Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

Abstract

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.

High-Throughput Phenotypic Screening of Kinase Inhibitors to Identify Drug Targets for Polycystic Kidney Disease

“…5D ). We therefore considered whether PI3K, which is aberrantly active in PKD, 22 , 23 was not involved in cyst growth in our model, or whether the PI3K inhibition could exert synergistic effects together with mTOR inhibitors. To test this, we selected one mTOR inhibitor, torin 1 26 that displayed potent activity in our previous screens and has a 1000-fold selectivity for mTOR over PI3K 27 ( Suppl.…”

Crystal deposition triggers tubule dilation that accelerates cystogenesis in polycystic kidney disease

“…Since female Cy/+ rats were exposed to the same ethylene glycol regimen as male Cy/+ rats but were resistant to renal CaOx autosomal-recessive PKD caused by a mutation in the Pkhd1 gene (46). In addition to liver cysts, PCK rats develop renal cysts predominantly of collecting duct origin that exhibit increased mTOR (47,48), SRC (49), and STAT3 (50) activity. In contrast to CaOx nephrolithiasis, which affects male humans and rats more severely than female humans and rats, the opposite is true for CaP nephrolithiasis (23,51).…”

Vasopressin and disruption of calcium signalling in polycystic kidney disease

“…140 On the other hand, enhanced phosphorylation of PI3K and Akt have been found in cystic tissues from patients and rodent models of PKD. 141–145 Activation of mammalian target of rapamycin (mTOR) signaling downstream of PKA through ERK-mediated phosphorylation of tuberin 146,147 has been linked to transcriptional activation of aerobic glycolysis, increased levels of ATP, and together with ERK-dependent inhibition of liver kinase B1, inhibition of AMP kinase, which may further enhance mTOR signaling. 148–151 Phosphorylation and inhibition of glycogen synthase kinase 3β and direct phosphorylation and stabilization of β-catenin by PKA enhance Wnt/β-catenin signaling may contribute to the development of PKD.…”