45era-edta 377-SP Anaemia 1 Elisio Costa ERYTHROCYTE MEMBRANE PROTEIN COMPOSITION IN CHRONIC KIDNEY DISEASE PATIENTS UNDER HAEMODIALYSIS AND RECOMBINANT HUMAN ERYTHROPOIETIN THERAPY Elísio Costa (1,2,3), Susana Rocha (1,2), Petronila Rocha-Pereira (4), Elisabeth Castro (1,2), Vasco Miranda (5), Maria do Sameiro Faria (5), Alfredo Loureiro (6), Alexandre Quintanilha (2,7), Luís Belo (1,2), Alice Santos-Silva (1,2) (1) Faculdade de Farmácia, Serviço de Bioquímica, Universidade do Porto; (2) Instituto de Biologia Molecular e Celular, Universidade do Porto; (3) Escola Superior de Saúde, Instituto Politécnico de Bragança; (4) Universidade da Beira Interior, Covilhã; (5) FMC, Dinefro – Diálises e Nefrologia, SA. (6) Uninefro – Sociedade Prestadora de Cuidados Médicos e de Diálise, SA. (7) Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto. Portugal. Introduction Anaemia, common in chronic kidney disease (CKD), contributes markedly to morbidity of CKD patients. A deficient renal erythropoietin secretion is probably the most important cause of the anaemia, though its multifactor origin. A decreased erythrocyte survival due to changes in its membrane proteins may be one of the other factors that could contribute to the development of the anaemia (1-5). Changes in red blood cell (RBC) membrane protein composition may account for changes in the deformability of the cell, compromising its circulation in the microvasculature and its survival (6,7). Aims Our aim was to evaluate the erythrocyte membrane protein profile (% of the major RBC membrane proteins) in CKD patients under haemodialysis and recombinant human erythropoietin (rhEPO) therapies. Materials and Methods Subjects and samples We studied 54 individuals, including 63 CKD patients (32 responders and 31 non-responders to rhEPO therapy) and 18 healthy controls. Peripheral blood samples were collected into EDTA containing tubes. The causes of renal failure in patients were as follows: diabetic nephropathy (n=19), chronic glomerulonephritis (n=11), polycystic kidney disease (n=3), hypertensive nephrosclerosis (n=3), obstructive nephropathy (n=3), pyelonephritis associated with neurogenic bladder (n=1), nephrolithiasis (n=1), chronic interstitial nephritis (n=1), Alport syndrome (n=1), renal vascular disease due to polyarteritis (n=1) and chronic renal failure of uncertain aetiology (n=19). diabetic nephropathy (n=9), hypertensive nephrosclerosis (n=5), chronic glomerulonephritis (n=4), polycystic kidney disease (n=2), obstructive nephropathy (n=2), pyelonephritis associated with neurogenic bladder (n=1), nephrolithiasis (n=1), chronic interstitial nephritis (n=1), Alport syndrome (n=1), renal vascular disease due to polyarteritis (n=1) and chronic renal failure aetiology uncertain (n=9). Patients with autoimmune disease, malignancy, haematological disorders, and acute or chronic infection were excluded. All patients gave their informed consent to participate in this study. Classification of CKD patients in responders or non-responders, was performed in accordance with the European Best Practice Guidelines (5), that defines resistance to rhEPO as a failure to achieve target haemoglobin levels with doses of rhEPO more than 300 IU/Kg/week or 1.5 mg/Kg/week of darbopoietin-alfa. CKD patients were under regular haemodialysis for 12h/week, for a median period of time of 65 months, ranging from 4 to 324 months. All patients used the high-flux polysulfone FX-class dialysers of Fresenius. Age and gender-matched individuals, with normal haematological and biochemical values, without any history of renal or inflammatory