Establishment Of Combined Model Of Hepatorenal Fibrosis In Rats And Initial Investigation Of The Underlying Mechanism

Introduction:It's already a proven fact that there exists a relationship between CLD(Chronic Liver Disease)and kidney disease but still there is no available combined model of liver fibrosis and kidney fibrosis on the same animal.An animal model is one of the important research tool in the field of medical science because it is important to build a model that simulates the condition so that the disease can be studied.Therefore,our aim in this experiment is to build a less expensive,less time consuming,and reproducible model of heaptorenal fibrosis on rats.Methods:In this experiment,we used 25 female Wistar rats divided into four groups with 10 rats in model group,5 each rat in BSA group,CCl4 group and Normal group respectively.Model group was administered combined injection of CCl4 and BSA,BSA group was injected BSA only,CCl4 group was injected CCl4 only and Normal group received nothing.CCl4 dose was 30%and injected intraperitoneally twice a week for 10 weeks,BSA dose was 1 gram and injected intraperitoneally 7 days a week for 8 weeks and then five days a week through week 9 and week 10.Animals were weighed and recorded every two weeks.At the end,all the animals were killed and livers and kidneys were send for Histopathology(HE stains,Picrosirius Red stain),Immunohistochemistry for a-SMA(a-Smooth Muscle Actin),Immunofluorescence for IgG,IgM,IgA,C3 and Clq,Electron microscopy etc.Blood samples were also collected and send for liver and renal biochemistry.Urine were also collected and send for total protein content.Results:All the animals gained weight during the course of experiment but the weight gain was highest in the BSA group and lowest in the CCl4 group.Grossly livers from CCl4 group showed more prominent sign of fibrosis than the other groups,whereas kidneys were unremarkable.Histopathology of the liver tissues showed marked liver fibrosis in the CCl4 group,the Model group had lesser fibrosis than that of CCl4 group.BSA group and Normal group had no difference.Histopathology of Kidney showed marked interstitial inflammation and focal connective tissue proliferation in the Model group and BSAgroup.Collagen deposition in the liver showed no difference between Model group and CCl4 but was higher than rest of the group,whereas collagen deposition in the kidneys were insignificant.Immunohistochemistry for ?-SMA were positive in the fibrotic region and other areas of normal distribution.Immunofluorescence in the Model were positive for both IgG and IgM whereas other group showed positivity for IgM only.IgA,C3 and Clq were negative in all groups.Electron microscopy showed intramembranous as well as sub epithelial electron dense deposits in the Model group,whereas there was only sub epithelial deposits in the BSA group.Biochemical parameters for liver injury was highest in the CCl4 group and lowest in the BSA group.Biochemical parameters for renal functions showed no significant differences.Urine output was highest in the Model group and Urinary protein was also highest in the Model group.Conclusion:The results from this experiment showed that BSA had protective effects against the hepatotoxic effect of CCl4.Both CCl4 and BSA combinedly affected liver and kidney to produce a hepatorenal lesion,both histological and biochemical lesion were prominent in the liver whereas in the kidney the changes were mild with no biochemical lesions.The IHC results suggested myofibroblast involvement whereas immunofluorescence showed immunoglobin deposits in the glomerulus.Ultrastructural study of renal tissues showed GBM(glomerular basement membrane)disruption and increased senescent epithelial cells.Therefore,the possible mechanism involved in this model could be due to oxidative stress generated by CCl4 and due to GBM disruption by BSA overload and subsequent immunological injury followed by thereafter.Therefore,we can conclude that we had a considerable success in establishing the model.The result demonstrated CCl4 and BSA can be used in conjunction to induce a hepatorenal injury but we encountered many difficulties and questions which should be addressed by doing further studies to conduct a successful model of hepatorenal fibrosis in rats.We hope this study will serve as an important reference for the future attempt to establish the model.