Multi-thiol assessment upon the Peritoneal Equilibration Test (PET) reveals solute-specific membrane transport profiles

Abstract

Peritoneal membrane (PM) dysfunction remains a key limitation to the long-term efficacy of peritoneal dialysis (PD) [1]. The Peritoneal Equilibration Test (PET), widely used to clinically assess PM function, primarily evaluates creatinine transport but may overlook distinct membrane response patterns. Aminothiols such as cysteine (Cys), homocysteine (HCys), cysteinylglycine (CysGly), and glutathione (GSH), a thiol profile we refer to as the “thiolome” [2], may offer insights into PM membrane integrity and redox balance. Extracellular vesicles (EVs), which carry antioxidant molecules, may also contribute to local protective mechanisms against injury [3]; however, the thiol content of EVs in the context of PET has yet to be investigated. Therefore, we hypothesized that thiol responses to PET vary among patients, and that these molecules are present in the cargo of EVs isolated from PET dialysate. We aimed to characterize patient thiol responses during PET and to investigate the presence of thiols in EVs derived from PET dialysate. Methods: A cross-sectional, single-center study was conducted in 25 PD patients at Unidade Local de Saúde de Lisboa Ocidental (Ethical approval: NMS 120/2023/CEFCM; ULSLO 2024-63). PD effluents samples were collected at 0, 2, and 4 hours during a standard PET. Alongside, plasma samples were collected before PET started. Free total aminothiol fractions were quantified via HPLC with fluorescence detection (HPLC-FD) [4,5]. Area under the curve (AUC) for dialysate-to-plasma (D/P) ratios of aminothiols were calculated to assess their peritoneal transport characteristics during PET. Furthermore, EVs were isolated from PET dialysate by filtration followed by an ultracentrifugation step. EVs successful isolation was confirmed by the presence of CD63, an EV-specific marker, through western blot analysis, and GSH content was assessed by HPLC-FD [4,5]. Results: Compared to creatinine, the AUC values for Cys and CysGly were significantly lower (p = 0.001 and p < 0.0001, respectively), while the AUC for HCys did not differ. Additionally, GSH was detected in isolated EVs obtained from PET dialysate. Conclusion: This exploratory and preliminary data indicate that transport across the PM varies significantly among different aminothiols, highlighting potential differences in underlying transport mechanisms and membrane selectivity.