Elevated Akt phosphorylation as an indicator of renal tubular epithelial cell stress.
Characterization of the phosphoinositide 3-kinase-signaling pathway in a human renal tubular epithelial cell (TEC) line HKC-8 revealed high levels of Akt phosphorylation in serum-starved cultures. In contrast to Erk1/2, little additional phosphorylation of Akt was observed after cytokine or serum stimulation. Replacement of the conditioned medium attenuated Akt phosphorylation such that 90 min after the addition of warmed serum-free media, Akt phosphorylation had fallen sufficiently to allow an epidermal growth factor-stimulated increase to be detected readily. Although the mechanism by which the phosphoinositide 3-kinase/Akt pathway is activated in serum-starved TEC is unknown, the mediator responsible is secreted from these cells. Thus, conditioned media removed from a dish of quiescent TECs stimulated Akt phosphorylation in washed TEC cultures within 10 min. Biochemical characterization of the bioactive agent identified a heat labile factor of small apparent molecular mass. The basal level of Akt phosphorylation observed in serum-starved cultures was inhibited by wortmannin at concentrations that demonstrated its dependence on 3-phosphoinositide synthesis (IC(50) = 8 nm). Regular removal of conditioned media from TEC cultures and its replacement with serum free media resulted in a sustained attenuation of Akt phosphorylation. Interestingly, after 5 days of this treatment, washed TEC cultures contained a greater number of viable cells than cultures maintained in conditioned media throughout. This observation was not explained by a difference in the rate of DNA synthesis. Instead, the number of cells undergoing apoptosis increased markedly in the unwashed cultures. Consequently, we propose that in HKC-8 cells Akt phosphorylation is up-regulated in an effort to minimize cell death. This stress-activated response is initiated by a factor secreted into the conditioned medium that stimulates the phosphoinositide 3-kinase signaling pathway.
Renal Section, Faculty of Medicine, Imperial College, Du Cane Road, London W12 0NN, United Kingdom.
Toshihiko
T
Nishino
Charles D
CD
Pusey
Jan
J
Domin
D002460:Cell Line; D002470:Cell Survival; D017077:Culture Media, Conditioned; D004847:Epithelial Cells; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D007684:Kidney Tubules / Q000166:cytology* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt
D017077:Culture Media, Conditioned; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C494918:AKT1 protein, human