Exploratory study of dialysis membrane impact on epigenetic methylation patterns in hemodialysis patients using genomic and synchrotron investigations.

Hemodialysis (HD) patients suffer from chronic inflammation which is a serious complication and contributes significantly to morbidity and mortality. Dialysis technology has advanced over many years but despite advances, its underlying molecular mechanism remains poorly understood. We have designed an exploratory study and investigated how hemodialysis influences epigenetic changes by understanding the DNA methylation patterns which are associated with inflammation. By using the same polysulfone (PS) membrane dialyzer, we have collected the blood samples from the patients before and after dialysis. For DNA methylation analysis, we have extracted genomic DNA using the Monarch® Genomic DNA Purification Kit. Bisulfite conversion was done using the QIAGEN Epitect Bisulfite Kit. Genome Studio, which is a computational software, was used to identify the DNA methylation profiles. These profiles revealed significant epigenetic changes across the hemodialysis patients’ samples. From these DNA methylation profiles, we identified the differential methylated regions (DMRs) and focused on the genes that are associated with these DMRs with the change greater than 80% to confirm robustness. We identified the chromosomal mapping of DMR-associated genes on CpG islands and shore localization, promotor or enhancer regions, to evaluate the functional impact of methylation changes. One of the bioinformatics tools, named Discovery Annotation, Visualization, Integration Database (DAVID) was used to perform functional enrichment analysis to identify the affected biological pathways involved in neuronal signaling, immune regulation, inflammation, and metabolism. Protein- Protein Interaction network was constructed using another bioinformatics tool, named String Database, and It analysis was performed using a bioinformatics desktop-based tool called Cytoscape, which helped in identifying critical hub genes including TWIST1, SHANK3, FGF20, MEF2C, RUNX2, CAV1, CDKN2A, WNT3A, and CXCL12, which play crucial roles in immune system compromise and inflammatory regulation. A total of 93 genes, across the samples of HD patients, showed significant methylation alterations. Of which, 41 genes showed changes exceeding 100% and mostly mapped on Promoter/enhancer regions at CpG islands and shores. In HD patients, these functional consequences suggest changes in their role in impaired vascular function, cognitive decline and immune dysregulation. Fibrinogen (FB) behaviour during dialysis, particularly its adsorption onto the dialysis membranes, was understood using Synchrotron Imaging at the Canadian Light Source (CLS) which provided us a high-resolution visualization. To confirm significant FB fouling between initial and intermediate membrane layers, these targeted regions were identified using raw CT scan images and their magnified views. Similarly, we identified that the level of inflammatory biomarkers, which were elevated in post-dialysis samples of HD patients including vWF, CRP, Serpin C1, Properdin, and PF4 indicating an amplified inflammatory response. Particularly, the critical hub genes that are associated with vascular regulation including CAV1, TWIST1, CDKN2A, and CXCL1, were reported as hypermethylated after dialysis, meaning their expression may reduce which affects their contribution in endothelial dysfunction. These findings suggest a self-perpetuating inflammatory loop, where FB adsorption initiates molecular and immunological responses that reinforce epigenetic dysregulation. One of the important limiting factors is sample size, which influenced the reliability of findings across the molecular and clinical levels, and this underlines that further investigation is needed. This study helped us designed the foundational insights to target the anti-inflammatory strategies that may lead us to the development of more hemocompatible dialysis membranes to reduce long-term complications in HD patients.