BACKGROUND

Estradiol-17β-D-glucuronide (E17G) induces cholestasis in vivo, endocytic internalization of the canalicular transporters multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11) being a key pathomechanism. Cyclic AMP (cAMP) prevents cholestasis by targeting these transporters back to the canalicular membrane. In hepatocyte couplets, glucagon and salbutamol, both of which increase cAMP, prevented E17G action by stimulating the trafficking of these transporters by different mechanisms, namely: glucagon activates a protein kinase A-dependent pathway, whereas salbutamol activates an exchange-protein activated by cAMP (Epac)-mediated, microtubule-dependent pathway.

METHODS

The present study evaluated whether glucagon and salbutamol prevent E17G-induced cholestasis in a more physiological model, i.e., the perfused rat liver (PRL). Additionally, the preventive effect of in vivo alanine administration, which induces pancreatic glucagon secretion, was evaluated.

RESULTS

In PRLs, glucagon and salbutamol prevented E17G-induced decrease in both bile flow and the secretory activity of Abcc2 and Abcb11. Salbutamol prevention fully depended on microtubule integrity. On the other hand, glucagon prevention was microtubule-independent only at early time periods after E17G administration, but it was ultimately affected by the microtubule disrupter colchicine. Cholestasis was associated with endocytic internalization of Abcb11 and Abcc2, the intracellular carriers being partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained colocalized with Rab11a after glucagon treatment. In vivo, alanine administration increased hepatic cAMP and accelerated the recovery of bile flow and Abcb11/Abcc2 transport function after E17G administration. The initial recovery afforded by alanine was microtubule-independent, but microtubule integrity was required to sustain this protective effect.

CONCLUSIONS

We conclude that modulation of cAMP levels either by direct administration of cAMP modulators or by physiological manipulations leadings to hormone-mediated increase of cAMP levels (alanine administration), prevents estrogen-induced cholestasis in models with preserved liver architecture, through mechanisms similar to those arisen from in vitro studies.

Objective: Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder of pregnancy characterized by pruritus, elevated liver enzymes and fasting serum bile acids. Genetic predisposition has been suggested to play a role in its etiology and mutations in the ATP8B1(OMIM ∗602397) (FIC1), ABCB11(OMIM ∗603201) (BSEP), and ABCB4(OMIM ∗171060) (MDR3) genes have been implicated. In the present study, we aimed to investigate the possible role of ATP8B1, ABCB11, and ABCB4 gene mutations in the patients with ICP.

Materials and methods: A total of 25 patients who were diagnosed with ICP were included in the study. Genetic test results and mutation status of the patients as assessed by the next-generation sequencing technology were retrospectively retrieved from the hospital database.

Results: Of all patients, significant alterations in the ATP8B1 (n = 2), ABCB11 (n = 1), and ABCB4 (n = 7) genes were observed in 10 patients using the molecular analysis testing. All these alterations were heterozygous. Of these alterations, four were reported in the literature previously, while six were not. Using the in-silico parameters, there was a pathogenic alteration in the ABCB4 gene in one patient, while there was no clinically relevant alteration in the other gene mutations in the remaining nine patients.

Conclusion: Considering the fact that the alterations were compatible with clinical presentations of the ICP patients and the incidence of these mutations is low in the general population, we believe that our study results are clinically relevant. Further molecular genetic tests in ICP patients and functional studies supporting the results would shed light into the clinical importance of these alterations.

Keywords: ABCB11; ABCB4; ATP8B1; Intrahepatic cholestasis of pregnancy; Mutation.

