COMMD1-deficient dogs accumulate copper in hepatocytes and provide a good model for chronic hepatitis and fibrosis

Abstract

New therapeutic concepts developed in rodent models should ideally be evaluated in large animal models prior to human clinical application. COMMD1-deficiency in dogs leads to hepatic copper accumulation and chronic hepatitis representing a Wilson's disease like phenotype. Detailed understanding of the pathogenesis and time course of this animal model is required to test its feasibility as a large animal model for chronic hepatitis. In addition to mouse models, true longitudinal studies are possible due to the size of these dogs permitting detailed analysis of the sequence of events from initial insult to final cirrhosis. Therefore, liver biopsies were taken each half year from five new born COMMD1-deficient dogs over a period of 42 months. Biopsies were used for H&E, reticulin, and rubeanic acid (copper) staining. Immunohistochemistry was performed on hepatic stellate cell (HSC) activation marker (alpha-smooth muscle actin, α-SMA), proliferation (Ki67), apoptosis (caspase-3), and bile duct and liver progenitor cell (LPC) markers keratin (K) 19 and 7. Quantitative RT-PCR and Western Blots were performed on gene products involved in the regenerative and fibrotic pathways. Maximum copper accumulation was reached at 12 months of age, which coincided with the first signs of hepatitis. HSCs were activated (α-SMA) from 18 months onwards, with increasing reticulin deposition and hepatocytic proliferation in later stages. Hepatitis and caspase-3 activity (first noticed at 18 months) increased over time. Both HGF and TGF-β1 gene expression peaked at 24 months, and thereafter decreased gradually. Both STAT3 and c-MET showed an increased time-dependent activation. Smad2/3 phosphorylation, indicative for fibrogenesis, was present at all time-points. COMMD1-deficient dogs develop chronic liver disease and cirrhosis comparable to human chronic hepatitis, although at much higher pace. Therefore they represent a genetically-defined large animal model to test clinical applicability of new therapeutics developed in rodent models.

Figure 1. Histological description.

COMMD1-deficient dog livers were stained with H&E and RA to assess inflammation and copper accumulation, respectively, and stained for α-SMA and reticulin (collagen type III) to assess fibrosis. Representative pictures of a COMMD1-deficient dog over a period of 42 months are shown. Numbers indicate age in months.

Figure 2. TGF-β1 pathway.

(A) Gene-expression profiling. Interaction plots of Q-PCR data of important mediators of fibrogenesis in a time-dependent copper-induced hepatitis in five COMMD1-deficient dogs. Q-PCR results were normalized against the expression of six control dogs (one to three years of age). Linear mixed-effect modeling was used; * significant difference corrected for multiple testing. (B) Western blot analysis on the activation of TGF-beta signalling (total Smad2 and phosphorylated Smad2 (Ser727)) of five COMMD1-deficient dogs at 12, 30, and 42 months. β-actin (ACTB) served as loading control.

Figure 3. HGF pathway.

(A) Gene-expression profiling. Interaction plots of Q-PCR data of important mediators of regeneration in a time-dependent copper-induced hepatitis in five COMMD1-deficient dogs. Q-PCR results were normalized against the expression of six control dogs (one to three years of age). Linear mixed-effect modeling was used; * significant difference corrected for multiple testing. (B) Western blot analysis on the activation HGF-signalling (HGF, phosphorylated c-Met, (phosphorylated) STAT3) of five COMMD1-deficient dogs at 12, 30, and 42 months. β-actin (ACTB) served as loading control.