sirolimus and Cholestasis

Biliary atresia (BA) is a cholestatic liver disease in neonates with devastating obstructive intrahepatic and extrahepatic biliary ducts. Owing to the lack of an early diagnostic marker and limited understanding of its pathogenesis, BA often leads to death within 2 years. Therefore, this study aimed to develop early diagnostic methods and investigate the underlying pathogenesis of liver injury in BA using metabolomics. Metabolomics and organoid combined energy metabolism analysis was used to obtain new insights into BA diagnosis and pathobiology using patient samples, mice liver organoids, and a zebrafish model. Metabolomics revealed that D-2-hydroxyglutarate (D-2-HG) levels were significantly elevated in the plasma and liver of patients with BA and closely correlated with liver injuries and impaired liver regeneration. D-2-HG suppressed the growth and expansion of liver organoids derived from the intrahepatic biliary ducts. The energy metabolism analysis demonstrated that D-2-HG inhibited mitochondrial respiration and ATP synthase; however, it increased aerobic glycolysis in organoids. In addition, D-2-HG exposure caused liver degeneration in zebrafish larvae. Mechanistically, D-2-HG inhibited the activation of protein kinase B and the mammalian target of rapamycin signaling. These findings reveal that D-2-HG may represent a novel noninvasive diagnostic biomarker and a potential therapeutic target for infants with BA.

Topics: Animals; Biliary Atresia; Cholestasis; Liver; Mammals; Mice; Sirolimus; TOR Serine-Threonine Kinases; Zebrafish

Rapamycin is employed as an immunosuppressant following organ transplant and, in patients with hepatocellular carcinoma, to inhibit cancer cell regrowth following liver surgery. Preconditioning the liver with rapamycin to induce the expression of antioxidant enzymes is a potential strategy to reduce ischemia reperfusion (IR) injury. However, pre-treatment with rapamycin inhibits bile flow, especially following ischemia. The aim was to investigate the mechanisms involved in this inhibition. In a rat model of segmental hepatic ischemia and reperfusion, acute administration of rapamycin by intravenous injection did not inhibit the basal rate of bile flow. Pre-treatment of rats with rapamycin for 24 h by intraperitoneal injection inhibited the expression of mRNA encoding the sinusoidal influx transporters Ntcp, Oatp1 and 2 and the canalicular efflux transporter Bsep, and increased expression of canalicular Mrp2. Dose-response curves for the actions of rapamycin on the expression of Bsep and Ntcp in cultured rat hepatocytes were biphasic, and monophasic for effects on Oatp1. In cultured tumorigenic H4IIE liver cells, several bile acid transporters were not expressed, or were expressed at very low levels compared to hepatocytes. In H4IIE cells, rapamycin increased expression of Ntcp, Oatp1 and Mrp2, but decreased expression of Oatp2. It is concluded that the inhibition of bile flow recovery following ischemia observed in rapamycin-treated livers is principally due to inhibition of the expression of sinusoidal bile acid transporters. Moreover, in tumorigenic liver tissue the contribution of tumorigenic hepatocytes to total liver bile flow is likely to be small and is unlikely to be greatly affected by rapamycin.

Topics: Animals; Bile; Bile Ducts; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cholestasis; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatocytes; Humans; Immunosuppressive Agents; Ischemia; Liver; Liver Neoplasms; Liver Transplantation; Male; Membrane Glycoproteins; Rats; Rats, Sprague-Dawley; Rats, Zucker; Reperfusion Injury; Sirolimus; Transplantation Conditioning

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Cholestasis; Drug Resistance, Neoplasm; Everolimus; Humans; Kidney Neoplasms; Neoplasm Metastasis; Protein Kinase Inhibitors; Salvage Therapy; Sirolimus

Hepatotoxicity, including cholestasis, is a rare but significant complication of treatment with calcineurin inhibitors. Timely life-saving therapy with revision of immunosuppression is mandatory. A 43-year-old woman with pulmonary hypertension was found to have severe cholestasis (serum bilirubin up to 35 mg/dL) after a living-donor lobar lung transplantation. Calcineurin-inhibitor cholestasis markedly improved after withdrawal of the calcineurin inhibitor, initiation of sirolimus, and interleukin-2 receptor blockade. Awareness of the diagnostic criteria of this rare posttransplant complication is important to initiate timely therapy.

