An Attempt to Establish a Common Animal Model for Hepatorenal Fibrosis in Rats

Abstract

It is already a proven fact that there exists a relationship between CLD (chronic liver disease) and kidney disease but still there is no available combined animal model of liver and kidney fibrosis on the same animal. An animal model is one of the important research tools in the field of medical science because it is important to build a model that can simulate the disease condition so that the particular disease can be studied. Therefore, the aim of this study is to build a less expensive, less time consuming, and reproducible model of hepatorenal fibrosis on rats. We administered combined intraperitoneal injection of CCl₄ (Carbon Tetrachloride) and BSA (Bovine Serum Albumin) on a female Wistar rats. At the end, the liver and kidney tissues were examined under microscope to see whether we were successful in establishing the model or not. The results show that liver fibrosis was marked but the changes on the kidneys were mild. In this study, we were able to induce significant fibrosis in the liver and early stages of fibrosis in the kidneys. The result also demonstrated that the addition of BSA conferred a liver protective effect against CCl₄ induced hepatotoxicity, whereas combination of CCl₄ and BSA proved to be detrimental for kidneys.

Figure 1

Graphical presentation of weight changes among different groups at different weeks throughout the duration of study. Weight gain is highest in the BSA group and lowest in the CCl₄ group.

Figure 2

Comparison of histopathology among the groups. (a) Liver histopathology shows marked liver fibrosis in the model group and CCl₄ group. HE stain in the Model group shows periportal fibrosis and fibrous septa formation. Anti-α-SMA antibody positive staining is observed in the fibrotic areas and periportal/perivascular areas. Picro-Sirius red staining corresponds to the changes seen in HE stain. Histopathology of CCl₄ group also shows similar changes as that of Model group along with fatty changes. BSA group and Normal group show no changes except for edema and mild connective tissue proliferation in the BSA group. (b) Histopathology of kidney from Model group and BSA group shows focal areas of connective tissue proliferation and inflammatory cell infiltrates. Anti-α-SMA antibody positive staining is observed in the fibrotic regions and perivascular regions of Model, CCl₄, and BSA group. Picro-Sirius red staining shows similar intensity of collagen staining between Model, CCl₄, and BSA group.

Figure 3

Graphical presentation of collagen deposition among various groups. The amount of collagen deposition in the livers of Model group and CCl₄ group is significantly higher than that of other groups but difference is insignificant in comparison to each other. Collagen deposition in the kidneys is insignificant (Note. a: significant in comparison to Normal group, b: significant in comparison to BSA group).

Figure 4

Immunofluorescent microscopy of renal tissues. (a) CCl₄ group shows granular deposits of IgM in the mesangium. (b) BSA group shows granular deposits of IgM in the mesangiocapillary region. (c) Model group shows granular deposits of IgM in the mesangiocapillary region. (d) Model group shows granular deposits of IgG in the mesangium.

Figure 5

Graphical presentation of biochemical index of various groups. (a) There is no significant difference in the serum albumin levels among all the groups. (b) Serum ALT/AST shows similar trend among various groups. (c and d) Serum BUN and creatinine levels show no significant differences among the groups. (Notes. c: significant in comparison to CCl₄ group, b: significant in comparison to BSA group, and a: significant in comparison to Normal group.)

Figure 6

Graphical presentation of mean 24-hour urinary output (a) and mean proteinuria (b) in different groups. Model group shows significant increase in urine output as well as increased urinary protein in comparison to other groups. (Note. a: statistically significant in comparison to Normal group, b: statistically significant in comparison to BSA group, and c: statistically significant in comparison to CCl₄ group).

Figure 7

Electron microscopy of renal tissues. (a and b) BSA group shows subepithelial electron-dense deposits, GBM loss of trilaminar structure, podocyte swelling, and loss of foot processes. (c and d) Model group shows electron-dense deposits in the subepithelial as well as intramembranous region. (e) Proximal tubular cell of Model group shows senescent mitochondria, autophagosomes, and increased endocytosomes.