Parthenolide Alleviates Peritoneal Fibrosis by Blocking Smad2/3 Phosphorylation via Smad Anchor for Receptor Activation

ABSTRACT Progressive peritoneal fibrosis is a severe complication of peritoneal dialysis (PD) with a complex pathogenesis. Our previous studies demonstrated that parthenolide (PTL) alleviates peritoneal fibrosis by suppressing the transforming growth factor (TGF)‐β/Smad pathway. Smad anchor for receptor activation (SARA) acts as an adaptor protein for Smad2 and Smad3; however, its role in PD‐associated peritoneal fibrosis and the relationship between PTL and SARA remain to be elucidated. In this study, single‐cell sequencing (scRNA‐seq) data and long‐dwell PD fluid samples were collected. PD mice, a TGF‐β1‐induced mesothelial–mesenchymal transition (MMT) model, and CRISPR/Cas9‐engineered SARA gene ( ZFYVE9 ) knockout MeT‐5A cells were established. The results revealed that SARA activates Smad2/3, whereas Smad3 promotes SARA degradation, leading to the downregulation of SARA expression during fibrosis progression in long‐term‐retained PD fluid samples, PD mice, TGF‐β1‐treated MeT‐5A cells, and HMrSV5 cells. PTL does not regulate SARA protein stability, but luciferin reporter gene experiments revealed that the transcriptional recovery of the effects of PTL on SARA is consistent with its known antifibrotic effects. PTL was further confirmed to inhibit the SARA‐Smad3 interaction through both in vitro and in vivo coimmunoprecipitation experiments. Molecular docking and PTL‐biotin pulldown assays confirmed that PTL directly binds to the Smad3‐binding interface of SARA at the Pro788 and Ser795 residues. In summary, SARA promotes PD‐related peritoneal fibrosis through the phosphorylation of Smad2/3. PTL specifically binds to ZFYVE9 (P788/S795), disrupting the interaction between SARA and Smad3, thereby improving MMT and alleviating peritoneal fibrosis, which in turn restores SARA expression. This study provides a theoretical foundation for the clinical diagnosis and treatment of peritoneal fibrosis.