Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice

Abstract

The asialoglycoprotein (ASGP) receptor is an abundant hepatocyte-specific receptor involved in receptor-mediated endocytosis. This receptor's abundance and function is decreased by chronic ethanol administration prior to the appearance of pathology such as necrosis or inflammation. Hence, this study aimed to determine if ASGP receptor function is required to protect against liver injury by utilizing a knockout mouse model lacking functional ASGP receptor in the setting of carbon tetrachloride (CCl(4)) hepatotoxicity. Briefly, ASGP receptor-deficient (RD) mice and wild-type (WT) mice were injected with 1ml/kg body weight of CCl(4). In the subsequent week, mice were monitored for liver damage and pathology (aspartate transaminase (AST), alanine transaminase (ALT) and light microscopy). The consequences of CCl(4) injection were examined by measuring alpha-smooth muscle actin (alpha-SMA) deposition, contents of malondialdehyde and the percentage of apoptotic hepatocytes. After CCl(4) injection, RD mice showed increased liver pathology together with significantly increased activities of AST and ALT compared to that in WT mice. There were also significantly more apoptotic bodies, lipid peroxidation and deposition of alpha-SMA in RD mice versus WT mice following CCl(4) injection. Since these two mouse strains only differ in whether or not they have the ASGP receptor, it can be concluded that proper ASGP receptor function exerted a protective effect against CCl(4) toxicity. Thus, receptor-mediated endocytosis by the ASGP receptor could represent a novel molecular mechanism that is responsible for subsequent liver health or injury.

Fig. 1

Activities (U l^-1) of (A) aspartate transaminase (AST) and (B) alanine transaminase (ALT), in the serum of wild-type (WT) and ASGP receptor-deficient (RD) mice after injection with CCl₄. WT values are represented in open bars and RD values in filled bars. Controls were injected with an equivalent amount of olive oil. Values represent means ± S.E.M. with N = 3 – 7. *Significantly different from the corresponding WT condition, P < 0.05. ^#Significantly different from the corresponding control condition, P < 0.05.

Fig. 2

Histology of livers from wild-type (WT) and ASGP receptor-deficient (RD) mice after injection with CCl₄. Paraffin embedded sections were prepared and stained with H & E. Controls were injected with an equivalent amount of olive oil. Photomicrographs show representative liver sections at 100 times magnification. (A) WT, control, (B) WT, 48 h after CCl₄ injection, (C) WT, 96 h after CCl₄ injection, (D) RD, control, (E) RD, 48 h after CCl₄ injection and (F) RD, 96 h after CCl₄ injection. Note, the more severe necrosis in the RD (E) versus WT (B) mice at 48 h and even at 96 h after CCl₄ injection (F versus C).

Fig. 3

Content (nmol mg protein^-1) of malondialdehyde (MDA) in the liver of wild-type (WT) and ASGP receptor-deficient (RD) mice after injection with CCl₄. WT values are represented in open bars and RD values in filled bars. Controls were injected with an equivalent amount of olive oil. Values represent means ± S.E.M. with N = 3 – 7. *Significantly different from the corresponding WT condition, P < 0.05. ^#Significantly different from the corresponding control condition, P < 0.05.

Fig. 4

Content (O.D. μg DNA^-1) of α-smooth muscle actin (α-SMA) in the liver of wild-type (WT) and ASGP receptor-deficient (RD) mice after injection with CCl₄. WT values are represented in open bars and RD values in filled bars. Controls were injected with an equivalent amount of olive oil. Values represent means ± S.E.M. with N = 3 – 9. *Significantly different from the corresponding WT condition, P < 0.05. ^#Significantly different from the corresponding control condition, P < 0.05.

Fig. 5

Apoptotic hepatocytes (%) in the liver of wild-type (WT) and ASGP receptor-deficient (RD) mice after injection with CCl₄. WT values are represented in open bars and RD values in filled bars. Controls were injected with an equivalent amount of olive oil. Values represent means ± S.E.M. with N = 3 – 4. *Significantly different from the corresponding WT condition, P < 0.05. ^#Significantly different from the corresponding control condition, P < 0.05.