ZFYVE19 deficiency: a ciliopathy involving failure of cell division, with cell death.

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

Hydrophilic bile acids prevent liver damage caused by lack of biliary phospholipid in Mdr2-/- mice.

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3), severe liver diseases associated with genetic defects in the biliary bile acid transporter bile salt export pump (BSEP; ABCB11) or phosphatidylcholine transporter multidrug resistance protein 3 (MDR3; ABCB4), respectively. Mdr2-/- mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the nonmicelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep-/- mice could protect Mdr2-/- mice from progressive liver damage. We generated double-KO (DKO: Bsep-/- and Mdr2-/- ) mice. Their bile acid composition resembles that of Bsep-/- mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBAs), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2-/- littermates. The livers of DKO mice have gene expression profiles very similar to Bsep-/- mice, with 4,410 of 6,134 gene expression changes associated with the Mdr2-/- mutation being suppressed. Feeding with THBAs partially alleviates liver damage in the Mdr2-/- mice. Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2-/- (PFIC3) mutation.

Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis.

Hereditary cholestasis in childhood and infancy with normal serum gamma-glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low-GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow-up. Liver biopsy specimens revealed giant-cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP-deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated-cholestasis patients (11 of 38 vs. 0 of 13). CONCLUSION: MYO5B deficiency may underlie 20% of previously undiagnosed low-GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655-1669).

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

BACKGROUND & AIMS: Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. METHODS: We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. RESULTS: The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p < 0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p < 0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p < 0.0000001). CONCLUSIONS: Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.

Metabolic profiling of bile acids in human and mouse blood by LC-MS/MS in combination with phospholipid-depletion solid-phase extraction.

To obtain a more comprehensive profile of bile acids (BAs) in blood, we developed an ultrahigh performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC-MRM-MS) method for the separation and detection of 50 known BAs. This method utilizes phospholipid-depletion solid-phase extraction as a new high-efficiency sample preparation procedure for BA assay. UPLC/scheduled MRM-MS with negative ion electrospray ionization enabled targeted quantitation of 43 and 44 BAs, respectively, in serum samples from seven individuals with and without fasting, as well as in plasma samples from six cholestatic gene knockout mice and six age- and gender-matched wild-type (FVB/NJ) animals. Many minor BAs were identified and quantitated in the blood for the first time. Method validation indicated good quantitation precision with intraday and interday relative standard deviations of ≤9.3% and ≤10.8%, respectively. Using a pooled human serum sample and a pooled mouse plasma sample as the two representative test samples, the quantitation accuracy was measured to be 80% to 120% for most of the BAs, using two standard-substance spiking approaches. To profile other potential BAs not included in the 50 known targets from the knockout versus wild-type mouse plasma, class-specific precursor/fragment ion transitions were used to perform UPLC-MRM-MS for untargeted detection of the structural isomers of glycine- and taurine-conjugated BAs and unconjugated tetra-hydroxy BAs. As a result, as many as 36 such compounds were detected. In summary, this UPLC-MRM-MS method has enabled the quantitation of the largest number of BAs in the blood thus far, and the results presented have revealed an unexpectedly complex BA profile in mouse plasma.

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep (-/-) mice.

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2ß-, 6ß-, and 15ß-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Comprehensive Bile Acid Profiling of ABCB4-mutated Patients and the Prognostic Role of Taurine-conjugated 3α,6α,7α,12α-Tetrahydroxylated Bile Acid in Cholestasis.

