Bufalin‐Induced Epithelial‐to‐Mesenchymal Transition in Kidney Epithelial Cells

The kidney plays a central role in fluid, electrolyte, and blood pressure regulation, processes tightly coupled to Na⁺/K⁺-ATPase activity. Beyond its canonical transport function, Na⁺/K⁺-ATPase also acts as a signaling receptor for cardiotonic steroids (CTSs) such as bufalin, which have been implicated in fibrosis and epithelial-to-mesenchymal transition (EMT). Here, we investigated the effects of serial passages on porcine kidney epithelial LLC-PK1 cells and their response to the endogenous CTS bufalin. High-passage cells (P > 80) displayed increased proliferation (1.7x), viability (1.5x), and migration (2.2x) compared to low-passage cells (P < 40), concomitant with elevated ERK1/2 phosphorylation (2.5x), while NKA activity and expression remained unchanged. Bufalin treatment (20 nM, 48 h) induced striking morphological changes consistent with EMT in P > 80 cells, including a transition from cuboidal to elongated shapes with cytoplasmic extensions, whereas P < 40 cells were largely resistant. In high-passage cells, bufalin reduced pan-cadherin, E-cadherin, occludin, claudin-1, ZO-1, and ZO-2 expression, with redistribution of adhesion proteins from membrane to cytoplasm. β-catenin and ZEB-1 were excluded from the nucleus, indicating altered transcriptional regulation during EMT. In contrast, low-passage cells exhibited only modest reductions in E-cadherin, claudin-1, and ZEB-1, along with increased ZO-2, and β-catenin expression. For comparison, TGF-β1 induced partial EMT features in bufalin-resistant LLC-PK1 cells, including striking cell elongation, increased vimentin expression, and appearance of E-cadherin aggregates. Together, these results demonstrate that bufalin induces EMT-like changes in LLC-PK1 cells in a passage-dependent manner, possibly through ERK1/2 activation, disruption of intercellular adhesion, and modulation of transcription factor localization. These findings highlight bufalin as a regulator of epithelial plasticity with potential implications for renal pathophysiology.