Elastase release during the hemodialysis procedure seems to induce changes in red blood cell membrane proteins

Abstract

To the Editor: Elastase is a serine proteinase, expressed mainly by neutrophils, and also by monocytes and mast cells. It is a component of the primary azurophil granules of the neutrophils. Within the cell, it is involved in the degradation of microorganisms that are phagocytosed, and, outside the cell, it may degrade local extracellular matrix proteins, favoring neutrophil migration into or through tissues. 1,2 Recently, we showed that the red blood cell (RBC) membrane protein composition is altered in hemodialysis (HD) patients, particularly in nonresponders to recombinant human erythropoietin (rhEPO) therapy. 3,4 We also showed that the HD procedure induces neutrophil activation. 5,6 Therefore, we hypothesized that the RBC changes could be due to the release of elastase by neutrophils during HD. Our aim was to study the effect of elastase in RBC membrane protein composition from healthy controls and from HD patients under rhEPO therapy (responders and non-responders) to strengthen this hypothesis. We performed in vitro assays using RBC from 18 HD patients (10 responders and 8 nonresponders) and of 8 healthy controls. Classification of the patients, as responders or non-responders, was performed in accordance with the European Best Practice Guidelines, which defines resistance to rhEPO as a failure to achieve target Hb levels (between 11 and 12g/dL) with maintained doses of rhEPO higher than 300IU/kg/wk of epoietin or 1.5mg/kg/wk of darbopoietin-a. The two groups of patients were matched for age, gender, weight, body mass index, mean time under HD, urea reduction ratio, urea Ktv, and parathyroid hormone serum levels. Hemodialysis patients (10 males, 8 females; mean age 62.1 15.7 years) were under therapeutic HD 3 times per week, for 3‐5 hours, for a median period of time of 35 months. All patients used the highflux polysulfone FX-class dialysers of Fresenius (Fresenius Medical Care, Bad Homburg, Germany). The causes of renal failure in patient’s population were as follows: diabetic nephropathy (n=5), chronic glomerulonephritis (n=3), polycystic kidney disease (n=2), hypertensive nephrosclerosis (n=2), obstructive nephropathy (n=1), and chronic renal failure of uncertain etiology (n=5). Patients with autoimmune disease, malignancy, hematological disorders, and acute or chronic infection were excluded. All patients gave their informed consent to participate in this study. In HD patients, this assay used RBCs collected before and immediately after the HD procedure. Red blood cell suspensions (5 10 8 cell/mL; 10mL PBS, pH 7.4) were incubated for 3 hours at 371C, under the following assay conditions: without elastase and with 0.03, 0.1, and 0.5mg/mL of elastase. After incubation, the relative amount of each major RBC membrane protein was evaluated by densitometry. 3 We found no significant differences in the membrane protein composition of the RBCs from healthy controls and from responders and nonresponders after the HD procedure, when incubated without and with different elastase concentrations. However, the RBCs from responders and nonresponders HD patients, collected before the HD procedure, showed some susceptibility to elastase; the RBCs from responders incubated with 0.5mg/mL of elastase showed a significant decrease in ankyrin [7.0 (6.5‐7.5%) vs. 6.0 (5.9‐6.5%), P=0.024] and trends toward a decrease in spectrin [25.6 (25.1‐ 26.9%) vs. 24.7 (24.4‐25.6%), P=0.073) and an increase in band 3 [36.6 (34.8‐37.6%) vs. 39.1 (36.9‐39.4%), P=0.077) as compared with RBCs incubated without elastase. Similar changes were found for the RBCs incubated with 0.1mg/mL of elastase. In nonresponder patients, the RBCs incubated with 0.1 and 0.5mg/mL of elastase showed a significant decrease in spectrin [25.5 (24.9‐25.9%) and 25.3 (24.8‐26.2%), respectively, vs.