Increased eryptosis and inflammatory cytokines are associated with low hemoglobin levels in hemodialysis patients.

Renal anemia is prevalent at some point in time in most hemodialysis (HD) patients. In addition to absolute or relative erythropoietin deficiency, research suggests premature red blood cell (RBC) death (eryptosis) and inflammation are key pathogenetic factors. However, a comprehensive understanding of the interplay between eryptosis, hemoglobin (Hb) levels, and the concentration of inflammatory mediators is still missing. Our goal was to explore the relationship between eryptosis and cytokine levels with Hb levels in hemodialysis (HD) patients.

Blood samples were collected pre-dialysis in 19 chronic HD patients. The exposure of phosphatidylserine (PS) on the RBC membrane, an indicator of eryptosis, was quantified using the Annexin V-PE-conjugated and assessed by flow cytometry. Serum cytokine levels of IL-6, IL-1b, IFN-g, and TNF-a were measured using ELISA. Samples from seven healthy individuals were used as controls. We compared PS and cytokine levels between HD patients and controls using the unpaired Student's t-test. The correlation among PS expression, cytokine concentrations, and Hb levels in HD patients was assessed using Spearman's rank correlation test.

The patients’ mean age was 58.3 years; 63% were males. The controls’ mean age was 48.4 years, and 57% were males (Fig 1A). In HD patients, PS expression (mean±SD) was increased compared to controls (2.9±1.3 vs 8.8±3.1; p<0.0001; Fig 1B). Compared to controls, HD patients showed higher levels of IL-6 (35.6±8.8 vs. 192.4±86.2 pg/mL); IFN-g (89.4±19.6 vs. 243.3±117 pg/mL); IL-1β (468.6±76.6 vs. 732.6±150.7); and TNF-a (585.7±81.8 vs. 837.6±119.6) (all p<0.001) (Fig 1 C-F). Lower Hb levels, defined as 8 to 11 g/dL, correlated with increased PS exposure and high (Fig 1G-J, red dots) and median (green dots) cytokine concentrations. Lower Hb levels were also associated with increased PS exposure in samples with high (red dots) and median (green dots) cytokine concentrations (Fig 1G-J). However, patients with Hb levels above 11 g/dL were associated with both high and low PS expression in the presence of lower cytokine levels (blue dots) (Fig 1G-J)