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.

Heterogeneity of the MDCK Cell Line and Its Applicability for Influenza Virus Research

“…It was also reported that cryoprotective agents, such as DMSO and DMF, might induce dome formation [55], [66], [84]. In addition, it was shown that the dome formation could be reversed under conditions of mechanical stimulation (shear stress by apical flow) [81]. One of the recent reports provided additional evidence that the cyst formation induced by specific chemical agents is a property of cells with Type-1 phenotype [85].…”

“…The liquid-filled structures formed by MDCK cells were first described in 1969 and named “domes” [65], and the phenomenon was well investigated as a model of epithelial cell function. It was determined that dome formation is sustained by tight intercellular junctions and associated with a unidirectional ion transport into a lumen formed between a solid substratum and the basolateral cell surface, resulting in change of the osmotic pressure, followed by the flow of water into the lumen and inflation of blister-like domes [54], [55], [66], [81]–[84]. The published material regarding which type of cell (Type-1 or Type-2) has a tendency of dome formation is inconsistent, especially taking into account the fact that in many cases dome formation was investigated using specific chemical stimulants, hormones, or special culture medium composition affecting the salt balance [21], [24], [48], [53], [55], [57], [60], [66], [83]–[88].…”

Physics of lumen growth

“…Our simplifying assumptions minimize the specific biological details that have yet to be accounted for to perform a quantitative comparison with experimental data. In particular, tight junctions act as diffusive barriers for different classes of ions across claudin pores (20,21). For the sake of simplicity, we account for their activity as a steady factor included in the hydrodynamic resistance of the paracellular cleft.…”

The Leak Pathway: A Pathway in Flux

“…Osmotic stress increased the paracellular permeability of Caco-2 intestinal epithelial cells [130]. Cattaneo et al [131] attributed collapse of domes in MDCK Type II renal epithelial cell cultures exposed to shear stress to increased paracellular permeability although they did not measure paracellular permeability directly. Cavanaugh et al [99] and Cohen et al [126] concluded that the effect of stretch increased the number of large pores without significantly changing the pore radius.…”