Metabolomic analysis of swainsonine poisoning in renal tubular epithelial cells.

Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To understand the mechanism of SW-induced toxicity and to delineate the metabolic profile of locoweed poisoning we performed the LC-MS/MS untargeted metabolomic study to analyze metabolites in SW-treated renal tubular epithelial cells (0.8 mg/mL, 12 h) and in order to identify the SW-induced metabolomic changes. The analysis identified 2,563 metabolites in positive ion mode and 1,990 metabolites in negative ion mode. Our results showed that the metabolites were mainly benzenoids, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids, and derivatives. The differential metabolites were primarily enriched in pathways involving bile secretion, primary bile acid biosynthesis, riboflavin metabolism, ferroptosis, drug metabolism-cytochrome P450, and primidine metabolism. We have screened out substances such as swainsonine, 3alpha,7alpha-Dihydroxy-5beta-cholestanate, 2-Hydroxyiminostilbene, and glycochenodeoxycholate, which may have the potential to serve as biomarkers for swainsonine poisoning. This study provides insights into the types of metabolomic alteration in renal tubular epithelial cells induced by swainsonine.

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases.

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

Construction of a predictive nomogram and bioinformatic investigation of the potential mechanism of postoperative early recurrence of hepatocellular carcinoma meeting the Milan criteria.

Background: Hepatectomy is the most common treatment for hepatocellular carcinoma (HCC) meeting the Milan criteria; however, postoperative early recurrence (PER) compromises the survival time. This study aimed to construct a predictive nomogram for PER of HCC patients within the Milan criteria. And the underlying mechanism related to PER may associate with the independent risk factors used to construct the nomogram, therefore, we preliminarily investigated the potential mechanism of PER using The Cancer Genome Atlas (TCGA) database to provide an idea for preventing PER. Methods: Patients with HCC meeting the Milan criteria receiving hepatectomy in our center between 2009 and 2015 were enrolled. The clinical and histological data of all participants were collected. Follow-up was performed at outpatient and PER was defined as recurrence within 2 years after resection. All participants were randomly assigned to the training or validation cohort at a 4:1 ratio. A nomogram was constructed based on the independent risk factors in the training cohort. The accuracy and clinical utility of this nomogram were evaluated using the C-index, calibration plot, and decision curve analysis (DCA). The differentially-expressed genes (DEGs) between early-stage HCC patients with and without PER in TCGA database were identified. Enrichment analysis was performed to determine the potential relapse-related mechanism. Results: The independent risk factors were alpha-fetoprotein (AFP) ≥400 ng/mL, gamma-glutamyl transpeptidase (GGT) ≥60 U/L, Glisson's capsule invasion, microvascular invasion (MVI), and satellite lesions. The C-index value of the nomogram was 0.693 [95% confidence interval (CI): 0.632-0.754; P<0.001] in the training cohort and 0.658 (95% CI: 0.529-0.787; P=0.016) in the validation cohort. The calibration and decision curves demonstrated good accuracy and clinical utility of this nomogram respectively. 133 DEGs were identified and enrichment analysis showed the bile secretion pathway related to PER and two bile secretion pathway-related genes {ATP1A2 [P=0.027; hazard ratio (HR) =2.086, 95% CI: 0.916-4.749] and SLC5A1 (P=0.0016; HR =0.361, 95% CI: 0.145-0.898)} were significantly associated with disease free survival (DFS). Conclusions: Our nomogram has satisfactory accuracy and clinical utility in predicting the PER of patients with HCC meeting the Milan criteria. Aberrant bile secretion may be an important mechanism of PER.

MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Interventional treatment of biliodigestive anastomosis leaks with a modified percutaneous transhepatic cholangiodrainage.

INTRODUCTION: Biliodigestive leaks are typically caused by an insufficiency at the surgical anastomosis. Biliodigestive anastomosis (BDA) insufficiencies can lead to bilomas, abscesses and vascular erosion in chronic conditions. MATERIAL AND METHODS: We performed a retrospective analysis of the medical and radiological records of all patients with biliodigestive insufficiency who received interventional treatment between July 2015 and February 2021. Nine patients (three with unilateral drainage and six with bilateral drainage) were treated with a modified percutaneous transhepatic cholangiodrainage (PTCD). Clinical success was considered after complete resolution of the peribiliary collections, absence of bile within the surgical drains, radiological patency of the BDA (contrast medium flowing properly through the BDA and no signs of leakage) and haemodynamic stability of the patient without signs of sepsis. RESULTS: Clinical success was achieved in all nine patients. No patients required revision surgery to repair their BDA. The mean indwelling drainage time was 34.8±16.5 days. The mean number of interventional procedures performed per patient was 6.6±2.0. CONCLUSION: Patients who present with BDA insufficiency may benefit from interventional radiological techniques. Our modified PTCD resolved the BDA leak in all nine cases and should be considered as a valuable option for the treatment of patients with this complication. Our technique demonstrated to be feasible and effective.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Molecular Regulation of Canalicular ABC Transporters.

