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

## TL;DR

This study shows that the compound bufalin can trigger a change in kidney cells from epithelial to mesenchymal, a process linked to disease, and this effect depends on how many times the cells have been grown in the lab.

## Contribution

The study reveals that bufalin induces epithelial-to-mesenchymal transition (EMT) in high-passage kidney epithelial cells via ERK1/2 activation and adhesion disruption.

## Key findings

- High-passage LLC-PK1 cells showed increased proliferation, viability, and migration with elevated ERK1/2 phosphorylation.
- Bufalin treatment caused EMT in high-passage cells, marked by morphological changes and reduced adhesion proteins.
- Low-passage cells were resistant to bufalin-induced EMT but showed partial changes with TGF-β1 treatment.

## Abstract

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.