Shear Stress Reverses Dome Formation in Confluent Renal Tubular Cells

Abstract

Background/Aims. It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. Methods. A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm² to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca²⁺ blockade and cell deciliation on SS exposure were also investigated. Results. Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm², p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. Conclusions. These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca²⁺ entry in to the cell. SS induced Ca²⁺ entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.