Background and Aims: We asked if comprehensive bile acid profiling could provide insights into the physiopathology of ABCB4-mutated patients and evaluated the prognostic value of taurine-conjugated tetrahydroxylated bile acid (tauro-THBA) in cholestasis. Methods: Serum bile acid profiles were evaluated in 13 ABCB4-mutated patients with 65 healthy controls by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). The concentration of tauro-THBA was compared between ABCB4-mutated patients with different prognoses. The areas under the curve (AUCs) of tauro-THBA were compared between ABCB11-mutated patients with native liver survival and those who died or underwent liver transplantation before 3 years of age by receiver operating characteristic curve (ROC), with another patient cohort for further verification. Results: The overall hydrophobicity indices of bile acids in ABCB4-mutated patients (12.99±3.25 m) were significantly lower than those of healthy controls (14.02±1.74 m, p<0.000). That was due to markedly increased bile acid modifications including conjugation, sulfation, and ketonization. Differences in the tauro-THBA concentration in ABCB4-mutated patients with different prognoses were not significant. ROC analysis indicated that levels of tauro-THBA of <60 nM yielded an AUC of 0.900 with a sensitivity of 80% and specificity of 87.5% for ABCB11-mutated patients with different prognoses (p=0.0192). Of the 15 patients with good prognosis, 14 were classified correctly and four of the five patients with a poor prognosis were classified correctly (14:15 vs. 1:5, p=0.005) with tauro-THBA as a classifier. Conclusions: Tauro-THBA concentration may be a biomarker for predicting the clinical outcome in low gamma-glutamyl transferase intrahepatic cholestasis patients.

CWF19L2 is Essential for Male Fertility and Spermatogenesis by Regulating Alternative Splicing.

The progression of spermatogenesis along specific developmental trajectories depends on the coordinated regulation of pre-mRNA alternative splicing (AS) at the post-transcriptional level. However, the fundamental mechanism of AS in spermatogenesis remains to be investigated. Here, it is demonstrated that CWF19L2 plays a pivotal role in spermatogenesis and male fertility. In germline conditional Cwf19l2 knockout mice exhibiting male sterility, impaired spermatogenesis characterized by increased apoptosis and decreased differentiated spermatogonia and spermatocytes is observed. That CWF19L2 interacted with several spliceosome proteins to participate in the proper assembly and stability of the spliceosome is discovered. By integrating RNA-seq and LACE-seq data, it is further confirmed CWF19L2 directly bound and regulated the splicing of genes related to spermatogenesis (Znhit1, Btrc, and Fbxw7) and RNA splicing (Rbfox1, Celf1, and Rbm10). Additionally, CWF19L2 can indirectly amplify its effect on splicing regulation through modulating RBFOX1. Collectively, this research establishes that CWF19L2 orchestrates a splicing factor network to ensure accurate pre-mRNA splicing during the early steps of spermatogenesis.

Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

Neonatal Dubin-Johnson Syndrome and its Differentiation from Biliary Atresia.

Background and Aims: The aim was to determine if liver biochemistry indices can be used as biomarkers to help differentiate patients with neonatal Dubin-Johnson syndrome (nDJS) from those with biliary atresia (BA). Methods: Patients with genetically-confirmed nDJS or cholangiographically confirmed BA were retrospectively enrolled and randomly assigned to discovery or verification cohorts. Their liver chemistries, measured during the neonatal period, were compared. Predictive values were calculated by receiver operating characteristic curve analysis. Results: A cohort of 53 nDJS patients was recruited, of whom 13 presented with acholic stools, and 14 underwent diagnostic cholangiography or needle liver biopsy to differentiate from BA. Thirty-five patients in the cohort, with complete biochemical information measured during the neonatal period, were compared with 133 infants with cholangiographically confirmed BA. Total and direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acids, alkaline phosphatase, and gamma-glutamyl transferase were significantly lower in nDJS than in BA. In the discovery cohort, the areas under the curve for ALT and AST were 0.908 and 0.943, respectively. In the validation cohort, 13/15 patients in the nDJS group were classified as nDJS, and 10/53 in the BA control group were positive (p<0.00001) with an ALT biomarker cutoff value of 75 IU/L. Thirteen of 15 patients were classified as nDJS and none were classified positive in the BA group (13/15 vs. 0/53, p<0.00001) with an AST cutoff of 87 IU/L. Conclusions: Having assembled and investigated the largest cohort of nDJS patients reported to date, we found that nDJS patients could be distinguished from BA patients using the serum AST level as a biomarker. The finding may be clinically useful to spare cholestatic nDJS patients unnecessary invasive procedures.

