C135. PERITONEAL DIALYSIS EFFLUENT-DERIVED MESOTHELIAL CELLS AS A MODEL TO STUDY ARYL HYDROCARBON RECEPTOR

Abstract

Introduction: Peritoneal dialysis (PD) is a home-based renal replacement therapy that relies on the dialytic properties of the peritoneal membrane [1]. However, progressive membrane dysfunction, mesothelial-to-mesenchymal transition events and fibrosis remain major limitations to long-term PD success [2]. The aryl hydrocarbon receptor (AhR) is a ligand activator transcription factor that has been implicated in fibrotic processes [3], including in the peritoneal membrane in animal models [4]. Our aim was to investigate AhR activation in human primary mesothelial cells (HPMCs) and its relation to patients' clinical variables. The underlying concept is that PD effluent, collected after each dwell, reflects the peritoneal membrane microenvironment and serves as a non-invasive liquid biopsy, providing access to patient-specific cellular and molecular information. Materials & Methods: HPMCs were isolated from PDE obtained from nine PD patients at Unidade Local de Saúde de Lisboa Ocidental (Ethics Committee approval:094/2025/CEFCM). The total volume of effluent in each PDE bag was centrifuged at 430×g, the supernatant discarded, and the cellular fraction washed with PBS 1X, pH 7.4 and plated in 48-well plates. Cells reached adequate confluence up to 30 days after plating and their morphology was assessed by phase-contrast microscopy and classified as epithelial-like or non-epithelial-like features by blind evaluation, as previously described [5,6]. Total cell lysates were prepared and CYP1A1 protein expression was assessed by Western blot as a readout of AhR activation. GAPDH was used as loading control. Associations between CYP1A1 expression and clinical and anthropometric variables (age, time on PD, sex, peritoneal transport type, and medication use) were explored using appropriate statistical analyses, with statistical significance was set at p<0.05. Results: Nine adult PD patients were included (mean age 60 ± 16 years), of whom 6 were men and 3 were women. Five patients were incident (PD duration <1 year) and four were prevalent. Four patients were treated with the drug class X, whereas five were not. PDE-derived HPMCs were successfully isolated and cultured from PD patients, demonstrating the feasibility of this approach as a non-invasive window into peritoneal membrane biology. At confluence, HPMCs displayed either epithelial-like (n=5) or non-epithelial (n = 4) morphologies. Most epithelial-like cultures were derived from incident PD patients (4 of 5), whereas only one incident patient exhibited a non-epithelial phenotype. This distribution suggests a possible association between HPMC morphology and earlier stages of PD, although no causal inference can be made. CYP1A1 protein levels did not differ between phenotypes. Inter-individual variability in CYP1A1 protein expression was observable (CV=20%), with an inverse correlation with patient age (r=-0.780; p=0.013). No significant associations were detected for sex, time on PD, or peritoneal transport type. X treatment was associated with lower CYP1A1 levels (p=0.039). Conclusions: This hypothesis-generating pilot study establishes PD effluent-derived primary HPMC as a human experimental model to explore AhR pharmacology and its association with PD long-term success.