Effects of mTOR Inhibitors on the Biliary Cystogenesis of the Polycystic Kidney Rat, an Animal Model of Caroli's Disease.
Xiang Shan Ren, Yasunori Sato, Kenichi Harada, Motoko Sasaki, Yasuni Nakanuma. Kanazawa University Graduate School of Medicine, Japan
Background: The polycystic kidney (PCK) rat is an animal model of Caroli's disease as well as autosomal recessive polycystic kidney disease (ARPKD). The activation of the AKT-mTOR signaling pathway has been well documented in the patients with autosomal dominant PKD (ADPKD), and the mTOR inhibitors, sirolimus(SL) and everolimus(EL), have been indicated as potential therapeutic agents. However, the involvement of the AKT-mTOR signaling pathway in the biliary dysgenesis of Caroli's disease has not been fully clarified.
Design: In vivo, immunostaining of p-AKT and p-mTOR was performed using liver sections of the PCK and control (SD) rats. In vitro, biliary epithelial cells (BECs) of the PCK and SD rats were treated with the mTOR inhibitors (SL, EL and PP242). The mTOR have two complexes TORC1 and TORC2, and the both complexes are known to act independently in association with AKT. SL and EL are inhibitors of TORC1, while PP242 is a dual inhibitor of TORC1 and 2. The cell proliferative activity was measured using the WST-1 assay. Phospholyration of AKT, mTOR and P70S6K was examined using Western blotting. The effects of the inhibitors on the biliary cyst formation were determined using the 3-D cell culture system. Apoptosis and autophagy of the BECs were evaluated using the ssDNA ELISA kit and the Western blotting of cleaved caspase 3 for apoptosis, and the Western blotting of the light chain 3B for autophagy.
Results: Both in vivo and in vitro, the phosphorylation of AKT and mTOR of the cholangiocytes was increased in the PCK rat compared to those of the SD rat. In vitro, treatment with mTOR inhibitors induced significant reduction in the cell proliferative activity of the BECs of the PCK rat. Western blot analysis showed that the phosphorylation of AKT, mTOR and P70S6K was inhibited following the treatment. Notably, PP242, but not SL and EL, showed significant inhibitory effects on the biliary cyst formation of the PCK rat in the 3-D cell culture system. PP242 induced both apoptotic and autophagic cell death in the BECs, while EL induced only apoptotic cell death.
Conclusions: A recent study showed that in vivo administration of SL, failed to improve the biliary cystogenesis of the PCK rat (Reken C et al. Nephrol Dial Transplant 2010). Our data suggest that the dual inhibition of TORC1 and TORC2 may be required for the inhibition of the biliary cystogenesis of the PCK rat, and PP242 may be an effective therapeutic agent.
Category: Liver & Pancreas
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 181, Wednesday Morning