Wall shear stress (WSS) is a tangential, frictional force per unit area generated by urine flow along the urinary tract, while the urothelium, a tight epithelial layer, is the first line of defense. The urothelium is in direct contact with urine and therefore exposed to WSS. Increasing evidence suggests that WSS is a key regulator of urothelial function and pathology [1]. Under physiological conditions, urothelial cells are exposed to WSS generally below 0.2 Pa [1]. However, pathological conditions and/or treatments (e.g., fluid irrigation during ureteroscopy) can locally elevate WSS [2]. The resulting WSS may have unintended effects on the surrounding tissue and cells. In this study, we aimed to investigate whether abnormally elevated WSS differentially affects urothelial cell viability and the balance between cytoskeletal and nuclear proteins by adapting a nutation-based WSS model originally established for endothelial mechanobiology [3].

TEU-2 human urothelial cells were cultured as an undifferentiated monolayer or differentiated into a tight multilayered epithelium by adding fetal bovine serum and Ca²⁺. Confluent TEU-2 cells, either undifferentiated or differentiated, were subjected to static conditions or to nutation-induced WSS (ranging from 0 to 1 Pa) for 48 hours using the methods reported in the nutation-based model [3] (Fig 1). At the end of the exposure period, cell viability was assessed using the AlamarBlue assay, and β-actin (cytoskeletal marker) and histone H3 (nuclear marker) levels were quantified by Western blot. While housekeeping proteins such as β-actin and histone H3 are routinely used for normalization, their stability under mechanical stimulation remains untested. The β-actin/histone H3 ratio was used to assess relative changes between cytoskeletal and nuclear proteins under WSS and analyzed using two-way ANOVA. In total, three independent experiments were performed, each with triplicate samples (n = 9 per condition).

WSS elicited a differentiation-dependent response in urothelial cells. The AlamarBlue assay showed no significant change in viability in either undifferentiated or differentiated TEU-2 cells following WSS exposure. Despite the absence of viability effects and the lack of a significant overall effect in the two-way ANOVA, post hoc analysis revealed a significant reduction in the β-actin/histone H3 ratio in undifferentiated TEU-2 cells exposed to WSS (p = 0.0121), whereas differentiated cells showed no significant change. These findings indicate that undifferentiated urothelial cells exhibit molecular sensitivity to WSS in the absence of overt viability loss, while differentiated cells remain comparatively stable.

These preliminary findings indicate that WSS can induce subcellular remodeling in undifferentiated urothelial cells without compromising viability. This suggests that cellular differentiation state can be a key determinant of urothelial mechanosensitivity and may influence vulnerability to WSS-related perturbations.

To our knowledge, this study is the first to show that WSS alters the balance between cytoskeletal and nuclear protein levels in undifferentiated urothelial cells, indicating that β-actin and histone H3 may not serve as stable normalization controls under mechanical stimulation. These preliminary findings reveal a potential source of bias in mechanobiology studies. Importantly, differentiated urothelium—representing the intact umbrella cell layer—appears comparatively resilient to elevated WSS. However, exposure of underlying undifferentiated cells, such as in injured or denuded urothelium, may increase susceptibility to WSS-induced alterations. Further validation is needed to confirm these observations and refine normalization strategies.

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