Introduction: Legalization of medicinal cannabis around the world has led to an increase in the use of commercial cannabis-based products in the community. These cannabis-based products are being used in combination with conventional drugs to treat a variety of health conditions. Moreover, recreational cannabis-based products may be used in combination with other drugs. In this setting, there is increased potential for drug-drug interactions (DDIs) involving commercial cannabis-based products. Since DDIs can lead to serious adverse events, drug regulatory bodies require that every investigational drug be evaluated for DDI potential at metabolic enzymes and transporters. However, this seldom occurs for cannabis-based products due to legislation in many jurisdictions allowing a direct pathway to market. This study aimed to examine the inhibitory potential of three commercially available cannabis-based products at human ATP-binding cassette (ABC) and solute-carrier (SLC) transporters. Materials and Methods: Three commercial cannabis-based products (Spectrum Yellow™, Tweed Argyle, and Spectrum Red™) that contain differing concentrations of cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) were evaluated for DDI potential at 12 drug transporters. HEK293 cells or vesicles expressing human ABC transporters (ABCB1, ABCC2, ABCG2, or ABCB11) and SLC transporters (SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLCO1B1, SLCO1B3, SLC47A1, and SLC47A2) were used to measure transporter function. Results: Spectrum Yellow and Tweed Argyle inhibited ABCG2 transporter function. The IC₅₀ value of Spectrum Yellow based on CBD and Δ⁹-THC content was 4.5 μM for CBD and 0.20 μM for Δ⁹-THC, and the IC₅₀ value of Tweed Argyle was 9.3 μM for CBD and 6.0 μM for Δ⁹-THC. Tweed Argyle also inhibited ABCB11 transporter function with an IC₅₀ value of 11.9 μM for CBD and 7.7 μM for Δ⁹-THC. SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 transporter functions were modestly inhibited by high concentrations of the cannabis-based products. The three cannabis-based products did not inhibit ABCB1, ABCC2, SLC47A1, SLC47A2, or SLC22A8 transporters. Discussion: Novel findings were that the cannabis-based products inhibited ABCB11, SLC22A6, SLC22A1, SLC22A2, SLCO1B1, and SLCO1B3 (although modestly in most instances). Spectrum Yellow and Tweed Argyle potently inhibited ABCG2, and future in vivo DDI studies could be conducted to assess whether cannabis products affect the pharmacokinetics of medications that are ABCG2 substrates.

Keywords: ABC transporters; SLC transporters; cannabis; cannabis-based products.

Biliary atresia (BA) is the most severe form of obstructive cholangiopathy occurring in infants. Definitive diagnosis of BA usually relies on operative findings together with supporting pathological patterns found in the extrahepatic bile duct. In infancy, overlapping clinical patterns of cholestasis can be found in other diseases including biliary hypoplasia and progressive familial intrahepatic cholestasis. In addition, BA has been reported as a phenotype in some rare genetic syndromes. Unlike BA, other cholangiopathic phenotypes have their own established genetic markers. In this study, we used these markers to look for other cholestasis entities in cases diagnosed with BA. DNA from 20 cases of BA, diagnosed by operative findings and histopathology, were subjected to a study of 19 genes associated with infantile cholestasis syndromes, using whole exome sequencing. Variant selection focused on those with allele frequencies in dbSNP150 of less than 0.01. All selected variants were verified by polymerase chain reaction-direct sequencing. Of the 20 cases studied, 13 rare variants were detected in 9 genes: 4 in JAG1 (Alagille syndrome), 2 in MYO5B (progressive familial intrahepatic cholestasis [PFIC] type 6), and one each in ABCC2 (Dubin-Johnson syndrome), ABCB11 (PFIC type 2), UG1A1 (Crigler-Najjar syndrome), MLL2 (Kabuki syndrome), RFX6 (Mitchell-Riley syndrome), ERCC4 (Fanconi anemia), and KCNH1 (Zimmermann-Laband syndrome). Genetic lesions associated with various cholestatic syndromes detected in cases diagnosed with BA raised the hypothesis that severe inflammatory cholangiopathy in BA may not be a distinct disease entity, but a shared pathology among several infantile cholestatic syndromes.

Progressive familial intrahepatic cholestasis type 2 (PFIC 2) results from mutations in ABCB11 gene coding bile salt export pump (BSEP). Medical treatment is usually unsuccessful and surgery intervention is necessary. Partial external biliary diversion (PEBD) is regarded as the first choice of surgical treatment. Ileal exclusion (IE) is an alternative operation if external stoma is not tolerated; however, a favorable outcome is uncertain. In chronic liver diseases pregnancy brings additional risk of deterioration of liver function and generally is not recommended. We present the first case report of successful pregnancy in a genetically confirmed PFIC 2 patient after surgical conversion from PEBD to IE.