Poly-hydroxylated bile acids and their prognostic roles in Alagille syndrome.

BACKGROUND: The liver manifestations of Alagille syndrome (ALGS) are highly variable, and factors affecting its prognosis are poorly understood. We asked whether the composition of bile acids in ALGS patients with good clinical outcomes differs from that in patients with poor outcomes and whether bile acids could be used as prognostic biomarkers. METHODS: Blood for bile acid profiling was collected from genetically confirmed JAG1-associated ALGS patients before one year of age. A good prognosis was defined as survival with native liver and total bilirubin (TB) < 85.5 µmol/L, while a poor prognosis was defined as either liver transplantation, death from liver failure, or TB ≥ 85.5 µmol/L at the last follow-up. RESULTS: We found that the concentrations of two poly-hydroxylated bile acids, tauro-2ß,3α,7α,12α-tetrahydroxylated bile acid (THBA) and glyco-hyocholic acid (GHCA), were significantly increased in patients with good prognosis compared to those with poor prognosis [area under curve (AUC) = 0.836 and 0.782, respectively] in the discovery cohort. The same trend was also observed in the molar ratios of GHCA to glyco- chenodeoxycholic acid (GCDCA) and tetrahydroxylated bile acid (THCA) to tauro-chenodeoxycholic acid (TCDCA) (both AUC = 0.836). A validation cohort confirmed these findings. Notably, tauro-2ß,3α,7α,12α-THBA achieved the highest prediction accuracy of 88.00% (92.31% sensitivity and 83.33% specificity); GHCA at > 607.69 nmol/L was associated with native liver survival [hazard ratio: 13.03, 95% confidence interval (CI): (2.662-63.753), P = 0.002]. CONCLUSIONS: We identified two poly-hydroxylated bile acids as liver prognostic biomarkers of ALGS patients. Enhanced hydroxylation of bile acids may result in better clinical outcomes.

Hepatocyte transplantation in bile salt export pump-deficient mice: selective growth advantage of donor hepatocytes under bile acid stress.

The bile salt export pump (Bsep) mediates the hepatic excretion of bile acids, and its deficiency causes progressive familial intrahepatic cholestasis. The current study aimed to induce bile acid stress in Bsep(-/-) mice and to test the efficacy of hepatocyte transplantation in this disease model. We fed Bsep(-/-) and wild-type mice cholic acid (CA) or ursodeoxycholic acid (UDCA). Both CA and UDCA caused cholestasis and apoptosis in the Bsep(-/-) mouse liver. Wild-type mice had minimal liver injury and apoptosis when fed CA or UDCA, yet had increased proliferative activity. On the basis of the differential cytotoxicity of bile acids on the livers of wild-type and Bsep(-/-) mice, we transplanted wild-type hepatocytes into the liver of Bsep(-/-) mice fed CA or CA + UDCA. After 1-6 weeks, the donor cell repopulation and canalicular Bsep distribution were documented. An improved repopulation efficiency in the CA + UDCA-supplemented group was found at 2 weeks (4.76 ± 5.93% vs. 1.32 ± 1.48%, P = 0.0026) and at 4-6 weeks (12.09 ± 14.67% vs. 1.55 ± 1.28%, P < 0.001) compared with the CA-supplemented group. Normal-appearing hepatocytes with prominent nuclear staining for FXR were noted in the repopulated donor nodules. After hepatocyte transplantation, biliary total bile acids increased from 24% to 82% of the wild-type levels, among which trihydroxylated bile acids increased from 41% to 79% in the Bsep(-/-) mice. We conclude that bile acid stress triggers differential injury responses in the Bsep(-/-) and wild-type hepatocytes. This strategy changed the balance of the donor-recipient growth capacities and was critical for successful donor repopulation.

