THE ACTIN CYTOSKELETON AND INTEGRIN EXPRESSION IN THE RECOVERY OF CELL-ADHESION AFTER OXIDANT STRESS TO A PROXIMAL TUBULE CELL-LINE (JTC-12)

This study examines the role of the actin cytoskeleton and integrin ex pression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, o r 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viab ility measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide . ATP levels fell immediately after the peroxide incubation in all gro ups to approximately 10% of normal, but already showed some recovery b y 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhes ion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Im mediately after exposure to 10 mM peroxide, there were subtle abnormal ities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the peri phery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened micro filaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cy toskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha 6 subunit on cells exposed t o 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increas ed to 118.5 +/- 4.5% and by 24 h to 146 +/- 13.4% of control levels. T he expression of the beta 1 and alpha V beta 3 integrins remained unch anged. Thus, despite coarsening of the actin cytoskeleton and depresse d ATP levels, cell adhesion recovered from oxidant stress. Abnormal ce ll adhesion after injury was not a consequence of a decrease in integr in expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.