Speaker
Description
Mechanical forces generated by fluid flow are essential regulators of epithelial physiology in vivo, particularly in renal tubules where cells experience sustained yet dynamically varying fluid shear stress (FSS). While the influence of shear magnitude on epithelial behavior has been extensively studied, how temporal variations in shear independent of spatial heterogeneity and geometric complexity modulate epithelial responses remains poorly understood. In particular, it is unclear how epithelial cells respond to continuous versus time-varying mechanical inputs when subjected to identical mean shear levels.
Here, we investigate renal epithelial mechanotransduction in a microfluidic channel specifically designed to impose the same average FSS under two distinct flow regimes-continuous and intermittent Swith static culture serving as a baseline control. The curvature-free channel architecture ensures spatially uniform shear, enabling isolation of temporal mechanical effects without confounding contributions from geometry-induced gradients. The device is fabricated using a hybrid workflow combining 3D-printed molds with soft-lithography-based PDMS replication, allowing rapid prototyping with precise control over channel dimensions and flow uniformity.
Live-cell calcium imaging is employed as a sensitive and rapid readout of mechanosensitive signaling, enabling quantitative comparison of intracellular calcium responses across flow regimes and exposure durations. Our results show that, despite identical mean shear magnitudes, intermittent flow induces more heterogeneous and spatially non-uniform calcium activation compared to continuous flow, with a larger fraction of cells exhibiting elevated calcium responses. In contrast, continuous shear produces a more uniform but comparatively attenuated calcium signaling profile across the epithelial monolayer. These differences persist across exposure durations, indicating that temporal modulation of shear influences both the magnitude and variability of mechanosensitive signaling.
These signaling responses are complemented by fixed-cell analyses of actin cytoskeletal organization, tight junction integrity (ZO-1), and nuclear morphology. Intermittent FSS is associated with increased cytoskeletal remodeling and altered junctional patterning relative to continuous shear, suggesting differences in tension distribution and cell-cell coupling. Additionally, flow-induced changes in nuclear shape and orientation are observed under intermittent loading, pointing to a coupling between temporal mechanical cues and nuclear mechanotransduction.
We hypothesize that intermittent FSS permits partial mechanical recovery between loading phases, limiting full adaptation to shear and thereby sustaining mechanosensitive signaling relative to steady exposure. Importantly, these effects emerge in the absence of spatial shear gradients, demonstrating that temporal dynamics alone are sufficient to regulate epithelial mechanotransduction. Overall, this study establishes a minimal yet mechanistically informative framework for probing time-dependent mechanical regulation in epithelial systems, with direct relevance to microfluidic kidney-on-chip platforms incorporating controlled, time-varying flow cues.