The protective role of hydrophilic tetrahydroxylated bile acids (THBA).

Bile acids are key components of bile required for human health. In humans and mice, conditions of reduced bile flow, cholestasis, induce bile acid detoxification by producing tetrahydroxylated bile acids (THBA), more hydrophilic and less cytotoxic than the usual bile acids, which are typically di- or tri-hydroxylated. Mice deficient in the Bile Salt Export Pump (Bsep, or Abcb11), the primary bile acid transporter in liver cells, produce high levels of THBA, and avoid the severe liver damage typically seen in humans with BSEP deficiencies. THBA can suppress bile acid-induced liver damage in Mdr2-deficient mice, caused by their lack of phospholipids in bile exposing their biliary tracts to unbound bile acids. Here we review THBA-related works in both animals and humans, and discuss their potential relevance and applications as a class of functional bile acids.

Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump.

UNLABELLED: Bile salt export pump (BSEP; ATP-binding cassette, subfamily B, member 11) mutations in humans result in progressive familial intrahepatic cholestasis type 2, a fatal liver disease with greatly reduced bile flow. However in mice, Bsep knockout leads only to mild cholestasis with substantial bile flow and up-regulated P-glycoprotein genes (multidrug resistance protein 1a [Mdr1a] and Mdr1b). To determine whether P-glycoprotein is responsible for the relatively mild phenotype observed in Bsep knockout mice, we have crossed mouse strains knocked out for Bsep and the two P-glycoprotein genes and generated a triple knockout mouse. We found that a knockout of the three genes leads to a significantly more severe phenotype with impaired bile formation, jaundice, flaccid gallbladder, and increased mortality. The triple knockout mouse is the most severe genetic model of intrahepatic cholestasis yet developed. CONCLUSION: P-glycoprotein functions as a critical compensatory mechanism, which reduces the severity of cholestasis in Bsep knockout mice.

Changes in plasma bile acid profiles after partial internal biliary diversion in PFIC2 patients.

BACKGROUND: We ask if plasma bile acid profiles can be used to monitor the effectiveness of partial internal biliary diversion (PIBD) for treating uncontrolled cholestasis in progressive familial intrahepatic cholestasis type 2 (PFIC2) patients. METHODS: Plasma bile acids were profiled in 3 cases of ATP-binding cassette, sub-family B member 11 (ABCB11)-mutated PFIC2 children before and after PIBD compared to healthy controls and 8 PFIC2 patients. The quantitation of bile acids was performed by reversed-phase ultrahigh-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS) with negative ion detection. RESULTS: Before PIBD, all three patients presented with >50-fold higher levels of total plasma bile acids, 2-7 folds higher ratios of taurine: glycine conjugated primary bile acids, and unchanged secondary bile acids levels compared to healthy controls. After PIBD, only one of the three patients (P3) showed relief of cholestasis. The bile acid profiles of the two nonresponding patients showed little change while that of the responding patient showed a 5-fold reduction in total plasma primary bile acids, a reduced taurine: glycine conjugate ratio, and an unexpected 26- and 12-fold increase in secondary bile acids DCA and LCA respectively. One year later, the responder suffered a recurrence of cholestasis, and the bile acid profile shifted back to a more pre-PIBD-like profile. CONCLUSIONS: Plasma bile acid profiles may potentially be useful as sensitive biomarkers for monitoring the clinical course of PIBD patients. Relief of cholestasis after PIBD appears to be associated with significantly increased circulating toxic secondary bile acids and this may limit the utility of PIBD in PFIC2 patients in the long run.

Bone marrow transplantation results in donor-derived hepatocytes in an animal model of inherited cholestatic liver disease.

