Role of the phosphatidylinositol 3-kinase and mTOR pathways in the regulation of renal fibroblast function and differentiation.

Tubulointerstitial fibrosis is largely mediated by (myo)fibroblasts present in the interstitium. In this study, we investigated the role of mTOR and phosphatidylinositol 3-kinase in the regulation of fibroblast kinetics, fibroblast differentiation, and collagen synthesis. Rat renal fibroblasts were propagated from kidneys 3 days post-ureteric obstruction and specific inhibitors of mTOR (RAD) and phosphatidylinositol 3-kinase (LY294002) were used to examine the regulation of fibrogenesis. LY294002 but not RAD completely inhibited phosphorylation of Akt, while both inhibitors decreased phosphorylation of the S6 ribosomal protein. RAD and LY decreased foetal calf serum stimulated proliferation and DNA synthesis. In addition to their individual effects, treatment with both RAD and LY294002 decreased serum-induced fibroblast proliferation and DNA synthesis significantly more than either drug alone. TUNEL positive cells (apoptosis) in RAD and LY294002 treated groups were not different from control groups. In addition to their effect on proliferation, both inhibitors also reduced total collagen synthesis. Differentiation studies indicated an increase in alpha-smooth muscle actin expression relative to beta-actin (western blotting), with cytochemistry confirming that all doses of RAD and LY294002 increased the proportion of alpha-smooth muscle actin positive cells, and hence myofibroblasts. Effects were independent of cell toxicity. These results highlight the potential significance of PI3K and mTOR, in the regulation of renal (myo)fibroblast activity. The synergistic effects of LY and RAD on proliferation suggest that mTOR signalling involves pathways other than phosphatidylinositol 3-kinase. These results provide a novel insight into the mechanisms of fibroblast regulation during fibrogenesis.