Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo

Abstract

Background. Exogenous growth factors administered during unilateral ureteral obstruction (UUO) in neonatal rats significantly reduce apoptosis and tubular atrophy. Because the mechanism underlying these salutary effects is largely unknown, we investigated signaling pathways potentially activated by growth factors to determine their roles in therapeutic action. Methods. Mechanical strain was applied to confluent cultures of immortalized rat proximal tubule cells to simulate obstruction-induced stretch injury in vivo. Growth factors, inhibitory antibodies or pharmacological inhibitors were added to cultures that were subsequently processed for TUNEL analysis or immunoblots to identify signaling pathways that could be modulating cell survival. For in vivo studies, kidneys harvested from rats ± UUO ± epidermal growth factor (EGF) were fixed or frozen for immunohistochemistry or immunoblot analysis. Results. Treatment with EGF or insulin-like growth factor-1 (IGF-1) during stretch decreased apoptosis by 50% (P < 0.001). Neutralizing antibodies (Abs) directed against either growth factor or its receptor blocked the reduction in apoptosis. Stretch decreased BAD phosphorylation by ∼50% (P < 0.001) relative to unstretched cells and each growth factor restored phosphorylation to basal levels. Kinase-specific inhibitors that blocked growth factor-mediated BAD phosphorylation promoted apoptosis in vitro. BAD phosphorylation decreased by ∼50% (P < 0.001) in the tubules of obstructed hydronephrotic rat kidneys and administration of EGF restored BAD phosphorylation to basal levels. Conclusions. Signaling pathways converging at BAD phosphorylation are key to growth factor-mediated attenuation of stretch-induced apoptosis in vitro and in vivo.