Topics: Adult; Calcineurin; Calcineurin Inhibitors; Cholestasis; Disease Progression; Fatal Outcome; Female; Graft Rejection; Humans; Hypertension, Pulmonary; Immunosuppressive Agents; Living Donors; Lung Transplantation; Methylprednisolone; Pneumonia, Bacterial; Postoperative Complications; Pseudomonas Infections; Risk Assessment; Sirolimus; Tacrolimus; Transplantation Immunology

Cyclosporine A and sirolimus are used alone or in combination as immunosuppressants in organ transplantation. To elucidate hepatic side effects, we examined hepatic mRNA of proteins involved in biliary and hepatocellular transport of drugs, formation of glutathione (GSH) and drug metabolising cytochrome P-450 enzymes (CYPs) in rats treated orally for 2 weeks with cyclosporine A (15 mg/kg/day), sirolimus (0.4 mg/kg/day), their combination (same doses), or vehicle. Liver function tests (alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and bilirubin) in blood were then analysed as were hepatic mRNA levels of canalicular transport proteins (Mrp2, Bsep, Mdr1b and Mdr2), sinusoidal transport proteins (Ntcp, Oatp1 and Oatp2), GSH related enzymes (gamma-glutamylcysteine synthetase light (GCSlc) and heavy (GCShc) chain subunits and glutathione-S-transferase) and CYPs (CYP3A9, CYP1A2, CYP2E1 and CYP2BI/II). Cyclosporine A caused moderate cholestatic changes in liver enzymes, which was synergistically exacerbated by sirolimus. The data suggest that the underlying mechanisms behind cholestasis were not totally identical in the different treatment regimens. Cholestasis secondary to cyclosporine A could be related to reduction in mRNA expression of GSH synthesising enzymes and Mrp2, leading to reduced protection against oxidative stress and reduced bile acid-independent bile flow. After sirolimus treatment, Mrp2 mRNA was also reduced together with reduced levels of most CYPs and increased Oatp2, possibly leading to accumulation of toxic metabolites in the hepatocytes. The enhanced cholestatic effect of the combination treatment could be related to reduced GSH synthesising enzymes and even more pronounced reduction in Mrp2 mRNA and increase of Oatp2 mRNA.

Topics: Animals; Carrier Proteins; Cholestasis; Cyclosporine; Cytochrome P-450 Enzyme System; Drug Interactions; Gene Expression Regulation; Glutathione; Immunosuppressive Agents; Liver; Liver Function Tests; Liver Transplantation; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus

The new immunosuppressive agent sirolimus is combined in transplant patients with the cholestatic substances cyclosporin and tacrolimus. Nothing is known about possible cholestatic effects of these combinations. Therefore, we compared their effects on bile flow and on important bile parameters in an acute bile fistula model in rats. Cyclosporin reduced bile flow, biliary excretion of bile salts, cholesterol, and GSH to 20-40% of basal values. Sirolimus decreased bile flow to 50% and excretion of GSH to 30% of the initial conditions but had no effect on cholesterol and bile salt excretion. In contrast, tacrolimus increased bile flow to 120% and GSH excretion to 220% of the basal levels. Sirolimus/cyclosporin decreased bile flow and bile parameters to the same extent as cyclosporin alone. Sirolimus/tacrolimus reversed sirolimus-induced reduction of bile flow and GSH excretion and resulted in a normal bile salt and cholesterol excretion, thus it may be the better alternative in cholestatic patients.

Topics: Animals; Bile Acids and Salts; Biliary Fistula; Biliary Tract; Cholestasis; Cyclosporine; Disease Models, Animal; Drug Combinations; Immunosuppressive Agents; Male; Rats; Rats, Wistar; Sirolimus; Tacrolimus