Drug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80μM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20μM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP.

OBJECTIVE

To assess whether prolonged neonatal cholestasis, described in congenital hypopituitarism and septo-optic dysplasia (SOD), is associated with altered expression of selected canalicular ectoenzymes and canalicular transport proteins.

METHODS

Children with congenital hypopituitarism (n = 21), SOD (n = 18), and cholestasis seen in our center over 26 years were reviewed. Histopathologic findings in archival liver biopsy specimens were assessed (n = 10) and in those with low/normal levels of serum γ-glutamyltransferase (GGT) activity despite conjugated hyperbilirubinemia, expression of canalicular ectoenzymes and canalicular transport proteins was evaluated immunohistochemically.

RESULTS

Patients presented at a median age of 8 weeks (range 3-20 weeks) with median total bilirubin 116 µmol/L (45-287 µmol/L), GGT 95 IU/L (25-707 UI/L), and serum cortisol 51 nmol/L (17-240 nmol/L). All but 3 had low free thyroxin (median 9.6 pmol/L [6.8-26.9]) with increased thyroid-stimulating hormone levels (median 5.95 mU/L [<0.1-9.24]). Liver histologic features included moderate-to-severe intralobular cholestasis with nonspecific hepatitis, giant-cell transformation of hepatocytes, and fibrosis. In all, immunohistochemical staining for canalicular ectoenzymes and canalicular transport proteins revealed a degree of reduced expression, associated with normal serum GGT values in 6 of the 10 patients, and another 6 nonbiopsied infants with cholestasis also had low/normal serum GGT activity. Sequencing of ABCB11 and ATP8B1 performed in 6 of the biopsied patients did not identify pathogenic mutations. Following replacement therapy, biochemical evidence of hepatobiliary injury resolved in all children within a median period of 6 months.

CONCLUSIONS

Hepatobiliary involvement in congenital hypopituitarism associated with SOD has a good prognosis, but its etiology remains uncertain. Immunohistochemical expression of canalicular transport proteins was reduced in available liver samples.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

Bile secretion by hepatocytes is an osmotic process. The output of bile salts and other organic anions (e.g. glutathione), through the bile salt transporter BSEP/ABCB11 and the organic anion transporter MRP2/ABCC2, respectively, are considered to be the major osmotic driving forces for water secretion into bile canaliculi mainly via aquaporin-8 (AQP8) channels. The down-regulated canalicular expression of these key solute transporters and AQP8 would be a primary event in the establishment of hepatocellular cholestasis. Recent studies in animal models of hepatocellular cholestasis show that the hepatic delivery of AdhAQP1, an adenovector encoding for the archetypical water channel human aquaporin-1 (hAQP1), improves bile secretion and restores to normal the elevated serum bile salt levels. AdhAQP1-transduced hepatocytes show that the canalicularly-expressed hAQP1 not only enhances osmotic membrane water permeability but also induces the transport activities of BSEP/ABCB11 and MRP2/ABCC2 by redistribution in canalicular cholesterol-rich microdomains likely through interactions with the cholesterol-binding protein caveolin-1. Thus, the hepatic gene transfer of hAQP1 improves the bile secretory failure in hepatocellular cholestasis by increasing both biliary output and choleretic efficiency of key osmotic solutes, such as, bile salts and glutathione. The study of hepatocyte aquaporins has provided new insights into the mechanisms of bile formation and cholestasis, and may lead to innovative treatments for cholestatic liver diseases.

Keywords: Adenoviral gene transfer; Aquaporin-1; Bile salts; Biliary glutathione excretion; Cholestasis; Liver.

Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B1*1B and*5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A5*3. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response.

Keywords: Pharmacogenetics; familial hypercholesterolemia (FH); myalgia; precision medicine; statins.

