Expression Of Bile Transporters Research Articles

Tectorigenin alleviates intrahepatic cholestasis by inhibiting hepatic inflammation and bile accumulation via activation of PPARγ.

Increasing evidence suggests that human cholestasis is closely associated with the accumulation and activation of hepatic macrophages. Research indicates that activation of PPARγ exerts liver protective effects in cholestatic liver disease (CLD), particularly by ameliorating inflammation and fibrosis, thus limiting disease progression. However, existing PPARγ agonists, such as troglitazone and rosiglitazone, have significant side effects that prevent their clinical application in the treatment of CLD. In this study, we found that tectorigenin alleviates intrahepatic cholestasis in mice by activating PPARγ. Wild-type mice were intragastrically administered α-naphthylisothiocyanate (ANIT) or fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to simultaneously establish an experimental model of intrahepatic cholestasis and tectorigenin intervention, followed by determination of intrahepatic cholestasis and the mechanisms involved. In addition, PPARγ-deficient mice were administered ANIT and/or tectorigenin to determine whether tectorigenin exerts its liver protective effect by activating PPARγ. Treatment with tectorigenin alleviated intrahepatic cholestasis by inhibiting the recruitment and activation of hepatic macrophages and by promoting the expression of bile transporters via activation of PPARγ. Furthermore, tectorigenin increased expression of the bile salt export pump (BSEP) through enhanced PPARγ binding to the BSEP promoter. In PPARγ-deficient mice, the hepatoprotective effect of tectorigenin during cholestasis was blocked. In conclusion, tectorigenin reduced the recruitment and activation of hepatic macrophages and enhanced the export of bile acids by activating PPARγ. Taken together, our results suggest that tectorigenin is a candidate compound for cholestasis treatment.

Altered Bile Transporter Expression and Cholesterol Metabolism in Children With Cholesterol and Pigment Gallstones.

We elucidated pathophysiology of pediatric gallstone disease by assessing liver expression of bile transporters in relation to bile acids and surrogates of cholesterol absorption and synthesis in serum and gallstones. RNA expression of canalicular bile transporters in liver biopsies from 32 pediatric gallstone patients and from 6 liver donors (controls) was measured by qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction). Concentrations of cholesterol and precursors, plant sterols and bile acids in gallstones, and in serum of the patients and 82 healthy children were measured. Primary outcomes were the difference in RNA expressions and serum sterol profiles between patients and controls. Cholesterol stones (CS; n = 15) contained cholesterol >42% and pigment stones (PS; n = 17) <9% of weight. CS patients had markedly lower serum plant sterols (absorption) and higher cholesterol precursors (synthesis) than PS patients or healthy controls. CS contained several times more cholesterol precursors and less plant sterols relative to cholesterol than PS, which were enriched by primary bile acids (12-5.2-fold, P < 0.001). Liver RNA expression of ABCG5/G8 was similarly increased 2.5- to 1.8-fold (P < 0.002) in CS and PS patients, whereas PS patients had higher ABCB11 expression (P < 0.05). In PS bile acid concentration correlated with gallstone plant sterols (R = 0.83, P < 0.0001), and ABCG5 expression with ABCB11 expression (R = 0.27, P = 0.03). In CS, upregulation of ABCG5/G8 expression associates with low absorption and high gallstone content of cholesterol. In PS, activation of bile acid transport by ACBC11 interconnects with hepatic upregulation of ABCG5/G8 enriching PS with bile acids and plant sterols.

Effects of Hepatic Ischemia–Reperfusion Injuries and NRF2 on Transcriptional Activities of Bile Transporters in Rats

