Long-term peritoneal dialysis (PD) frequently leads to peritoneal fibrosis (PF), resulting in ultrafiltration failure. This study aims to elucidate the molecular mechanisms underlying PD-associated PF and provide new insights for its treatment. Single-cell RNA sequencing (scRNA-seq) data from PD patients in the GEO database were analyzed to examine the cellular composition and gene expression profiles of peritoneal tissues. Human peritoneal mesothelial cells (PMCs) were treated with high glucose (2.5%) to simulate the PD microenvironment. Cellular senescence and related signaling pathways were assessed using SA-β-gal staining, ELISA and Western blot. Regulatory mechanisms were further explored through RNA interference, dual-luciferase reporter assays, and Transwell co-culture experiments. Key findings were validated in a mouse model. scRNA-seq analysis revealed a reduced proportion of PMCs and an increased number of fibroblasts in long-term PD patients, along with activated senescence pathways and elevated RRAS expression in PMCs. In vitro experiments confirmed that high glucose induced PMCs senescence, while RRAS knockdown delayed this process by inhibiting the MEK/ERK pathway. The transcription factor KLF6 was found to directly bind to the RRAS promoter and enhance its transcription. Co-culture experiments further demonstrated that senescent PMCs activated fibroblasts through the paracrine secretion of TGF-β, upregulating the expression of α-SMA, fibronectin, and COL1A1 in fibroblasts. This effect was blocked by KLF6 knockdown or TGF-β receptor inhibition. Animal experiments confirmed that KLF6 knockdown suppressed the RRAS/MEK/ERK pathway, reduced the release of senescence-associated secretory phenotype (SASP) factors, and thereby alleviated peritoneal fibrosis. High-glucose PD fluid induces PMCs senescence by activating the KLF6/RRAS/MEK/ERK axis. Senescent PMCs, promote fibroblast activation via paracrine TGF-β, ultimately driving peritoneal fibrosis. This pathway may serve as a potential therapeutic target for intervening in PF.
