TRPV4-dependent PI3K-Akt activation mediates hyperosmotic stress-induced cell contraction and myofibroblast...

The osmotic environment surrounding the tubular epithelial cells fluctuates drastically. We have demonstrated that sustained hyperosmotic stress in tubular epithelial cells induces epithelial-mesenchymal transition, and that cell contraction induced by hyperosmotic stress is essential for their differentiation into α-smooth muscle actin (α-SMA)-positive myofibroblasts. However, the mechanism linking hyperosmolarity-induced cell contraction to the differentiation of epithelial cells into α-SMA-positive myofibroblasts remains unclear. To elucidate the mechanisms underlying hyperosmotic contraction in NRK-52E cells, we conducted a targeted pharmacological screening of mechanosensitive receptor modulators, identifying transient receptor potential vanilloid 4 (TRPV4) channel antagonists (HC-067047, RN-1665, and GSK205) as potent inhibitors of cell contraction in response to 200 mM mannitol, highlighting TRPV4 as a key upstream mechanosensor. We performed RNA sequencing to profile gene expression under hyperosmotic stress. Using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, we identified 3137 differentially expressed genes (adjusted p < 0.05, |log2FoldChange| > 1), which were most significantly enriched in the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, exhibiting the highest gene ratio. Finally, to connect the mechanosensor to this pathway, we focused on TRPV4 and PI3K-Akt signaling. Hyperosmotic stress increased Akt phosphorylation, which was suppressed by TRPV4 inhibition. TRPV4 or PI3K inhibition reduced α-SMA expression and attenuated the upregulation of extracellular matrix-related genes, indicating that TRPV4-mediated activation of the PI3K-Akt pathway drives α-SMA-positive myofibroblast differentiation. Collectively, the pharmacological and transcriptomic analyses identified TRPV4-dependent PI3K-Akt activation as the central driver of osmotic stress-induced contraction and myofibroblast differentiation.