Multiple extracellular vesicle types in peritoneal dialysis effluent are prominent and contain known biomarkers

Abstract

Peritoneal dialysis inevitability results in activation of inflammatory processes and its efficiency is highly variable between patients. An improved method to isolate biomarkers and study pathophysiological mechanisms in peritoneal dialysis effluent (PDE) is expected to be of much benefit for the development of this treatment approach and help with patient management. Extracellular vesicles (EVs) are released as part of normal cellular processes. Their proteome is expected to reflect both type and health of their cell of origin. Although there is a significant interest in using EVs for “liquid biopsies”, little is reported of their presence or composition in plentiful dialysis waste fluids, including peritoneal dialysis effluent (PDE). Here we determined the presence of EVs in PDE and subsequently characterized their proteome. EVs were first isolated from PDE using differential centrifugation, then a further enrichment using size exclusion chromatography (SEC) was performed. The presence of EVs was demonstrated using transmission electron microscopy, and their particle counts were investigated using nanoparticle tracking analysis and dynamic light scattering. Using tandem mass spectrometry, marker proteins from three types of EVs i.e. apoptotic bodies, ectosomes, and exosomes were identified. The proteomic results demonstrated that the isolation of EVs by differential centrifugation helped enrich for over 2,000 proteins normally masked by abundant proteins in PDE such as albumin and SEC markedly further improved the isolation of low abundant proteins. Gene ontology analysis of all identified proteins showed the marked enrichment of exosome and membrane-associated proteins. Over 3,700 proteins were identified in total, including many proteins with known roles in peritoneal pathophysiology. This study demonstrated the prominence of EVs in PDE and their potential value as a source of biomarkers for peritoneal dialysis patients.

Proteomic Research in Peritoneal Dialysis

“…Later, Corciulo and co-workers [99] described aquaporin (AQP1)-containing exosomes in PDE as biomarkers of dialysis efficiency. AQP-1 had also been found in samples from Pearson's study [98]. On the basis of a study of 30 patients, the authors found that levels of AQP1 in PDE-EVs positively correlated with the efficiency of PD.…”

“…Only a few weeks later, Pearson et al published another study on the proteomics of EVs and PD [98]. In this case, samples were segregated in terms of EV purification, as some of them were enriched by ultracentrifugation (UC) and some others by UC plus an additional size exclusion chromatography enrichment.…”

“…As before, the results of gene ontology analysis confirmed the definition of an exosome enrichment component, and many of the proteins found in both studies were coincided. While both studies had the limitation of a small number of patients recruited (nine in [97] and eight patients in [98]), importantly both groups demonstrated that EVs could be isolated from peritoneal fluid, and that these EVs could be used as a source of biomarkers in a non-invasive fashion. Later, Corciulo and co-workers [99] described aquaporin (AQP1)-containing exosomes in PDE as biomarkers of dialysis efficiency.…”

Extracellular Vesicle Quantification and Characterization: Common Methods and Emerging Approaches

“…However, due to the large number of assumptions about the nature of the particle size distribution (e.g., mono- or multi-modal) that is necessary for appropriate fitting of the measured intensity correlation function, DLS is best suited for quantitative analysis of relatively monodisperse samples. In a biological and biomedical context, it is often applied for determining the size of isolated EVs and the synthetic variants (e.g., liposomes) [92,93,94,95]. However, because DLS detects all scattering objects in solution, it has limited utility in the analysis of minimally-processed biofluids.…”

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures

“…A literature search in NCBI Pubmed utilizing the query string (proteomics AND peritoneal dialysis) OR (proteome AND peritoneal dialysis) OR (proteomics AND peritoneal fluid) OR (proteome AND peritoneal fluid) OR (proteomics AND peritoneal effluent) OR (proteome AND peritoneal effluent) on Oct 26, 2017 resulted in 83 hits. These were restricted to clinical peritoneal dialysis effluent from humans, to return only 15 studies ( 7 – 19 , 38 , 39 ) of which two were excluded because they focused solely on the exosomal fraction ( 38 , 39 ) and one did not provide any protein accessions ( 19 ), resulting in 12 studies and 171 unique proteins for analysis ( supplemental Table S1 ) ( 7 – 18 ). Manual query of protein names presented by Zavvos et al ( 19 ) yielded 19 protein names not contained in the 171, of which 11 were unreviewed and 8 were reviewed entries in UniProt.…”