Dyslipidemia enhances progression of atherosclerosis. Coagonist of GLP-1 and glucagon are under clinical investigation for the treatment of obesity and diabetes. Earlier, we have observed that coagonist reduced circulating and hepatic lipids, independent of its anorexic effects. Here, we investigated the role of coagonist of GLP-1 and glucagon receptors in complications of diet-induced dyslipidemia in hamsters and humanized double transgenic mice. Hamsters fed on high fat high cholesterol diet were treated for 8 weeks with coagonist of GLP-1 and glucagon receptors (75 and 150 μg/kg). Pair-fed control was maintained. Cholesterol fed transgenic mice overexpressing hApoB100 and hCETP with coagonist (300 μg/kg) for 4 weeks. After the completion of treatment, biochemical estimations were done. Coagonist treatment reduced triglycerides in plasma, liver and aorta, plasma cholesterol and hepatic triglyceride secretion rate. Expressions of HMG-CoA reductase and SBREBP-1C were reduced and expressions of LDLR, CYP7A1, ABCA1 and ABCB11 were increased in liver, due to coagonist treatment. Coagonist treatment increased bile flow rate and biliary cholesterol excretion. IL-6 and TNF-α were reduced in plasma and expression of TNF-α, MCP-1, MMP-9 and TIMP-1 decreased in liver. Treatment with coagonist reduced oxidative stress in liver and aorta. Energy expenditure was increased and respiratory quotient was reduced by coagonist treatment. These changes were correlated with reduced hepatic inflammation and lipids in liver and aorta in coagonist treated hamsters. Coagonist treatment also reduced lipids in cholesterol-fed transgenic mice. These changes were independent of glycaemia and anorexia observed after coagonist treatment. Long term treatment with coagonist of GLP-1 and glucagon receptor ameliorated diet-induced dyslipidemia and atherosclerosis by regulating bile homeostasis, liver inflammation and energy expenditure.

<AbstractText>This study aimed to investigate the effect of single nucleotide polymorphisms (SNPs) of genes involved in ribavirin (RBV) transport (SLC28A2 gene, ABCB1 gene and <em>ABCB11</em> gene) on the clinical outcome and pharmacokinetics of ribavirin in HCV- 4 Egyptian patients.</AbstractText><AbstractText>100 patients treated with sofosbuvir/daclatasvir and ribavirin for 12 weeks. The SNP genotyping was performed by real-time PCR using high resolution melting analysis. Ribavirin plasma trough concentrations were determined at week 4 of therapy using a liquid chromatography/tandem mass spectrometry (LC-MS/MS). For clinical outcomes, sustained virological response (SVR), liver function tests (ALT and AST), total bilirubin, albumin, serum creatinine, hemoglobin, leukocyte count, and platelet count were measured.</AbstractText><p><div><b>RESULTS</b></div>Concerning RBV pharmacokinetics, ABCB1 2677 G > T SNP and <em>ABCB11</em> 1331 T > C SNP were statistically associated with RBV C_trough levels after 4 weeks of therapy. <em>ABCB11</em> 1331 T > C SNP revealed significant association with clinical outcomes (SVR). SLC28A2-146 A > T SNP has not showed any statistically significant association with RBV plasma levels or response.</p><AbstractText>SNP genotyping for ABCB1 and <em>ABCB11</em> genes can help in better personalized medicine for maximizing response for ribavirin as explored by the significant association between polymorphism in ABCB1 and <em>ABCB11</em> genes and ribavirin pharmacokinetics and the significant association of <em>ABCB11</em> 1331 T > C SNP with clinical response.</AbstractText>

