Renal tubular epithelial cells are continuously exposed to osmotic pressure fluctuations, which are a fundamental aspect of their physiological environment. Under sustained hyperosmotic stress, these cells undergo epithelial–mesenchymal transition (EMT), transforming into α-smooth muscle actin (α-SMA)-positive myofibroblasts that contribute to tubulointerstitial fibrosis and renal dysfunction [1,2]. Our previous study identified hyperosmotic stress–induced cell contraction as an early trigger of EMT, linking transient morphological contractions to long-term phenotypic reprogramming [3]. However, the molecular mechanisms that couple hyperosmotic stress to cell contraction and the subsequent differentiation into α-SMA–positive myofibroblasts remain poorly understood. Although calcium influx and mechanical signaling have been implicated in osmotic adaptation [4], how these events integrate to drive fibrogenic transformation has not been clearly delineated.

Cellular responses to hyperosmotic stress, including changes in cell shape and volume, differ markedly among the different cell types [[5], [6], [7]]. In renal tubular epithelial cells, exposure to hyperosmotic media, such as 200 mM mannitol, induces a pronounced reduction in both the projected cell area and cell volume [3,8,9]. These volume changes are often attributed to rapid water efflux through aquaporins [10]. However, increasing evidence suggests that mechanosensitive ion channels also play critical roles in the detection and transduction of osmotic stimuli [11]. Transient receptor potential (TRP) channels have emerged as key osmosensors that mediate Ca2+ entry and downstream signaling cascades in response to changes in membrane tension [12,13]. Although TRP channels have been implicated in osmotic sensing [14], their specific contribution to hyperosmotic stress–induced cell contraction remains unclear.

This study aimed to elucidate the mechanisms by which hyperosmotic stress triggers cell contraction and promotes α-SMA-positive myofibroblast transformation in renal tubular epithelial cells. Therefore, we applied two complementary strategies: first, a targeted pharmacological screening to identify inhibitors of hyperosmolarity-induced contractions, and second, RNA sequencing to delineate the signaling pathways involved. These experiments identified TRPV4 as a critical mechanosensor and the PI3K–Akt pathway as a key mediator of downstream cytoskeletal and transcriptional responses.