Dedifferentiation and redifferentiation of renal proximal tubule cells following oxidant injury: Role of the epidermal growth factor receptor.

摘要

Repair of injured renal epithelium is thought to be mediated by surviving renal proximal tubular cells (RPTC) which must dedifferentiate to allow the proliferation and migration necessary for epithelial regeneration. RPTC then redifferentiate to restore tubular structure and function. Current models suggest that epidermal growth factor receptor (EGFR) activation is required for dedifferentiation characterized by enhanced vimentin expression, decreased N-cadherin expression, spindle morphology, and loss of apical-basal polarity after injury. Redifferentiation is stimulatied by collagen IV. Because an in vitro model of RPTC redifferentiation has not been reported and the mechanism(s) of redifferentiation has not been determined, we used rabbit RPTC in primary cultures to address these issues. H2O 2 induced the dedifferentiated phenotype which persisted >48 hr; redifferentiation occurred spontaneously in the absence of exogenous growth factors after 72-120 hr. Phosphorylation of multiple tyrosine residues of EGFR increased 12-24 hr, peaked at 24 hr, and declined to basal levels by 48 hr post-injury. EGFR inhibition during dedifferentiation restored epithelial morphology and apical-basal polarity and decreased vimentin expression to control levels 24 hr later. Conversely, exogenous EGF addition increased vimentin expression and potentiated spindle morphology. p38 mitogen activated protein kinase (MAPK) and transforming growth factor (TGF)-beta receptor inhibitors did not affect redifferentiation after H2O2 injury. Similar results were observed in a mechanical injury model. Interrogations of the possible signaling pathways downstream of the EGFR were conducted using pharmacologic inhibitors. These experiments demonstrated EGFR-dependent activation of PI3K/Akt/GSK3beta determined by increased phosphorylation of specific residues. Activation of this pathway at 24 hr post-injury was necessary for dedifferentiation, and it resulted in translocation of beta-catenin from the plasma membrane to the cytoplasm. Exogenous addition of collagen IV at 24 hr caused redifferentiation at 48 hr post-injury. This corresponded to dephosphorylation of GSK3beta and localization of beta-catenin to the cell membrane. These experiments represent a new model for the investigation of RPTC redifferentiation after acute injury and identify a key regulator of re-differentiation: EGFR, independent of p38 MAPK and the TGF-beta receptor. The EGFR causes dedifferentiation by activation of the PI3K/Akt/GSK3beta/beta-catenin pathway. Finally, collagen IV inhibits this pathway by GSK3beta dephosphorlyation.