The damage of tight junction proteins in elder MDR 2-/- knockout mice: A question of cholestasis?

Background and Aims:

In the Mdr2-/- knockout mouse model the secretion of phosphatidylcholin is impaired which results in tight junction leakage and destroyed bile duct epithelium by the toxic biliary bile acids. The macroscopic and microscopic image of the tissue damage resembles the primary sclerosing cholangitis (PSC). Aims: We investigated the dimension of the damage on tight junction proteins occludin and claudin 8 in liver of elder Mdr2-/- knockout mice.

Method:

In Mdr2-/- knockout mice (n = 10 with 14/15 months; n = 10 with 20/21 months) several cholestasis serum parameters were determined inter alia bilirubin. After RNA and protein isolation of the liver the proteins occludin and claudin 8 were analyzed by a real time PCR (microarray, Qiagen) and western blot analysis. Furthermore immunofluorescence microscopy was made of the livers of Mdr2-/- mice (n = 10) where occludin, claudin 8, ZO-1 (an intracellular tight junction protein) and e-cadherin (adhesion molecule and marker for bile ducts and hepatocytes in zone 1) were investigated. BalbC mice served as control in immunofluorescence microscopy as well as in real time PCR/western blot.

Results:

The results of the PCR Arrays showed an upregulation of occludin in both MDR 2 -/- knockout groups (BalbC vers high bilirubin group p < 0,01, BalbC vers low bilirubin group p < 0,01). However, the analysis evidenced a significant upregulation of claudin 8 in both mice groups (BalbC vers high bilirubin group p < 0,01, BalbC vers low bilirubin group p < 0,01). In western blot analysis no significant changes in occludin levels could be observed in all groups. Claudin 8 protein analysis showed a significant downregulation in both MDR groups to BalbC (high bilirubin group vers BalbC p < 0,001, low bilirubin group vers BalbC p < 0,005) and additionally a significant downregulation in the strong cholestasis group vers low bilirubin levels (p < 0,05). The immunofluorescence staining showed a relation between the amount of bilirubin and the expression of occludin and claudin 8. In mice with high bilirubin (> 0.5 mg/dL) occludin is strongly expressed while in mice with low bilirubin (< 0.5 mg/dL) occludin was clearly less discernible. Claudin 8 was detected in zone 2 and 3 as well as around the central veins in a patchy pattern. The higher the bilirubin value is the less claudin 8 was visible around the central veins.

Conclusion:

In immunofluorescence staining and western blot analysis elder mice with sclerosing cholangitis showed a change in the expression of the tight junction protein occludin and claudin 8 dependent on how strong the cholestasis is distinctive. In PCR claudin 8 and occludin were upregulated in both groups. The findings were not dependent on cholestasis. These results are not consistent with the western blot analysis and the staining pattern. This inconsistency could be put down to posttranscriptional processes and requires further studies on protein level.