Scaffold stiffening within the pathophysiological range disrupts tight junctions and promotes epithelial mesenchymal transition in renal epithelial cells

Chronic kidney disease (CKD) is characterized by progressive renal fibrosis and decreased kidney function. Epithelial - mesenchymal transitionition (EMT), a pathological process involving the disruption of tight junctions (TJs) and acquisition of mesenchymal phenotypes in epithelial cells, contributes to the progression of fibrosis in CKD. Extracellular matrix (ECM) deposition is accompanied by increased tissue stiffness in CKD; however, the direct effects of pathological stiffness on renal epithelial cells remain poorly understood. This study investigated whether substrate stiffening itself induces EMT in renal epithelial cells. A tunable polyacrylamide (PAA) gel-based system was utilized to simulate the stiffness of normal kidneys (0.9 kPa), fibrotic kidneys (5 kPa), and a rigid control (glass). To assess morphological changes, HEK293A cells were cultured on these substrates the localization of the TJ protein ZO-1, and the expression of EMT-related markers. The potential involvement of mechanosensitive signaling pathways was explored. Finally, observations were compared with the phenotypic changes in a kidney ischemic reperfusion injury (IRI) model in vivo. HEK293A cells cultured on stiff substrates exhibited EMT-like features, including disrupted membrane localization of ZO-1, increased paracellular permeability, upregulation of α-SMA, SNAIL1, ZEB1 and vimentin, and downregulation of E-cadherin. Increased stiffness within the pathophysiological fibrotic range selectively activated p38 MAPK pathway, and pharmacological inhibition of p38 MAPK reduced α-SMA expression and partially restored continuous ZO-1 localization. In fibrotic kidneys of IRI mice, increased α-SMA expression, p38 MAPK activation, and altered ZO-1 localization were observed, which were consistent with the in vitro findings. Our findings demonstrate that scaffold stiffness within the pathophysiological range is a critical factor influencing renal epithelial cell integrity and partial EMT. The p38 MAPK pathway appears to be involved in partial EMT and could serve as a therapeutic target. Although further validation in various types of kidney cells is required, this PAA gel-based model provides a more physiologically relevant platform for studying the mechanobiology of partial EMT compared to traditional rigid culture models.