Featuring molecular regulation of bile acid homeostasis in pediatric short bowel syndrome.

BACKGROUND: Disturbed bile acid homeostasis may foster development of short bowel syndrome (SBS) associated liver disease during and after weaning off parenteral nutrition (PN). Our aim was to study hepatic molecular regulation of bile acid homeostasis in relation to serum and fecal bile acid profiles in pediatric SBS. METHODS: Liver histopathology and mRNA expression of genes regulating synthesis, uptake and export of bile acids, and cellular receptors involved in bile acid signaling were measured in SBS patients (n = 33, median age 3.2 years). Simultaneously, serum (n = 24) and fecal (n = 10) bile acid profiles were assessed. Sixteen patients were currently on PN. Results of patients were compared to healthy control subjects. RESULTS: Nine of ten (90 %) patients with histological cholestasis received current PN, while portal inflammation was present in 60 % (6/10) of patients with cholestasis compared to 13 % (3/23) without cholestasis (P = 0.01). In all SBS patients, hepatic synthesis and uptake of bile acids was increased. Patients on current PN showed widespread repression of hepatic FXR target genes, including downregulated canalicular (BSEP, MDR3) and basolateral (MRP3) bile acid exporters. Serum and fecal primary bile acids were increased both during and after weaning off PN. CONCLUSIONS: Bile acid homeostasis in SBS is characterized by interrupted enterohepatic circulation promoting increased hepatic synthesis and conservation of bile acids. In PN dependent SBS patients with hepatic cholestasis and inflammation, the molecular fingerprint of downregulated hepatocyte canalicular and basolateral bile acid export with simultaneously increased synthesis and uptake of bile acids could favor their accumulation in hepatocytes and predispose to liver disease.

Human menstrual blood-derived stem cell transplantation suppresses liver injury in DDC-induced chronic cholestasis.

BACKGROUND: Cholestatic liver injury can lead to serious symptoms and prognoses in the clinic. Currently, an effective medical treatment is not available for cholestatic liver injury. Human menstrual blood-derived stem cells (MenSCs) are considered as an emerging treatment in various diseases. This study aimed to explore the treatment effect of MenSCs in cholestatic liver injury. METHODS: The treatment effect of MenSCs on chronic cholestatic liver injury was verified in 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC)-induced C57/BL6 mice. Pathological, fibrosis area in the liver tissue and serum liver enzymes were tested. Proteomics and western blot were used to explore the related targets and molecular mechanisms. Adeno-associated virus (AAV) 9-infected mice were applied for verification. RESULTS: MenSCs markedly improved the survival rate of the DDC-treated mice (60% vs. 100%), and decreased the mouse serum aspartate aminotransferase (AST) (169.4 vs. 108.0 U/L, p < 0.001), alanine aminotransferase (ALT) (279.0 vs. 228.9 U/L, p < 0.01), alkaline phosphatase (ALP) (45.6 vs. 10.6 U/L, p < 0.0001), direct bilirubin (DBIL) (108.3 vs. 14.0 µmol/L, p < 0.0001) and total bilirubin (TBIL) (179.2 vs. 43.3 µmol/L, p < 0.0001) levels as well as intrahepatic cholestasis, bile duct dilation and fibrotic areas (16.12 vs. 6.57%, p < 0.05). The results further indicated that MenSCs repaired the DDC-induced liver tight junction (TJ) pathway and bile transporter (OATP2, BSEP and NTCP1) injury, thereby inhibiting COL1A1, α-SMA and TGF-ß1 activation by upregulating liver ß-catenin expression. CONCLUSIONS: MenSC transplantation could be an effective treatment method for cholestatic liver injury in mice. MenSCs may exhibit therapeutic effects by regulating ß-catenin expression.

Clonorchis sinensis and clonorchiasis.

Clonorchis sinensis is a fish-borne trematode that inhabits the bile duct of mammals including humans. Clonorchiasis is prevalent in China, Korea, and Vietnam, and 15-20 million people are estimated to be infected by this fluke. Freshwater snails act as the first intermediate host for the proliferation of C. sinensis larvae and shed the cercariae into water. The cercariae penetrate the skin of freshwater fish and transform to metacercariae. Humans are infected by eating raw or undercooked freshwater fish as dishes of filet, "sashimi," or congee, which contain C. sinensis metacercariae. In humans, the C. sinensis metacercariae excyst in the duodenum, and juvenile flukes migrate up via bile chemotaxis into bile ducts. Once there, C. sinensis provokes hyperplasia of the bile duct epithelium, obstructive jaundice, ascites, liver enlargement and cirrhosis, and infrequent cholangiocarcinoma (CCA). Although the association between C. sinensis infection and CCA has been firmly established in past decades, the underlying mechanisms are not elucidated in detail. In the context of chronic clonorchiasis-associated hepatobiliary aberrations, the constitutive disruption of redox homeostasis and dysregulation of physiological signaling pathways may promote the malignant transformation of cholangiocytes, thus leading to substantial acquisition of a more aggressive phenotype by these cells: CCA. With advances of genomic and molecular biological approaches, diverse C. sinensis proteins that are essential for parasite physiology and pathogenicity have been identified and characterized. Some of the proteins have been considered as attractive targets for development of vaccines and chemotherapeutics. Candidate antigens for reliable serodiagnosis of clonorchiasis have been studied.

Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking.

BACKGROUND: Multidrug resistance-associated protein 4 (MRP4) is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC) transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host's bile duct, accumulation of bile juice can be toxic to the worm's tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body. RESULTS: We cloned MRP4 (CsMRP4) from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2) and two nucleotide-binding domains (NBD1 & 2) as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa) fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults. CONCLUSIONS: Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites' survival.

Pre-Hepatectomy Assessment of Bile Transporter Expression by Gadoxetic Acid-Enhanced MRI in a Rat Model of Liver Cirrhosis.

BACKGROUND: Gadoxetic acid is a liver-specific intravenous T1 magnetic resonance (MR) contrast agent that is excreted via the hepatobiliary system. We hypothesize that hepatocyte expressions of bile transporters (OATP1 and MRP2) correlate with dynamic profile of Gadoxetic acid enhanced (GE)-MR imaging (MRI). METHODS: Two groups of rats, control (n = 6) and cirrhosis (n = 12), received gadoxetic acid enhanced MRI followed by 70% hepatectomy. The change in MR signal intensity from the baseline before the contrast injection (ΔSI) was analyzed every minute for 30 min. Dynamic signal intensity retention ratio (DSR) was defined as the mean ΔSI of the third 10-minmin period divided by the first 10-minmin period. Real-time PCR was utilized to quantify mRNA expressions. RESULTS: Compared to the control, cirrhosis group demonstrated lower mRNA levels of OATP1 (0.038 ± 0.020 vs. 0.232 ± 0.0979; p = 0.004), MRP2 (0.201 ± 0.084 vs. 0.7567 ± 0.254; p = 0.002), and OATP1/MRP2 mRNA ratio (0.193 ± 0.065 vs. 0.342 ± 0.206; p = 0.032). DSR was higher in the cirrhosis group (0.678 ± 0.554 vs -0.125 ± 0.839; p = 0.033). In the cirrhosis group, there was an inverse correlation between the ratios of OATP1/MRP2 mRNA and DSR (R = -0.709, p = 0.01). CONCLUSION: Bile transporters OATP1/MRP2 mRNA expression ratio in rat liver tissue decreased with DMN-induced liver injury. The expressions of bile transporters correlated with GE-MRI DSR. The GE-MRI DSR has potential utility in qualifying OATP1/MRP2 mRNA expression.

Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.

The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury.

Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes.

Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.