Objective: To evaluate the clinical utility of panel-based NGS in the diagnostic approach of monogenic cholestatic liver diseases. Study design: Patients with diagnosis of chronic cholestatic liver disease of an unknown etiology underwent NGS of targeted genes panel. Group 1 included five patients (prospectively recruited) hospitalized from January to December 2017 while group 2 included seventeen patients (retrospectively recruited) hospitalized from 2010 to 2017 presenting with low-GGT PFIC phenotype (group 2a, 11 patients) or indeterminant cholestatic liver cirrhosis (group 2b, 6 patients). Results: Among 22 patients enrolled into the study, 21 various pathogenic variants (including 11 novel) in 5 different genes (including ABCB11, ABCB4, TJP2, DGUOK, CYP27A1) were identified. The molecular confirmation was obtained in 15 out of 22 patients (68%). In group 1, two out of five patients presented with low-GGT cholestasis, and were diagnosed with BSEP deficiency. Out of three patients presenting with high-GGT cholestasis, one patient was diagnosed with PFIC-3, and the remaining two were not molecularly diagnosed. In group 2a, seven out of eleven patients, were diagnosed with BSEP deficiency and two with TJP-2 deficiency. In group 2b, three out of six patients were molecularly diagnosed; one with PFIC-3, one with CYP27A1 deficiency, and one with DGUOK deficiency. Conclusions: Panel-based NGS appears to be a very useful tool in diagnosis of monogenic cholestatic liver disorders in cases when extrahepatic causes have been primarily excluded. NGS presented the highest diagnosis rate to identify the molecular background of cholestatic liver diseases presenting with a low-GGT PFIC phenotype.

Keywords: children; cholestasis; liver transplantation; next-generation sequencing; progressive familial intrahepatic cholestasis.

Background: Many studies indicate that gallstone formation has genetic components. The abnormal expression of lipid-related genes could be the basis for particular forms of cholesterol gallstone disease. The aim of this study was to obtain insight into lipid metabolism disorder during cholesterol gallstone formation and to evaluate the effect of ursodeoxycholic acid (UDCA) on the improvement of bile lithogenicity and its potential influence on the transcription of lipid-related genes.

Methods: Gallstone-susceptible mouse models were induced by feeding with a lithogenic diet (LD) for 8 weeks. Bile and liver tissues were obtained from these mouse models after 0, 4 and 8 weeks. Bile lipids were measured enzymatically, and the cholesterol saturation index (CSI) was calculated to evaluate the bile lithogenicity by using Carey's critical tables. Real-time polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of farnesoid X receptor (FXR), liver X receptor (LXR), adenosine triphosphate-binding cassette subfamily G member 5/8 (ABCG5/8), cholesterol 7-α hydroxylase (CYP7A1), oxysterol 7-α hydroxylase (CYP7B1), sterol 27-α hydroxylase (CYP27A1), peroxisome proliferator-activated receptor alpha (PPAR-α) and adenosine triphosphate-binding cassette subfamily B member 11 (ABCB11).

Results: The rate of gallstone formation was 100% in the 4-week group but only 30% in the UDCA-treated group. The UDCA-treated group had a significantly lower CSI compared with other groups. Of special note, the data on the effects of UDCA showed higher expression levels of ABCG8, ABCB11 and CYP27A1, as well as lower expression levels of LXR and PPAR-α, compared to the model control group.

Conclusions: UDCA exhibits tremendously potent activity in restraining lipid accumulation, thus reversing the lithogenic effect and protecting hepatocytes from serious pathological damage. The abnormal expression of ABCG8, CYP7A1, CYP27A1, LXR and PPAR-α might lead to high lithogenicity of bile. These results are helpful in exploring new lipid metabolism pathways and potential targets for the treatment of cholesterol stones and for providing some basis for the study of the pathogenesis and genetic characteristics of cholelithiasis. Research on the mechanism of UDCA in improving lipid metabolism and bile lithogenicity may be helpful for clinical treatment and for reducing the incidence of gallstones.

Keywords: ABCB11; ABCG8; CYP27A1; CYP7A1; Cholesterol gallstone; LXR; Lipid metabolism; PPAR-α; Ursodeoxycholic acid.

