Supplementary Material for: Exploring the Impact of Expanded Hemodialysis with Super High-Flux Dialyzer on Inflammation and Gene Expression: A Prospective Cohort Study in Prevalent Hemodialysis Patients

Introduction: End-stage kidney disease (ESKD) features chronic inflammation and immune dysregulation. The effects of long-term expanded hemodialysis (HDx) using a super high-flux (SHF) dialyzer on peripheral blood mononuclear cell (PBMC) transcriptomes and circulating inflammatory markers are unclear.
Methods: In a single-center pilot study, 10 prevalent hemodialysis patients were evaluated at baseline on standard high-flux hemodialysis (HF-HD) and after 12 months on high-efficiency HDx delivered with an SHF dialyzer (ELISIO-17Hx; Nipro, Osaka, Japan). PBMC RNA sequencing was performed by Macrogen (Seoul, South Korea). Serum cytokines/chemokines were quantified by bead-based multiplex immunoassay on the Luminex xMAP platform (MILLIPLEX; Merck, Darmstadt, Germany). Differential expression controlled the false-discovery rate; paired tests compared biomarker levels.
Results: Out of 43 genes analyzed, 12 showed significant upregulation. TNF and CCL4 genes were notably altered after 12 months of HDx (adjusted p=0.037 and p=0.025). Serum levels of pro-inflammatory markers—TNF-α, CCL4, CCL2, and MMP-9—significantly declined, while IL-10 increased (p<0.001). Specific changes included TNF-α (26.9 [23.7–30.9] to 31.5 [29.2–35.2] pg/mL; p=0.028), CCL4 (22.7±8.2 to 32.5±14.1 pg/mL; p=0.015), CCL2 (319.6±103.5 to 489.6±97.0 pg/mL; p<0.001), and MMP-9 (555.6 [202.0–709.3] to 5,241.5 [4,432.3–19,709.3] pg/mL; p=0.005). IL-10 increased from 2.80±1.86 to 5.15±2.10 pg/mL (p=0.001). TNF-β showed a non-significant change (mean difference -2.4; p=0.101). Other markers remained unchanged.
Conclusion: ESKD shows PBMC upregulation of TNF and CCL4, whereas 12-month high-efficiency HDx lowers circulating TNF-α, CCL4, CCL2, and MMP-9. This transcriptome–serum discordance highlights immune dysregulation, while HDx appears to attenuate inflammation; the shift toward anti-inflammatory signals suggests potential clinical benefit.