanism of the Stationary Canalicular Excretion of Tributylmethyl Ammonium in Rats with a CCl4-Induced Acute Hepatic Injury

The in vivo canalicular excretion clearance of tributylmethyl ammonium (TBuMA), a P-glycoprotein (P-gp) substrate, was previously reported to be unaffected by the induction of an experimental hepatic injury (EHI) by CCl4 despite the increased expression of P-gp in the EHI liver. The objective of this study, therefore, was to elucidate the mechanism for the unchanged canalicular excretion clearance of TBuMA in EHI rats. TBuMA uptake was increased in cLPM vesicles from EHI rats compared with that from control rats. The total bile salt concentration in EHI liver was significantly reduced compared with that in a control liver. Because, in our previous studies, the uptake of TBuMA by cLPM vesicles was found to be significantly enhanced in the presence of bile salts, the reduction in bile salt levels in the EHI liver may be related to the unaltered TBuMA clearance. Despite the fact that the uptake of TBuMA by cLPM vesicles was increased by the addition of an EHI liver extract, the extent of the increase was comparatively less compared to the addition of a control liver extract. The in vivo excretion clearance of TBuMA was increased in a taurodeoxycholate dose-dependent manner in EHI rats. These observations suggest, therefore, that despite the induction of P-gp expression by the EHI, the in vivo canalicular excretion clearance of TBuMA remains unaltered as the result of an offset by reduced levels of bile salt(s).

Hepatic function in hypothermically stored porcine livers: Comparison of hypothermic machine perfusion vs cold storage

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage ( n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.