#4454 PERITONEAL DIALYSIS (PD) EFFLUENT DERIVED EXTACELLULAR VESICLES TO ESTABLISH PD-INDUCED PERITONEAL ALTERATIONS
Authors: Karsten Micky , Pegtel Dirk , de Rooij Johan , Vervloet Marc , Jakulj Lily 
Published: June 2023

Abstract

Background and Aims
Peritoneal dialysis (PD) is a life-saving kidney replacement treatment in patients with end-stage kidney disease (ESKD). Yet, long-term exposure of the peritoneal membrane to glucose and its degradation products results in fibrosis and loss of membrane function. At present, detection of early and specific peritoneal injury is challenging. With evolving therapies, biomarkers to assess peritoneal vitality and response to interventions mitigating peritoneal injury in PD-treated patients is mandatory. Extracellular vesicles (EVs) are nano-sized structures containing proteins, micro-RNAs (miRNAs) and lipids reflecting their cells of origin and have a role in intercellular communication. EVs have been widely investigated as potential easy-accessible and stable biomarkers, particularly in inflammatory conditions. Here, we describe a clinically applicable and robust technique to isolate and analyze the molecular cargo of peritoneal dialysis effluent (PDE)-derived EVs (PDE-EVs).

Method
PDE was collected from adult patients treated with PD, excluding those with a recent peritonitis (&lt;6 weeks). First, cell-free PDE was obtained by centrifugation. As a filtration and concentration quality control step cel-miRNA39 packed in EV-sized liposomes was added to the cell-free PDE. PDE-EVs were isolated by subsequent filtration and size-exclusion chromatography (SEC). We used Western blot with the EV-markers Flotilin 1, 70 kilodalton heat shock protein (HSP70), and Syntenin to confirm the presence of EVs in the SEC-fractions. We used Calnexin as a marker of other intercellular membranes as an indication of purity. To confirm robustness of the filtration and concentration steps, exogenous cel-miRNA39 was quantified by qPCR. Endogenous miRNA21 and -10b were used to check if the PDE-EVs contain adequate amounts of small RNA for future analyses.

Results
PDE was collected from 13 patients (mean age 64,7 years (standard deviation 20,1 years); 62% female; 46% treated with APD; median PD-vintage 18 months (range 7–33 months)). Presence of PDE-EVs was confirmed by Western blot (Fig. 1). A weak staining was observed in PDE from a 1-hour dwell time. There was no difference in staining between 4-hour dwells, 24-hour PDE collection or a 10-hour dwell. No Calnexin staining was seen. Ct-values for exogenous cel-miRNA39 and endogenous miRNA21 and -10b, obtained by qPCR, showed robust signals for all types of PDE (Fig. 2). No relevant differences were seen between samples from different dwell duration or with different glucose concentrations of the instilled PD-fluids.

Conclusion
We present a reproducible and clinically applicable method to isolate and molecularly characterize PDE-derived EVs. Further characterization of the molecular cargo of PDE-EVs may serve as a novel means to monitor peritoneal changes over time during PD and as a potential future biomarker for risk stratification in terms of systemic (cardiovascular) sequelae by PD-induced inflammatory responses. This would fill two important caveats in contemporary PD-management.