Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells

Breakdown of proximal tubule cell apical membrane microvilli is an early-oc curring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is kno wn that actin cytoskeleton alterations play a critical role in this cellula r process. Our laboratory previously demonstrated that ischemia-induced cel l injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [( ADF); Schwartz, N, Hosford M, Sando val RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol R enal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postula ted that ischemia-induced ADF relocalization from the cytoplasm to the apic al microvillar microfilament core was an early event occurring before F-act in alterations. To directly investigate this hypothesis, we examined the in tracellular localization of ADF in ischemic rat cortical tissues by immunof luorescence and quantified the concentration of ADF in brush-border membran e vesicles prepared from ischemic rat kidneys by using Western blot techniq ues. Within 5 min of the induction of ischemia, ADF relocalized to the apic al membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depol ymerization of microvillar F-actin to G-actin was documented by using coloc alization studies for G-and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain be fore microvillar destruction. These data further suggest that ADF plays a c ritical role in microvillar microfilament destruction and apical membrane d amage during ischemia.