Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response

Abstract

Cholangiopathies such as primary sclerosing cholangitis (PSC) are chronic liver diseases characterized by increased cholestasis, biliary inflammation and oxidative stress. The objective of this study was to elucidate the impact of cholestatic injury on oxidative stress-related factors. Using hepatic tissue and whole cell liver extracts (LE) isolated from 11-week old C57BL/6J (WT) and Mdr2^KO mice, inflammation and oxidative stress was assessed. Concurrently, specific targets of carbonylation were assessed in LE prepared from murine groups as well as from normal and human patients with end-stage PSC. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Picrosirius red staining revealed extensive fibrosis in Mdr2^KO liver, and fibrosis colocalized with increased periportal inflammatory cells and both acrolein and 4-HNE staining. Western blot analysis revealed elevated periportal expression of antioxidant proteins Cbr3, GSTμ, Prdx5, TrxR1 and HO-1 but not GCLC, GSTπ or catalase in the Mdr2^KO group when compared to WT. From immunohistochemical analysis, increased periportal reactive aldehyde production colocalized with elevated staining of Cbr3, GSTμ and TrxR1 but surprisingly not with Nrf2. Mass spectrometric analysis revealed an increase in carbonylated proteins in the Mdr2^KO and PSC groups compared to respective controls. Gene ontology and KEGG pathway analysis of carbonylated proteins revealed a propensity for increased carbonylation of proteins broadly involved in metabolic processes as well more specifically in Rab-mediated signal transduction, lysosomes and the large ribosomal subunit in human PSC. Western blot analysis of Rab-GTPase expression revealed no significant differences in Mdr2^KO mice when compared to WT livers. In contrast, PSC tissue exhibited decreased levels of Rabs 4, 5 and increased abundance of Rabs 6 and 9a protein. Results herein reveal that cholestasis induces stage-dependent increases in periportal oxidative stress responses and protein carbonylation, potentially contributing to pathogenesis in Mdr2^KO. Furthermore, during early stage cholestasis, there is cell-specific upregulation of some but not all, antioxidant proteins.

Keywords: Cholestasis; Inflammation; Liver; Oxidative stress; Protein carbonylation; Reactive aldehyde.

Figure 1.. Basic Pathology in Mdr2^KO livers.

Liver sections from WT and Mdr2^KO mice were stained with picrosirius red or immunostained using antibodies directed against the smooth muscle actin (αSMA), Cytokeratin 7, or adipophilin. Representative images are shown; n = at least 3 mice per genotype (CV, central vein, PT, portal triad). Blue arrows indicate increased periportal staining (aSMA) and increased ADPH staining in Mdr2^KO liver sections.

Figure 2.. Inflammatory infiltrates in Mdr2^KO livers.

Liver sections from WT and Mdr2^KO mice were immunostained using antibodies directed against the indicated markers of inflammation. Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in Figure 1.

Figure 3.. Impact of Mdr2^KO on protein carbonylation, oxidative injury and cellular proliferation.

Tissue sections isolated from WT and Mdr2^KO mice were immunostained using antibodies directed against acrolein, 4-HNE, γH2A.X and Ki67 as indicated, 100X magnification. Representative images are shown; n = at least 3 mice per genotype. Blue arrows indicate increased periportal staining of each marker. Abbreviations as in Figure 1.

Figure 4.

Impact of cholestasis on hepatic cysteine redox status and GSH.A. Cys, B. CysSS, C. Eh CysSS/Cys, D. CySSG, E. GSH, F. GSSG, G. Eh GSSG/GSH were determined using dansyl chloride derivatization, HPLC, and fluorescence detection. Redox potentials (panels C., G.) were calculated using the Nernst equation. N=5 per group; *P>0.05, **P>0.01.

Figure 5.. Impact of Mdr2^KO on expression of antioxidant responses.

Western immunoblotting analysis of antioxidant responses in liver lysates prepared from WT and Mdr2^KO mice. All exposures were normalized using GAPDH expression. Data are means +/− SEM, n=6 per genotype. Statistical analysis was via students t-test, *p<0.05;**p<0.01;***p<0.001.

Figure 6.. Impact of Mdr2^KO on zonal expression of antioxidant response enzymes.

Liver sections from WT and Mdr2^KO mice were immunostained using polyclonal antibodies directed against the indicated markers. Representative images are shown; n = at least 3 mice per genotype. Blue arrows indicate the hepatozonal expression for each anti-oxidant protein in WT and Mdr2^KO mice as described in the results section. A. TrxR1, PRDX5, catalase and GSTπ. B. GSTμ, CBR3, HO-1 and Nrf2. Abbreviations as in Figure 1.

Figure 7.. VENN analysis of carbonylated proteins during cholestatic liver disease.

A. WT compared to Mdr2^KO. B. Normal human and end-stage PSC.

Figure 8.. KEGG analysis of carbonylated ribosomal proteins in human end-stage PSC.

Red asterisks indicate proteins within the KEGG pathway hsa03010 (large ribosomal subunits) that were identified as carbonylated in PSC hepatic tissue.

Figure 9.. KEGG analysis of carbonylated lysosomal proteins in human end-stage PSC.

Red asterisks indicate proteins within the KEGG pathway hsa04142 (lysosomes) that were identified as carbonylated in PSC hepatic tissue.

Figure 10.. Impact of cholestasis on Rab-GTPase expression.

A. Western immunoblotting analysis of Rab-GTPases and INPP4A in liver lysates prepared from control and Mdr2^KO mice. All exposures were normalized using GAPDH expression. Data are means +/− SEM, n=6 per genotype. Statistical analysis was via students t-test, *p<0.05;**p<0.01;***p<0.001. Data are means +/− SEM, n=6 per genotype. B. Western immunoblotting analysis of Rab-GTPases and INPP4A in liver lysates prepared from normal human and human end-stage PSC patients. Data are means +/− SEM, n=7 per condition. Statistical analysis was via students t-test, *p<0.05;**p<0.01;***p<0.001.