Dysregulation of Lipid Metabolism due to Hepatotoxic Potential of Tacrolimus in Rattus norvegicus

Abstract

One of the major problems after solid organ transplant is graft rejection, and the risk has been significantly reduced using immunosuppressive agents. Tacrolimus is one of the potent immunosuppressive drug which belongs to calcineurin inhibitor and widely used in acute graft rejection of liver. Despite its immunosuppressive properties, some adverse effects of tacrolimus have been reported which include neurotoxicity, nephrotoxicity, new onset diabetes, gastrointestinal toxicity, post transplant lymphoproliferative disease and other infections. Hepatotoxicity impairs cholesterol metabolism in liver that results in hyperlipidemia and ultimately dysregulation of lipid metabolism genes, which may be a contributory factor for liver damage. The present study was designed with an aim to study the effects of acute dose of tacrolimus on liver and lipid metabolism in Rattus norvegicus through hedgehog signaling. Rat primary hepatocytes and male Wistar rats were subjected to 36 µM and 3mg /kg dose of TAC, respectively, in a time dependent manner. .In vitro and in vivo experiments were performed. Serum parameters, tissue histopathology and gene expression analysis was done from in vivo experiments, however protein expression was performed from in vitro studies to analyze regulation of HH pathway. Serum sample was used to measure concentration of superoxide dismutase and glutathione. Tissue samples were used for histopathological studies, immunohistochemistry and gene expression. Liver tissue samples were used to extract RNA and cDNA was prepared using commercial kits followed by gene amplification by polymerase chain reaction. Various histopathological studies including H & E staining, PAS-D staining and reticulin staining were performed to detect liver injury. Furthermore, DNA xvii damage induced by tacrolimus was assessed by TUNEL assay. However, in vitro experiment was performed using rat hepatocytes isolated by liver perfusion method, for western blot analysis. Results revealed increased serum glutathione in treatment groups with no significant change in serum SOD level. Upregulation of gene and protein expression showed that TAC treatment upregulated HH pthway in liver tissue and cells. Histological studies highlighted change in liver architecture and hepatic injury marked by hazy appearance of cytoplasm, increased vacuolation, nuclear fragmentation and vesicular steatosis with change in liver parenchyma and collapsed reticulin fibers. Also, positive immunostaining of liver section exposed to TAC was noticed showing upregulation of HH genes. Furthermore, DNA damage and presence of apoptotic bodies were evident in liver sections of experimental groups analysed by TUNEL assay. It is, therefore, concluded that short term exposure of tacrolimus leads to liver injury and upregulation of HH pathway. These findings can serve as ready reference for studying lipid metabolism pathway for therapeutic purpose and to evaluate dyslipidemia associated with tacrolimus, which is not only a contributory factor for hepatic injury but may also be a risk factor for cardiovascular diseases, pancreatitis and other disorders