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Background High-density lipoprotein (HDL) is a heterogeneous group of particles with anti-atherogenic properties whose metabolism is alterated in chronic kidney disease (CKD). The aim of this study was to evaluate the particle size and mobility of HDL subpopulations in non-dialysis CKD patients. Methods The study involved 42 non-dialysis CKD patients (stages 3a–4) and 18 control subjects. HDL was separated by non-denaturing two-dimensional polyacrylamide gradient gel electrophoresis (2D-PAGGE) and eight HDL subpopulations; preβ1, preβ2a-c, and α1-4 were distinguished. The size and electrophoretic mobility of HDL subpopulation particles were compared between the groups, and a regression analysis was conducted. Results In CKD patients, the mean sizes of α-HDL and preβ2-HDL particles were significantly lower compared to the control group (8.42 ± 0.32 nm vs. 8.64 ± 0.26 nm, p = 0.014; 11.45 ± 0.51 vs. 12.34 ± 0.78 nm, p = 0.003, respectively). The electrophoretic mobility of preβ2-HDL relative to α-HDL was significantly higher in CKD patients compared to the control group (Rf 0.65 ± 0.06 vs. 0.53 ± 0.10, p = 0.002). The size and mobility of HDL subpopulations correlated with eGFR values (p Discussion CKD affects the size and mobility of HDL particles, which can be related to HDL dysfunction. The magnitude of HDL size and mobility changes depended on CKD stage and differed for individual HDL subpopulations, which indicates that some stages of HDL metabolism may be more affected by the presence of chronic kidney disease. Graphical abstract |