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters' folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

Postoperative intestinal obstruction in patients with biliary atresia impedes biliary excretion and results in subsequent liver transplantation.

PURPOSE: This study aimed to investigate the negative effects of intestinal obstruction for jaundice-free native liver survival after Kasai portoenterostomy (PE) for biliary atresia (BA). METHODS: We retrospectively reviewed the records of patients who underwent PE for BA between 2006 and 2019. We evaluated the postoperative morbidity of intestinal obstruction for up to 2 years after PE and the effects of intestinal obstruction on jaundice-free native liver survival. On the basis of their initial operation, patients were divided into open portoenterostomy (Open-PE) and laparoscopic portoenterostomy (Lap-PE) groups, and morbidity was compared. RESULTS: Of the 87 patients reviewed, 6 (6.9%) patients developed postoperative intestinal obstruction and underwent surgery to relieve the obstruction. The morbidity of early postoperative intestinal obstruction was 1.68 per 10,000 person days. The jaundice-free native liver survival rate among patients who once achieved jaundice-free status after PE was significantly lower in the patients with intestinal obstruction compared to in those without intestinal obstruction (0% vs. 73.8%; RR = 3.81, p = 0.007). No significant differences were seen in postoperative intestinal obstructions between the Open-PE and Lap-PE groups (p = 0.242). CONCLUSIONS: Intestinal obstruction negatively impact jaundice-free native liver survival, even in patients who once achieved jaundice-free status after PE for BA.

The involvement of genes related to bile secretion pathway in rat tooth germ development.

Tooth formation is accomplished under strict genetic control procedures. Therefore, exploring the gene network system of tooth development has a very positive practical significance for the study of tooth tissue regeneration and the prevention and treatment of tooth abnormalities. Early bell stage is the initial phase of odontoblast formation and dentin matrix deposition in the process of tooth development. Through RNA sequencing and differential gene analysis of the rat tooth germ samples at cap stage and early bell stage, we found that the bile secretion pathway was the most significant difference signal pathway during the development between cap stage and bell stage, which mainly included ABCC3, AQP4, SLC10A1, SLC2A1, SLC4A4, ADCY5, AQP9, CFTR, ATP1A2, ATP1B1 and ATP1A1, totally 11genes. Immunostaining revealed that SLC2A1, SLC4A4, ADCY5 and ATP1B1were mainly expressed in epithelium in bud stage and inner and outer enamel epithelium during the embryonic phase. In the postnatal 1 and postnatal 7, SLC2A1, SLC4A4 and ABCC3 were highly expressed in ameloblasts and odontoblasts while ADCY5, ATP1B1 and SLC10A1was expressed moderately only in odontoblasts. This finding illustrated that the bile secretion pathway related genes may participate in the development of tooth germ.

Systematic review of progressive familial intrahepatic cholestasis.

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of rare genetic disorders associated with bile acid secretion or transport defects. This is the first systematic review of the epidemiology, natural history and burden of PFIC. METHODS: MEDLINE and Embase were searched for publications on PFIC prevalence, incidence or natural history, and the economic burden or health-related quality of life (HRQoL) of patients with PFIC. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. RESULTS: Of 1269 records screened, 20 were eligible (epidemiology, 17; humanistic burden, 5; both, 2). Incidence of intrahepatic cholestasis, including but not limited to PFIC, was 1/18 000 live births in one study that did not use genetic testing. In two studies of infants and children (2-18 years) with cholestasis, 12-13% had genetically diagnosed PFIC. Of the three main PFIC subtypes, PFIC2 was the most common (21-91% of patients). Common symptoms (e.g. pruritus, jaundice, hepatomegaly, splenomegaly) generally appeared at about 3 months of age and tended to emerge earliest in patients with PFIC2. Patients reported that pruritus was often severe and led to dermal damage and reduced HRQoL. Disease progression led to complications including liver failure and hepatocellular carcinoma, with 20-83% of patients requiring liver transplantation. Mortality was 0-87% across 10 studies (treatment varied among studies), with a median age at death of ~4 years in one study. CONCLUSIONS: Patients with PFIC face debilitating symptoms and poor prognosis. Further research is needed to inform patient management and clinical trial design. Published data on the epidemiology and socioeconomic burden of PFIC is limited.