disease, were used as controls. Erythrocyte studies RBC count, haematocrit, haemoglobin concentration (Hb), haematimetric indices [mean cell volume (MCV), mean cell Hb (MCV) and mean cell Hb concentration (MCHC)] and red cell distribution width (RDW) were measured using an automatic blood cell counter (Sysmex K1000; sysmex, Hamburg, Germany). Reticulocyte count was made by microscopic counting on blood smears after vital staining with new methylene blue (reticulocyte stain; sigma, St Louis, MO, USA). Erythrocyte membrane protein profile RBCs were isolated and membranes prepared as described elsewhere (8). The electrophoretic analysis of the red cell membrane proteins was carried out on a discontinuous system of polyacrylamide in the presence of sodium dodecylsulfate (SDS-PAGE), using a 5–15% linear acrylamide gradient gel and a 3.5–17% exponential acrylamide gradient gel, according to the Laemmli and Fairbanks methods, respectively (8,9). The proteins were stained with Coomassie brilliant blue, and scanned (Darkroom CN UV/wl, BioCaptMW version 99; Vilber Lourmat, Marne-La-Vallée, France). The relative amount of each major RBC membrane protein (spectrin, ankyrin, bands 3, 4.1, 4.2, 5, 6 and 7 proteins), was quantified by densitometry (Bio1D++ version 99; Vilber Lourmat, France). The electrophoretic analysis for each RBC membrane sample was performed in duplicate gels, and, in each gel, duplicates of each sample were loaded. Data analysis Measurements are presented as mean  SD or as median values (inter-quartile range). For statistical analysis, we used the Statistical Package for Social Sciences, version 14.0. Kolmogorov Smirnov statistics were used to evaluate sample normality distribution. Multiple comparisons between groups were performed by one-way ANOVA supplemented with Turkey’s HSD post-hoc test. For data presenting a non-Gaussian distribution, we used Kuskl-Walls test and Mann-Whitney U test. Significance was accepted at p less than 0.05. Bibliography 1. Collins AJ, et al. Am J Kidney Dis 1998;32:S133-S142. 2. Macdougall IC. Nephrol Dial Transpl 2001;16(suppl 5):50-55. 3. Foley RN, et al. Am J Kidney Dis 1998;32(Suppl 3):S112-S119. 4. Foley RN, et al. Am J Kidney Dis 1996;28:53-61. 5. Smrzova J, et al. Nephrol Dial Transpant 2005;20(Suppl 8):viii2-viii7. 6. Martos MR, et al. Clinica Clinica Acta 1997;265:235-246. 7. Maduell F, et al. Nephol Dial Transplant 1990;5: 1018-22. 8. Laemmli UK. Nature 1970;227:680-685. 9.Fairbanks G, et al. Biochemistry 1971;10:2606-2616. Discussion CKD patients showed a slightly regenerative anaemia (decreased RBC, Hb and hematocrit, with an increase reticulocyte count), with high RDW values. When compared the haematological data between the two groups of patients, we showed that non-responders patients are more anaemic, with RBC more hypocromic (MCH and MCHC lower) and with an increased anisocitosis (higher RDW). A statistically significant decrease for spectrin was found in CKD patients when compared with controls. Spectrin is the major protein of the RBC cytoskeleton, and, therefore, the major responsible for RBC shape, integrity and deformability. It links the cytoskeleton to the lipid bilayer, by protein interactions with band 3 and glicophorin-C. The deficiency in spectrin observed in total CKD patients may, therefore, account for a poor linkage of the cytoskeleton to the membrane, favoring membrane vesiculation, and, probably, a reduction in the RBC lifespan of CKD patients (6,7). When comparing the erythrocyte membrane protein profile presented by responder and non-responder CKD patients, we found a