Cholestasis is a clinical condition resulting from the imapairment of bile flow. This condition could be caused by defects of the hepatocytes, which are responsible for the complex process of bile formation and secretion, and/or caused by defects in the secretory machinery of cholangiocytes. Several mutations and pathways that lead to cholestasis have been described. Progressive familial intrahepatic cholestasis (PFIC) is a group of rare diseases caused by autosomal recessive mutations in the genes that encode proteins expressed mainly in the apical membrane of the hepatocytes. PFIC 1, also known as Byler's disease, is caused by mutations of the ATP8B1 gene, which encodes the familial intrahepatic cholestasis 1 protein. PFIC 2 is characterized by the downregulation or absence of functional bile salt export pump (BSEP) expression via variations in the ABCB11 gene. Mutations of the ABCB4 gene result in lower expression of the multidrug resistance class 3 glycoprotein, leading to the third type of PFIC. Newer variations of this disease have been described. Loss of function of the tight junction protein 2 protein results in PFIC 4, while mutations of the NR1H4 gene, which encodes farnesoid X receptor, an important transcription factor for bile formation, cause PFIC 5. A recently described type of PFIC is associated with a mutation in the MYO5B gene, important for the trafficking of BSEP and hepatocyte membrane polarization. In this review, we provide a brief overview of the molecular mechanisms and clinical features associated with each type of PFIC based on peer reviewed journals published between 1993 and 2020.

Keywords: ABCB11/bile salt export pump; ABCB4/multidrug resistance class 3; ATP8B1/familial intrahepatic cholestasis 1; Bile; Intrahepatic cholestasis; Progressive familial intrahepatic cholestasis.

Benign recurrent intrahepatic cholestasis represents a rare class of autosomal recessive chronic cholestasis disorders, usually presenting with recurrent episodes of intense pruritus and jaundice. We report a 27-year-old woman presenting with benign recurrent intrahepatic cholestasis type 2 due to heterozygosity in ABCB11. Interestingly, she was also found to be heterozygous in cystic fibrosis transmembrane conductance regulator, NPHP4, and A1ATD (SERPINA1), which may explain the severe nature of her disease expression because heterozygosity in each of these genes has been associated with cholestasis. Finally, she exhibited a response to steroids that may have implications for future treatment of bile salt export pump-related diseases.

Benign recurrent intrahepatic cholestasis (BRIC) is characterised by recurrent episodes of jaundice, severe pruritus and low or normal serum γ-glutamyltransferase activity lasting from several weeks to months. BRIC is an autosomal recessive disorder caused by the mutation in either of the two hepatic transporter genes-ATP8B1 or ABCB11 gene. The disease is very well known for episodic flare of jaundice with cholestatic symptoms that are spontaneous or perpetuated by acute insults, followed by self-recovery. There is no proven medical therapy and rarely does it progress to progressive familial intrahepatic cholestasis (PFIC) or biliary cirrhosis. BRIC may be associated with nephrolithiasis, diabetes or pancreatitis. Here, we report a case of BRIC with spontaneous flare and further complicated by drug-induced liver injury with disabling cholestastic symptoms, who underwent endoscopic nasobiliary drainage and was completely relieved of the distressing symptoms.

Benign recurrent intrahepatic cholestasis is a genetic disorder with recurrent cholestatic jaundice due to ATP8B1 and ABCB11 gene mutations encoding for hepato-canalicular transporters. Herein, we firstly provide the evidence that a nonsense variant of ATP8B1 gene (c.1558A>T) in heterozygous form is involved in BRIC pathogenesis.

A 29-year-old male showed severe jaundice and laboratory tests consistent with intrahepatic cholestasis despite normal gamma-glutamyltranspeptidase. Acute and chronic liver diseases with viral, metabolic and autoimmune etiology were excluded. Normal intra/extra-hepatic bile ducts were demonstrated by magnetic resonance. Liver biopsy showed: Cholestasis in the centrilobular and intermediate zones with bile plugs and intra-hepatocyte pigment, Kupffer's cell activation/hyperplasia and preserved biliary ducts. Being satisfied benign recurrent intrahepatic cholestasis diagnostic criteria, ATP8B1 and ABCB11 gene analysis was performed. Surprisingly, we found a novel nonsense variant of ATP8B1 gene (c.1558A>T) in heterozygosis. The variant was confirmed by Sanger sequencing following a standard protocol and tested for familial segregation, showing a maternal inheritance. Immunohistochemistry confirmed a significant reduction of mutated gene related protein (familial intrahepatic cholestasis 1). The patient was treated with ursodeoxycholic acid 15 mg/kg per day and colestyramine 8 g daily with total bilirubin decrease and normalization at the 6^th and 12^th mo.