Cell transplantation is a potential therapy for acquired or inherited liver diseases. Donor-derived hepatocytes (DDH) have been found in humans and mice after bone marrow transplantation (BMT) but with highly variable frequencies in different disease models. To test the effect of liver repopulation after BMT in inherited cholestatic liver diseases, spgp (sister of P-glycoprotein, or bile salt export pump, abcb11) knockout mice, a model for human progressive intrahepatic cholestasis type 2 with defects in excreting bile salts across the hepatocyte canalicular membrane, were transplanted with bone marrow cells from enhanced green fluorescent protein (EGFP) transgenic donor mice after lethal irradiation. One to 6 months later, scattered EGFP-positive DDHs with positive spgp staining were observed in the liver. These hepatocytes had been incorporated into hepatic plates and stained positively with hepatocyte-specific marker albumin. RT-PCR for the spgp gene revealed positive expression in the liver of sgsp knockout mice that had received the transplant. Bile acid analysis of bile samples showed that these mice also had higher levels of total biliary bile acid and taurocholic acid concentration than knockout mice without transplantation, indicating that BMT partially improved biliary bile acid secretion. Our results indicate that bone marrow cells could serve as a potential source for restoration of hepatic functions in chronic metabolic liver disease.

ABC transporters, bile acids, and inflammatory stress in liver cancer.

The biliary secretion of bile acids is critical for multiple liver functions including digesting fatty nutrients and driving bile flow. When this process is impaired, the accumulating bile acids cause inflammatory liver injury. Multiple ABC transporters in the liver are key players to safeguard the hepatocyte and avoid toxicity due to bile acid over-accumulation. BSEP provides for efficient secretion of bile acids across the canalicular membrane against a steep concentration gradient. MDR3/Mdr2 and ABCG5/G8 secrete phosphatidylcholine and cholesterol, respectively, in coordination with BSEP-mediated bile acid secretion to mask the detergent/toxic effects of bile acids in the bile ductular space. Several lines of evidence indicate that when these critical steps are compromised, bile acid toxicity in vivo leads to inflammatory liver injury and liver cancer. In bsep-/- mice, liver cancer is rare. These mice display greatly increased expression of alternative bile acid transporters, such as Mdr1a/1b, Mrp3 and Mrp4. We believe these alternative transport systems provide an additional safeguard to avoid bile acid overload in liver. Such backup systems appear to be under-utilized in humans, as defects in BSEP and MDR3 lead to severe, often fatal childhood diseases. It is possible, therefore, that targeting ABC transporters and modulating the toxicity of the bile acid pool could be vital interventions to alleviate chronic inflammation and reduce the incidence of liver cancer in high-risk populations. The combination of an alternative ABC transporter with a novel substrate may prove an effective chemo-preventive or therapeutic strategy.

Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice.

In vertebrates, bile flow is essential for movement of water and solutes across liver canalicular membranes. In recent years, the molecular motor of canalicular bile acid secretion has been identified as a member of the ATP binding cassette transporter (ABC) superfamily, known as sister of P-glycoprotein (Spgp) or bile salt export pump (Bsep, ABCB11). In humans, mutations in the BSEP gene are associated with a very low level of bile acid secretion and severe cholestasis. However, as reported previously, because the spgp(-)(/)(-) knockout mice do not express severe cholestasis and have substantial bile acid secretion, we investigated the "alternative transport system" that allows these mice to be physiologically relatively normal. We examined the expression levels of several ABC transporters in spgp(-)(/)(-) mice and found that the level of multidrug resistance Mdr1 (P-glycoprotein) was strikingly increased while those of Mdr2, Mrp2, and Mrp3 were increased to only a moderate extent. We hypothesize that an elevated level of Mdr1 in the spgp(-)(/)(-) knockout mice functions as an alternative pathway to transport bile acids and protects hepatocytes from bile acid-induced cholestasis. In support of this hypothesis, we showed that plasma membrane vesicles isolated from a drug resistant cell line expressing high levels of P-glycoprotein were capable of transporting bile acids, albeit with a 5-fold lower affinity compared to Spgp. This finding is the first direct evidence that P-glycoprotein (Mdr1) is capable of transporting bile acids.

Analysis of the in vivo functions of Mrp3.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.