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Elevated Akt phosphorylation as an indicator of renal tubular epithelial cell stress
Toshihiko Nishino1, Charles D Pusey, Jan Domin
1Renal Section, Faculty of Medicine, Imperial College, Du Cane Road, London W12 0NN, United Kingdom.
Abstract:
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.
Insights
Serum-starved renal tubular epithelial cells (TECs) activate the phosphoinositide 3-kinase/Akt pathway via a secreted factor, suggesting this Akt phosphorylation is a stress response to minimize cell death.
Area of Science:
- Cell Biology
- Molecular Signaling
- Renal Physiology
Background:
- The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway regulates crucial cellular processes.
- Understanding its activation in renal tubular epithelial cells (TECs) is vital for kidney health.
- Basal Akt phosphorylation in serum-starved TECs suggests an endogenous activation mechanism.
Purpose of the Study:
- To characterize the PI3K/Akt pathway activation in HKC-8 TECs under serum-starved conditions.
- To identify the endogenous factor responsible for Akt activation in quiescent TECs.
- To investigate the role of Akt phosphorylation in TEC survival and apoptosis.
Main Methods:
- Utilized HKC-8 human renal TEC line.
- Stimulated cells with cytokines, serum, and conditioned media.
- Assessed Akt and Erk1/2 phosphorylation via Western blotting.
- Characterized the secreted factor biochemically and tested wortmannin inhibition.
- Monitored cell viability and apoptosis rates.
Main Results:
- Serum-starved HKC-8 cells exhibited high basal Akt phosphorylation, unlike Erk1/2.
- A heat-labile, small molecular weight factor secreted by TECs activated Akt.
- Wortmannin (IC50 = 8 nm) confirmed PI3K dependence.
- Regular medium replacement reduced Akt phosphorylation and decreased apoptosis.
- Unwashed cultures showed increased apoptosis compared to washed cultures.
Conclusions:
- Akt phosphorylation is upregulated in serum-starved HKC-8 cells by a secreted factor.
- This activation represents a stress response to minimize cell death.
- The PI3K/Akt pathway plays a protective role against apoptosis in TECs.