Detection of Secondary Metabolites as Biomarkers for the Early Diagnosis and Prevention of Type 2 Diabetes.

BACKGROUND: Type 2 diabetes, or T2D, is a metabolic disease that results in insulin resistance. In the present study, we hypothesize that metabolomic analysis in blood samples of T2D patients sharing the same ethnic background can recover new metabolic biomarkers and pathways that elucidate early diagnosis and predict the incidence of T2D. METHODS: The study included 34 T2D patients and 33 healthy volunteers recruited between the years 2012 and 2013; the secondary metabolites were extracted from blood samples and analyzed using HPLC. RESULTS: Principal coordinate analysis and hierarchical clustering patterns for the uncharacterized negatively and positively charged metabolites indicated that samples from healthy individuals and T2D patients were largely separated with only a few exceptions. The inspection of the top 10% secondary metabolites indicated an increase in fucose, tryptophan and choline levels in the T2D patients, while there was a reduction in carnitine, homoserine, allothreonine, serine and betaine as compared to healthy individuals. These metabolites participate mainly in three cross-talking pathways, namely "glucagon signaling", "glycine, serine and threonine" and "bile secretion". Reduced level of carnitine in T2D patients is known to participate in the impaired insulin-stimulated glucose utilization, while reduced betaine level in T2D patients is known as a common feature of this metabolic syndrome and can result in the reduced glycine production and the occurrence of insulin resistance. However, reduced levels of serine, homoserine and allothrionine, substrates for glycine production, indicate the depletion of glycine, thus possibly impair insulin sensitivity in T2D patients of the present study. CONCLUSION: We introduce serine, homoserine and allothrionine as new potential biomarkers of T2D.

Regulation of bile secretion by calcium signaling in health and disease.

Calcium (Ca2+) signaling controls secretion in many types of cells and tissues. In the liver, Ca2+ regulates secretion in both hepatocytes, which are responsible for primary formation of bile, and cholangiocytes, which line the biliary tree and further condition the bile before it is secreted. Cholestatic liver diseases, which are characterized by impaired bile secretion, may result from impaired Ca2+ signaling mechanisms in either hepatocytes or cholangiocytes. This review will discuss the Ca2+ signaling machinery and mechanisms responsible for regulation of secretion in both hepatocytes and cholangiocytes, and the pathophysiological changes in Ca2+ signaling that can occur in each of these cell types to result in cholestasis.

Biliary bile acids in hepatobiliary injury - What is the link?

The main trigger for liver injury in acquired cholestatic liver disease remains unclear. However, the accumulation of bile acids (BAs) undoubtedly plays a role. Recent progress in deciphering the pathomechanisms of inborn cholestatic liver diseases, decoding mechanisms of BA-induced cell death, and generating modern BA-derived drugs has improved the understanding of the regulation of BA synthesis and transport. Now is the appropriate time to reassess current knowledge about the specific role of BAs in hepatobiliary injury.

Targeted pharmacotherapies for defective ABC transporters.

Human ABC (ATP Binding Cassette) transporters form a superfamily of forty-eight transmembrane proteins, which transport their substrates across biological membranes against important concentration gradients, in an energy-dependent manner. Gene variations in approximately half of these transporters have been identified in subjects with rare and often severe genetic diseases, highlighting the importance of their biological function. For missense variations leading to defects in ABC transporters, the current challenge is to identify new molecules with therapeutic potential able to rescue the induced molecular deficiency. In this review, we first address the progress provided by emerging pharmacotherapies in cystic fibrosis, the most frequent monogenic disease caused by variations of an ABC transporter, i.e. ABCC7/CFTR. Then, we enlarge the topic to the other ABC transporters, more notably to canalicular ABC transporters, the variations of which cause rare hepatobiliary diseases, and we discuss the first promising attempts aiming to correct molecular defects of these proteins.

Type 2 inositol trisphosphate receptor gene expression in hepatocytes is regulated by cyclic AMP.

The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.