statistically significant decrease for ankyrin in responder patients. This decreased ankyrin contents in responders could be related to the lower reticulocyte count found in these patients. Actually, in non-responders presenting the higher reticulocyte count, the ankyrin deficiency may have been obscured. A lower median value for spectrin in non-responder patients was also found, although it was not statistically significant (p=0.138). This trend led us to think that, at least in part, the decreased haemoglobin found in non-responders may be associated to an enhanced RBC destruction/production. Alterations in erythrocyte membrane proteins have been previously described in literature, namely a reduction in both spectrin and band 3 protein, and an isolated reduction in band 3 (6,7), which have been associated with the type of dialysis membranes used. This is the first description of erythrocyte membrane protein analysis using the high-flux polysulfone FX-class dialysers of Fresenius. Table II – Erythrocyte membrane protein profile for the studied groups. Results a) p<0.05, vs controls; b) p<0.001, vs controls; c) p<0.05, vs responders. Table I – Haematological data for the studied groups. Fig. 1 – Illustration of RBC membrane protein profile in linear and exponential gradient SDS-PAGE gels. a) p<0.05, vs controls; b) p<0.001, vs controls; c) p<0.05, vs responders. Fig. 2 – Spectrin values for controls and responders and non- responders to rhEPO therapy patients. Acknowledgments We are very grateful to FMC, Dinefro – Diálises e Nefrologia, SA and Uninefro – Sociedade Prestadora de Cuidados Médicos e de Diálise, SA, and to their nurses for the technical support. This study was supported by a PhD grant (SFRH/BD/27688/2006) attributed to E. Costa by FCT and FSE. Controls (n=26) CKD stage 5 Patients (n=63) rhEPO Responders (n=32) rhEPO Non-responders (n=31) Hb (g/dL) 14.12  1.27 11.08  1.67 b) 11.77  1.41 b) 10.37  1.63 b)c) Haematocrit (%) 43.50  4.28 33.90  4.76 b) 35.51  3.92 b) 32.23  0.50 b)c) RBC (x10 12 /L) 4.72  0.59 3.68 0.54 b) 3.76 0.42 b) 3.58 0.64 b) MCV (fl) 92.00 (90.00-94.00) 93.80 (90.00-98.20) a) 95.80 (92.48-98.08) a) 92.30 (85.40-100.30) MCH (pg) 29.83  1.39 30.15 3.04 31.29 1.53 b) 28.97 3.73 c) MCHC (g/dl) 32.47  0.58 32.03 2.37 33.16 1.77 30.85 2.35 a)c) RDW (%) 12.79  0.52 15.92 2.56 b) 14.56 1.23 b) 17.32 2.83 b)c) Reticulocytes (x10 9 /L) 33.57  22.78 61.03  31.36 b) 55.12  30.98 a) 67.14  31.06 b) RPI 0.42 (0.19-0.66) 0.98 (0.58-1.40) b) 1.08 (0.72-1.51) b) 0.92 (0.52-1.24) a) Controls (n=26) CKD stage 5 Patients (n=63) rhEPO Responders (n=32) rhEPO Non-responders (n=31) Spectrin (%) 27.63 (26.41-28.79) 24.27 (19.39-26.13) b) 24.75 (22.38-26.63) a) 22.35 (18.95-25.92) b) Ankyrin (%) 6.971.62 6.53 1.90 6.092.07 6.971.60 c) Band 3 (%) 38.57  3.99 39.294.03 39.924.03 38.653.70 Protein 4.1 (%) 7.561.45 7.24 1.49 7.181.33 7.31 1.63 Protein 4.2 (%) 5.510.72 5.44 1.44 5.541.57 5.351.29 Band 5 (%) 6.820.86 6.87 1.03 6.701.02 7.041.00 Band 6 (%) 5.191.04 6.981.37 b) 6.611.30 a) 7.371.32 b) Band 7 (%) 2.200.65 3.32 1.24 b) 3.160.98 a) 3.491.43 b) Protein 4.1/Spectrin 0.276 0.624 0.330 0.120 a) 0.310 0.105 0.340 0.130 a) Protein 4.1/Band 3 0.192 (0.154–0.227) 0.183 (0.155-0.208) 0.183 (0.154-0.208) 0.183 (0.159-0.205) Protein 4.2/Band 3 0.149 (0.125-0.162) 0.138 (0.110-0.163) 0.135 (0.110-0.169) 0.142 (0.110-0.161) Spectrin/Band 3 0.707 (0.649-0.822) 0.569 (0.512 -0.686) b) 0.572 (0.541-0.685) b) 0.544 (0.486 -0.687) b) Ankyrin/Band 3 0.185 0.585 0.169 0.057 0.155 0.060 0.183 0.052 c) Spectrin/Ankirin 4.18 1.07 3.77 1.84 4.44 2.25 3.10 0.94 b)c)