Dataset Information

Proteome of hemodialysis filter membranes

ABSTRACT: Used hemodialysis filter membranes (HD filters) are discarded as waste products but represent a reservoir of valuable biological information. Numerous serum proteins are known to bind to HD filters, but whether this process selectively affects individual protein classes has not been adequately elucidated. Modern proteomics analyses offer the possibility to identify and quantify this therapy-specific subproteome and, in combination with bioinformatics methods, allow the analysis of the associated metabolic pathways. The description of the proteins at a HD filter membrane could provide insights into important modulators of the immune system or pathophysiological processes at the patient level. The aim of this project is to characterize the extracorporeal proteome of HD patients. HD filters were continuously rinsed with physiological saline immediately after the end of the HD session to remove most of the remaining blood from the capillaries. Then, a chaotropic buffer was circulated in the system for 1h by peristaltic pump to elute adsorbed proteins. Enzymatically digested proteins were desalted and purified, and separated in technical duplicate by liquid chromatography and analyzed by Orbitrap mass spectrometer, and identified bioinformatically.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Klaus Kratochwill

LAB HEAD: Dr Klaus Kratochwill

PROVIDER: PXD035836 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

In-Depth Analysis of the Extracorporeal Proteome Adsorbed to Dialysis Membranes during Hemodialysis.

Daniel-Fischer Lisa L Sobieszek Isabel J IJ Wagner Anja A Sacnun Juan Manuel JM Watschinger Bruno B Aufricht Christoph C Kratochwill Klaus K Herzog Rebecca R

Membranes 20221109 11

Used hemodialysis membranes (HD-M) are a valuable reservoir of biological information. Proteins bind to HD-M, but whether this process depends on the type of membrane or patient factors or selectively affects specific protein classes has not been adequately elucidated. State-of-the-art proteomics techniques are capable of identifying and quantifying this therapy-specific subproteome to enable the analysis of disease- or membrane-induced pathophysiologies. We demonstrate the feasibility of the de ...[more]

PMID: 36363675

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Project description:Several reports have focused on the identification of biological elements involved in the development of abnormal systemic biochemical alterations in chronic kidney disease, but this abundant literature results most of the time fragmented. To better define the cellular machinery associated to this condition, we employed an innovative high-throughput approach based on a whole transcriptomic analysis and classical biomolecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both chronic kidney disease patients in conservative treatment (CKD, n=9) and hemodialysis (HD, n=17) compared to healthy subjects (NORM) (p<0.001, FDR=1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system (OXPHOS). Western blotting for COXI and COXIV, key constituents of the complex IV of OXPHOS, performed on an independent testing-group (12 NORM, 10 CKD and 14 HD) confirmed the elevated synthesis of these subunits in CKD/HD patients. However, complex IV activity was significantly reduced in CKD/HD patients compared to NORM (p<0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to NORM. Taken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress. Experiment Overall Design: For microarray analysis, we studied subjects included in the training group. This population included 8 healthy subjects (NORM), 9 patients with Chronic kidney disease (CKD) on stage II-III (CKD II-III) (mean±SD of estimated GFR by MDRD formula: 41.4±4.3 ml/min) and 17 patients undergoing hemodialysis treatment (HD).All HD patients were stably treated, for at least 1 year, three times a week (4-5 hours per session), using synthetic membrane dialyzers. During the study period, no CKD patients received dialysis treatment. In addition, all patients suffering from infectious diseases, diabetes, chronic lung diseases, neoplasm, or inflammatory diseases and patients receiving antibiotics, corticosteroids, or nonsteroidal anti-inflammatory agents were excluded. No patients had symptomatic coronary artery diseases or a family history of premature cardiovascular diseases. For all subjects, 20 ml of whole blood were collected. For HD patient the biological material was obtained at the beginning of the second HD session of the week. PBMC were isolated by density separation over a Ficoll-PaqueTM (GE healthcare, Sweden) gradient (460g for 30 min). Cells were then counted and their viability was assessed by trypan blue exclusion (>90% PBMC were viable). Total RNA was isolated by RNeasy mini kit Qiagen (QIAGEN AG, Basel, Switzerland) from a minimum of 5 milion cryopreserved PBMC. RNA was, then, processed and hybridized to the GeneChip Human Genome Plus 2.0 or U133A oligonucleotide microarray (Affymetrix, Santa Clara, CA, USA)