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Beyond its canonical transport function, Na\u207a\/K\u207a\u2010ATPase also acts as a signaling receptor for cardiotonic steroids (CTSs) such as bufalin, which have been implicated in fibrosis and epithelial\u2010to\u2010mesenchymal transition (EMT). Here, we investigated the effects of serial passages on porcine kidney epithelial LLC\u2010PK1 cells and their response to the endogenous CTS bufalin. High\u2010passage cells (\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&gt;\u200980) displayed increased proliferation (1.7x), viability (1.5x), and migration (2.2x) compared to low\u2010passage cells (\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&lt;\u200940), concomitant with elevated ERK1\/2 phosphorylation (2.5x), while NKA activity and expression remained unchanged. Bufalin treatment (20\u2009nM, 48\u2009h) induced striking morphological changes consistent with EMT in\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&gt;\u200980 cells, including a transition from cuboidal to elongated shapes with cytoplasmic extensions, whereas\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&lt;\u200940 cells were largely resistant. In high\u2010passage cells, bufalin reduced pan\u2010cadherin, E\u2010cadherin, occludin, claudin\u20101, ZO\u20101, and ZO\u20102 expression, with redistribution of adhesion proteins from membrane to cytoplasm. \u03b2\u2010catenin and ZEB\u20101 were excluded from the nucleus, indicating altered transcriptional regulation during EMT. In contrast, low\u2010passage cells exhibited only modest reductions in E\u2010cadherin, claudin\u20101, and ZEB\u20101, along with increased ZO\u20102, and \u03b2\u2010catenin expression. For comparison, TGF\u2010\u03b21 induced partial EMT features in bufalin\u2010resistant LLC\u2010PK1 cells, including striking cell elongation, increased vimentin expression, and appearance of E\u2010cadherin aggregates. Together, these results demonstrate that bufalin induces EMT\u2010like changes in LLC\u2010PK1 cells in a passage\u2010dependent 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.\n                  <\/jats:p>","DOI":"10.1002\/cbin.70091","type":"journal-article","created":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T09:48:38Z","timestamp":1759830518000},"page":"1764-1786","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Bufalin\u2010Induced Epithelial\u2010to\u2010Mesenchymal Transition in Kidney Epithelial Cells"],"prefix":"10.1002","volume":"49","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8041-9757","authenticated-orcid":false,"given":"Gabriela Morais de Oliveira","family":"Barros","sequence":"first","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Universidade Federal do Rio de Janeiro  Rio de Janeiro Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kayo M.","family":"Bagri","sequence":"additional","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Universidade Federal do Rio de Janeiro  Rio de Janeiro Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"Mermelstein","sequence":"additional","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Universidade Federal do Rio de Janeiro  Rio de Janeiro Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0767-1240","authenticated-orcid":false,"given":"Luis Eduardo M.","family":"Quintas","sequence":"additional","affiliation":[{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Universidade Federal do Rio de Janeiro  Rio de Janeiro Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,10,7]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0031-6997(25)00068-7"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms19092769"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1152\/ajprenal.00130.2011"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1054\/tice.2000.0135"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1023\/a:1018903931777"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1093\/geronj\/48.2.b43"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1124\/pr.108.000711"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1113\/expphysiol"},{"key":"e_1_2_11_10_1","first-page":"306","article-title":"The Effect of Age on Active\u2010Transport and Sodium\u2010Potassium\u2010Activated Atpase Activity in Renal Tissue of Rats","volume":"20","author":"Beauchene R. 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