BackgroundThe effect of hepatic ischemia–reperfusion injury (IRI) on bile transporter (BT) gene expression is unknown. We hypothesized that abnormal expression of BTs during hepatic IRI is dependent on nuclear factor erythroid 2–related factor 2 (NRF2), which contributes to the cholestasis after reperfusion. MethodsSham surgery and short (60 min) or long (90 min) periods of warm ischemia time (WIT) with or without reperfusion for 24 h were applied to wild-type Sprague–Dawley rats and Nrf2 knockout rats (n = 5 per group). At each stage of IRI, the serum levels of aminotransferase, total bilirubin, and bile acids were measured. In addition, hepatic tissue was sampled to determine the histologic score of IRI (Suzuki score), measure adenosine triphosphate (ATP), and identify the quantitative real-time polymerase chain reactions of BTs (Oatp1, Ntcp, Mrp2, Bsep, and Mrp3). ResultsIn short periods of WIT, BT expression increased during the ischemia stage and returned to the baseline after reperfusion. However, in long periods of WIT, BT expression did not increase after ischemia and decreased further after reperfusion. Short WIT did not increase BT expression in Nrf2 knockout animals. The level of BT expression was correlated with the Suzuki score, the serum levels of aminotransferase, bilirubin, and bile acids, and tissue ATP level. Stepwise multiple regression analysis derived equations to predict the Suzuki score (R2 = 76.8, P < 0.001), serum total bilirubin (R2 = 61.2, P < 0.001), and tissue ATP (R2 = 61.1, P < 0.001). ConclusionsShort WIT induces the transcriptional activities of BT, whereas long WIT depresses them, and the effect was blunted by Nrf2 knockout status. BT expression can be considered a surrogate marker for hepatic IRI.

Liver Inflammation Relates to Decreased Canalicular Bile Transporter Expression in Pediatric Onset Intestinal Failure

Although liver disease is a major complication of parenteral nutrition (PN) for intestinal failure (IF), its pathogenesis remains unclear. We investigated potential molecular mechanisms of liver injury in pediatric onset IF. Liver expression of canalicular phospholipid (ABCB4), bile acid (ABCB11), and sterol (ABCG5/8) transporters, their upstream regulators LXR and FXR as well as pro-inflammatory cytokines interleukin-6 (IL6) and tumor necrosis factor (TNF) were investigated among patients with IF [age median 3.8 (IQR 1.2 to 11)] in relation to biochemical and histologic liver injury, PN, serum plant sterols, fibroblast growth factor 19, and α-tocopherol. Patients receiving PN currently (n = 18) showed more advanced liver injury than patients after weaning off PN (n = 30). Histologic portal inflammation strongly segregated PN-dependent (44%) from weaned off patients (3%, P = 0.001) and coupled with progression of cholestasis and liver fibrosis. Patients with portal inflammation demonstrated markedly induced liver RNA expression of IL6 and TNF, repression of FXR and its canalicular bile transporter target gene RNA expression, including ABCB4 and ABCB11 as well as decreased protein expression of ABCB11 and ABCB4. Furthermore, upregulation of LXR and ABCG5/8 RNA expression was suppressed in patients with portal inflammation. Current PN, increased serum levels of plant sterols stigmasterol, avenasterol, and sitosterol along with serum citrulline, a marker of enterocyte mass, predicted portal inflammation. In pediatric onset IF, current PN delivery synergistically with intestinal compromise promote liver inflammation, which associates with progression of biochemical and histologic liver injury, while reducing expression of canalicular bile transporters.

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

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

Antibody-Mediated Inhibition of Fibroblast Growth Factor 19 Results in Increased Bile Acids Synthesis and Ileal Malabsorption of Bile Acids in Cynomolgus Monkeys

Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-β) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.

Endogenous Bile Salts Are Associated With Bile Duct Injury in the Rat Liver Transplantation Model

Nonanastomotic biliary strictures are a serious complication after orthotopic liver transplantation (OLT) and are difficult to cure. We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. A total of 90 Sprague-Dawley rats were divided into three groups: Sham-operated group (Sham, n=30), OLT group with 1 hr donor liver preservation (OLT-1h, n=30), and OLT group with 12 hr donor liver preservation (OLT-12h, n=30). Bile was collected and analyzed biochemically. The histopathologic study was performed to determine the intrahepatic bile duct morphologic changes. Hepatic expressions of bile transporters Ntcp, Bsep, Mdr2, and Oatps were detected. During the first 2 weeks after transplantation, bile salt secretion was not parallel with phospholipid secretion, resulting in high biliary bile salt-to-phospholipid (BS:PL) ratio. The expression of bile transporters was consistent with the change of bile composition. Bile duct injury correlated significantly with bile salt secretion and BS:PL ratio. Moreover, OLT group with longer donor liver preservation time (OLT-12h) had significantly lower Mdr2 messenger RNA/protein level, higher BS:PL ratio, and better correlation between BS:PL ratio and bile duct injury compared with those of OLT-1h. The unparallel secretion of bile salts and phospholipids results in cytotoxic bile formation with high BS:PL ratio after liver transplantation. Longer donor liver preservation time will increase graft bile cytotoxicity. The results of this study suggest that endogenous bile salts play a role in the pathogenesis of bile duct injury after OLT.

Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures

Nonanastomotic biliary strictures are troublesome complications after liver transplantation. The pathogenesis of NAS is not completely clear, but experimental studies suggest that bile salt toxicity is involved. In one hundred and eleven adult liver transplants, bile samples were collected daily posttransplantation for determination of bile composition. Expression of bile transporters was studied perioperatively. Nonanastomotic biliary strictures were detected in 14 patients (13%) within one year after transplantation. Patient and donor characteristics and postoperative serum liver enzymes were similar between patients who developed nonanastomotic biliary strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter expression. Although patients who develop nonanastomotic biliary strictures are initially clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary strictures.

Dearterialization of the Liver Causes Intrahepatic Cholestasis due to Reduced Bile Transporter Expression

Bile duct injury after hepatic artery thrombosis (HAT) in liver transplantation is believed to be caused by ischemia predominantly. We aimed to define the involvement of bile secretory dysfunction in the pathogenesis of liver injury after HAT. In a murine model, the main hepatic artery, the extrahepatic peribiliary plexus, or both arterial connections to the liver were interrupted (n=5 for each group). After 1, 14, or 28 days, hepatobiliary function was assessed by analysis of bile transporter expression, serum bile acids and bilirubin, and hepatic ATP content. In addition, cellular injury was assessed by light microscopy and biochemical markers. There were no signs of hepatobiliary dysfunction or injury in sham-operated animals or in mice with interruption of the hepatic artery or the extrahepatic peribiliary plexus alone. However, as early as 24 hr after complete dearterialization, bile transporter expression was significantly reduced and intrahepatic cholestasis started to progress the following weeks. Histologic studies at 28 days after complete dearterialization showed severe hepatobiliary injury. This study indicates that arterial blood supply is critical for normal bile secretion. Bile duct injury after complete arterial deprivation is preceded by a loss of bile secretory function and subsequent intrahepatic cholestasis.

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation inMdr2+/− mice

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild-type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/-) were transplanted into wild-type recipient mice. Mdr2+/- mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2-/- mice, the Mdr2+/- mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild-type donors. Liver grafts from Mdr2+/- donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/- livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild-type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/- grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation.

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. In 28 liver transplant recipients, bile samples were collected daily posttransplantation for determination of bile composition. Hepatic expression of bile transporters was studied before and after transplantation. Histopathological criteria as well as biliary concentrations of alkaline phosphatase (ALP) and gamma-glutamyltransferase (gamma-GT) were used to quantify bile duct injury. Early after transplantation, bile salt secretion increased more rapidly than phospholipid secretion, resulting in high biliary bile salt/phospholipid ratio (BA/PL). In parallel with this, mRNA levels of the bile salt transporters NTCP and BSEP increased significantly after transplantation, whereas phospholipid translocator MDR3 mRNA levels remained unchanged. Bile duct injury correlated significantly with bile salt secretion and was associated with a high biliary BA/PL ratio. Bile salt secretion after human liver transplantation recovers more rapidly than phospholipid secretion. This results in cytotoxic bile formation and correlates with bile duct injury. These findings suggest that endogenous bile salts have a role in the pathogenesis of bile duct injury after liver transplantation.

Management of the biliary complications associated with liver transplantation: 13 years of experience

Nonanastomotic biliary strictures are troublesome complications after liver transplantation. The pathogenesis of NAS is not completely clear, but experimental studies suggest that bile salt toxicity is involved.In one hundred and eleven adult liver transplants, bile samples were collected daily posttransplantation for determination of bile composition. Expression of bile transporters was studied perioperatively.Nonanastomotic biliary strictures were detected in 14 patients (13%) within one year after transplantation. Patient and donor characteristics and postoperative serum liver enzymes were similar between patients who developed nonanastomotic biliary strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter expression.Although patients who develop nonanastomotic biliary strictures are initially clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary strictures.