Immune landscape of PBMC based on single-cell rna sequancing in End Stage Renal Disease

Although an increase in studies has revealed the potential mechanisms underlying immune dysfunction during CKD progression, there are still many significant components that need to be clarified, such as the machinery of T-cell phenotype switching and the plasticity levels of different subsets. Fortunately, in the last decade, novel technologies such as single-cell RNA sequencing have enabled us to investigate transcriptomic profiles at the single-cell level and have enabled us to unravel the potential mechanisms of the heterogeneity of the cell phenotype underlying different diseases. In this study, we investigated the immune cell composition, function, and interaction with other cells in PBMCs (peripheral blood mononuclear cells) during the ESRD period, especially CD4+ T-cell plasticity and the involved signaling pathways and mediators, which could be potential therapeutic targets for preventing CKD progression and complications. In this case‒control study, 14 male and 6 female ESRD patients who underwent standard hemodialysis were enrolled. Twenty healthy volunteers who were sex- and age-matched to ESRD patients comprised the healthy control group. All the participants were involved between June 1, 2022, and September 1, 2022. ESRD patients were required to meet the following inclusion/exclusion criteria. Inclusion criteria: ESRD patients receiving standard hemodialysis (HD) with arteriovenous fistulas (AVFs) for 5-10 years; the cause of kidney damage was primary kidney disease or diabetic nephropathy; and the discontinuation of corticosteroid and immunosuppression therapy for at least 1 year. Exclusion criteria: infection, tumor (including parenchymal tumor and hematological system tumor), autoimmune disease, acute infection, and other diseases; vaccination for COVID-19, influenza, and other viruses in the past 3 months; and an unwillingness or inability to cooperate due to individual reasons. Ficoll-Paque density gradient centrifugation was applied for PBMC isolation. Dead cells were removed using a Miltenyi ® Dead Cell Removal Kit (MACS, 130-090-101). Cell suspensions were resuspended in 1× PBS buffer with 0.04% BSA and centrifuged at 300 × g for 3 min at 4 °C (repeated twice). The cell pellet was resuspended in 50 μl of 1× PBS buffer with 0.04% BSA. The overall cell viability was confirmed by trypan blue exclusion. Cell viability above 85% was required for scRNA sequencing using a Countess II Automated Cell Counter. The PBMCs were diluted to a concentration of 1*106 cells/mL with sorting buffer. The 20 cellular suspensions from patients and controls were separately pooled with an equal volume.