Polarized release of hepatic microRNAs into bile and serum in response to cellular injury and impaired liver function.

BACKGROUND & AIMS: Extracellular microRNAs (miRNAs) in serum and bile are currently under intense investigation for biomarker purposes in liver disease. However, the directions and pathways by which miRNAs are released from hepatic cells remains largely unknown. Here, we investigated the release of hepatocyte and cholangiocyte-derived miRNAs (HDmiRs and CDmiRs) into blood and bile during various (patho)physiological hepatic conditions. METHODS: MiRNA release was analysed using longitudinally collected tissue and paired bile and serum samples (n = 124) that were obtained from liver transplant recipients during follow-up. RESULTS: Cell-type specificity of HDmiRs and CDmiRs was confirmed in liver and common bile duct biopsies (P < 0.001). Analysis of paired bile and serum samples showed up to 20-times higher miRNA-levels in bile compared to serum (P < 0.0001). Fractionation of bile showed the majority of miRNAs being present in the unpelletable supernatant, where protein conjunctions protect miRNAs against degradation (P < 0.0001). During episodes of liver injury and histologically proven rejection in liver transplant recipients, relative HDmiR-levels in bile decreased while its levels in serum increased (P ≤ 0.015). Simultaneously, relative CDmiR-levels in bile significantly increased, while their levels in serum decreased. Related to liver excretory function, a strong positive correlation was observed between HDmiR-122 levels and bilirubin excretion into bile (R = 0.694, P < 0.0001), whereas CDmiRs showed an inverse correlation (P < 0.05). CONCLUSION: During impaired excretory function and injury, the liver shows polarized release of extracellular HDmiRs and CDmiRs. This sheds new light on the biology of hepatic miRNA release which is relevant for the interpretation of hepatic miRNAs as biomarkers.

The Pharmacological Effects of Benachio-F(®) on Rat Gastrointestinal Functions.

Functional dyspepsia (FD) is a prevalent idiopathic upper gastrointestinal (GI) disorder characterized by diverse symptomatology including epigastric pain or discomfort, postprandial fullness, and early satiety. Although its pathophysiological mechanisms have not yet been fully established, the available studies suggest that the etiology of FD is invariably multifactorial. Benachio-F(®) (BF) is a proprietary liquid formulation of 7 herbal extracts that has been proposed to address this multifactorial etiology using multi-drug phytotherapy. The pharmacological effects of BF, in comparison with those of two other herbal products (Whalmyungsu(®); WM and Iberogast(®); IB) were evaluated in rats. In a laparotomy-induced rat model of delayed GI transit, BF significantly accelerated the delayed gastric emptying caused by morphine, apomorphine, and cisplatin, and also significantly increased mean gastric transit, as compared to the control animals. BF markedly increased gastric accommodation in rats and produced higher gastric volume values than did the control treatment. The effects of BF were generally comparable or superior to those of WM and IB in these models. Furthermore, BF significantly stimulated biliary flow, as compared to the control treatment. These results indicated that BF might have great potential as an effective phytotherapeutic agent capable of reducing GI symptoms and increasing quality of life in FD patients.

The Pharmacological Effects of Benachio-F(R) on Rat Gastrointestinal Functions

Functional dyspepsia (FD) is a prevalent idiopathic upper gastrointestinal (GI) disorder characterized by diverse symptomatology including epigastric pain or discomfort, postprandial fullness, and early satiety. Although its pathophysiological mechanisms have not yet been fully established, the available studies suggest that the etiology of FD is invariably multifactorial. Benachio-F(R) (BF) is a proprietary liquid formulation of 7 herbal extracts that has been proposed to address this multifactorial etiology using multi-drug phytotherapy. The pharmacological effects of BF, in comparison with those of two other herbal products (Whalmyungsu(R); WM and Iberogast(R); IB) were evaluated in rats. In a laparotomy-induced rat model of delayed GI transit, BF significantly accelerated the delayed gastric emptying caused by morphine, apomorphine, and cisplatin, and also significantly increased mean gastric transit, as compared to the control animals. BF markedly increased gastric accommodation in rats and produced higher gastric volume values than did the control treatment. The effects of BF were generally comparable or superior to those of WM and IB in these models. Furthermore, BF significantly stimulated biliary flow, as compared to the control treatment. These results indicated that BF might have great potential as an effective phytotherapeutic agent capable of reducing GI symptoms and increasing quality of life in FD patients.