Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli

To better define the role of Ca²⁺ in pathophysiological alterations of the proximal tubule microvillus actin cytoskeleton, we studied freshly isolated tubules in which intracellular free Ca²⁺ was equilibrated with highly buffered, precisely defined medium Ca²⁺ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage and resulting nonspecific changes. Increases of Ca²⁺ to ≥ 10 μM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca²⁺-dependent, villin-mediated actin cytoskeletal disruption in tubule cell microvillar damage under conditions conceivably present during pathophysiological states. However, despite prior evidence for cytosolic free Ca²⁺ increases of the same order of magnitude and similar structural microvillar alterations, Ca²⁺- and villin-mediated events did not appear to account for the initial microvillar damage that occurs during ATP depletion induced by antimycin alone or hypoxia.