A genetic abnormality, different from those already known, could be involved in familial intrahepatic cholestatic disorders and/or pro-cholestatic genetic predisposition, thus encouraging further mutation detection in this field.

We measured ceftriaxone pharmacokinetics in patients' plasma and cerebrospinal fluid (CSF) and assessed the influence of biometric, demographic, genetic (ABCB1, ABCC2, ABCB11, ABCG2, and SLCO1A2 polymorphisms) and pathological features. Adult patients with signs and symptoms of central nervous system infections, receiving intravenous ceftriaxone, were enrolled. Ceftriaxone plasma and CSF concentrations were measured by high-precision liquid chromatographic methods; allelic discrimination was performed by real-time polymerase chain reaction. Forty-three patients were included: median ceftriaxone maximal concentration was 15,713 ng/mL in plasma and 3512 ng/mL in CSF with a CSF-to-plasma ratio of 0.3. ABCC2 1249 rs2273697 (P = .027) and ABCG2 1194+928 rs13120400 (P = .015) variants were significantly associated with CSF concentrations and CSF-to-plasma ratios. At linear regression analysis, CSF-to-serum albumin ratio was an independent predictor of ceftriaxone CSF concentrations (P = .001; also in those with intact blood-brain barrier: P = .031) and CSF-to-plasma ratio (P = .001; also in those with blood-brain barrier impairment: P = .040). We here report the role of transporters' genetic variants as well as of blood-brain barrier permeability in predicting ceftriaxone exposure in the central nervous system.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Understanding the mechanisms underlying the metabolically unhealthy normal weight (MUHNW) and metabolically healthy obese (MHO) phenotypes is important for developing strategies to prevent cardiometabolic diseases. Here, we conducted genome-wide association studies (GWASs) to identify the MUHNW and MHO genetic indices. The study dataset comprised genome-wide single-nucleotide polymorphism genotypes and epidemiological data from 49,915 subjects categorised into four phenotypes-metabolically healthy normal weight (MHNW), MUHNW, MHO, and metabolically unhealthy obese (MUHO). We conducted two GWASs using logistic regression analyses and adjustments for confounding variables (model 1: MHNW versus MUHNW and model 2: MHO versus MUHO). GCKR, ABCB11, CDKAL1, LPL, CDKN2B, NT5C2, APOA5, CETP, and APOC1 were associated with metabolically unhealthy phenotypes among normal weight individuals (model 1). LPL, APOA5, and CETP were associated with metabolically unhealthy phenotypes among obese individuals (model 2). The genes common to both models are related to lipid metabolism (LPL, APOA5, and CETP), and those associated with model 1 are related to insulin or glucose metabolism (GCKR, CDKAL1, and CDKN2B). This study reveals the genetic architecture of the MUHNW and MHO phenotypes in a Korean population-based cohort. These findings could help identify individuals at a high metabolic risk in normal weight and obese populations and provide potential novel targets for the management of metabolically unhealthy phenotypes.

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

Keywords: ABCB11; BRIC; BSEP; PFIC2; bile salts; chaperones; intrahepatic cholestasis.

Progressive Familial Intrahepatic Cholestasis (PFIC) are inherited severe liver disorders presenting early in life, with high serum bile salt and bilirubin levels. Six types have been reported, two of these are caused by deficiency of an ABC transporter; ABCB11 (bile salt export pump) in type 2; ABCB4 (phosphatidylcholine floppase) in type 3. In addition, ABCB11 function is affected in 3 other types of PFIC. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejection, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alternative for liver transplantation for all forms of PFIC based on their pathological mechanism. Conclusion: Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality.

Keywords: AAV; PFIC; gene therapy.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.