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Major determinants of BMP-2 serum levels in hemodialysis patients
Manuscript ID:
Date Submitted by the Author:
LRNF-2012-LE-0278 Letter to the Editor 26-Jun-2012
Costa, Elísio; University Catholic, Institute of Health Sciences Coimbra, Joana; ICS, Catarino, Cristina; FF-UP, Ribeiro, Sandra; FF-UP, Reis, Flávio; FM-UC, Nascimento, Henrique; FF-UP, Fernandes, João; FF-UP, Miranda, Vasco; Fresenius Medical Center, Dinefro – Diálises e Nefrologia, SA., Sameiro-Faria, Maria; Fresenius Medical Center, Dinefro – Diálises e Nefrologia, SA., Belo, Luis; FF-UP, Santos-Silva, Alice; FF-UP,
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Complete List of Authors:
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Manuscript Type:
ee
Journal:
On
Keywords:
BMP-2, hemodialysis, Anemia, erythropoietin, vascular access
ly URL: http://mc.manuscriptcentral.com/lrnf Email:
[email protected]
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Major determinants of BMP-2 serum levels in hemodialysis patients
Running title: BMP-2 serum levels in HD patients
Elísio Costa (1,2), Joana Coimbra (1), Cristina Catarino (2,3), Sandra Ribeiro (2,3), Flávio Reis (4), Henrique Nascimento (2,3), João Fernandes (3,4), Vasco Miranda (5),
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Maria do Sameiro Faria (5), Luís Belo (2,3), Alice Santos-Silva (2,3)
1 - Instituto de Ciências da Saúde da Universidade Católica Portuguesa; 2-Instituto de
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Biologia Molecular e Celular (IBMC), Universidade do Porto; 3- Faculdade Farmácia,
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Serviço de Bioquímica, Universidade do Porto; 4-Instituto de Farmacologia e Terapêutica Experimental, IBILI, Faculdade de Medicina da Universidade de Coimbra;
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5- Fresenius Medical Center, Dinefro – Diálises e Nefrologia, SA.
Correspondence to: Elísio Costa
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Instituto de Ciências da Saúde Universidade Católica Portuguesa
4200-072 Porto
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Rua Dr. António Bernardino de Almeida
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Portugal Email:
[email protected] or
[email protected]
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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily, and more than 20 different isoforms have been identified in mammals and drosophila [1]. BMP-2, one of that isoforms, is a multifunctional regulator of cell growth and differentiation, and appears to play an important role in nephropathogenesis, by inhibiting epithelial growth factor-induced biological effects, such as DNA synthesis in kidney glomerular mesangial cells, cell proliferation of collecting tubule cells and by
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stimulating apoptosis. Moreover, it was reported that abnormal preurine calcium-oxalate triggers the expression of BMP-2 in long Henle´s loop cells, inducing differentiation of these cells towards the osteogenic lineage [2].
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The 25-amino acid peptide hepcidin has emerged as a central regulator of iron homeostasis, being up-regulated by pro-inflammatory cytokines, such as interleukin
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(IL)-1α, IL1-β, and IL-6, and by high levels of iron; it is down-regulated by hypoxia and anemia [3]. Recently, it was reported that BMP-2, BMP-4 and BMP-9 are also involved in hepcidin expression [4].
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As hemodialysis (HD) patients present high serum levels of hepcidin and inflammatory markers, as well as, a disturbed iron metabolism [5], we wondered how BMP-2 and all these factors interact in HD patients.
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We evaluated in CKD patients under HD and recombinant human erythropoietin (rhEPO) therapies, the serum levels of BMP-2, hematological data, inflammatory
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markers, iron metabolism; the values of rhEPO doses used to treat patients and the type of vascular access used for HD procedure were also considered in data analysis. Forty nine patients (28 males, 21 females; 67.4 ± 15.0 years old) were enrolled in the study. A group of twenty four healthy volunteers presenting normal haematological and biochemical values, without history of kidney or inflammatory diseases, matched as far as possible for age and gender with patients, was also studied.
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Patients were under therapeutic HD three times per week, for 3 to 5 h, for a median period of time of 28.3 months. All patients used the high-flux polysulfone FX-class dialysers of Fresenius. Fourteen patients used central venous catheter (CVC) and 35 arterio-venous fistula (AVF), as vascular access for HD. The causes of renal failure in these
patients were as follows: diabetic nephropathy (n=17),
hypertensive
nephrosclerosis (n=5), obstructive nephropathy (n=2), amyloidosis (n=1), chronic
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nephritis syndrome (n=3), tubular nephropathy (n=1), chronic glomerulonephritis (n=2), renal myeloma (n=1), tuberculosis (n=1) and uncertain aetiology (n=16). Patients with autoimmune disease, malignancy, haematological disorders, and acute or chronic infection, were excluded. All participants gave their informed consent to participate in this study.
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The HD patients included 34 responders [0.33 (0.12-0.55) µg darbepoietin /Kg/week] and 15 non-responders [1.98 (1.76-2.34) µg darbepoietin/Kg/week] to rhEPO therapy,
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in accordance with the European Best Practice Guidelines [6]. Blood sample collection was performed immediately before starting HD procedure.
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Red blood cell (RBC) count, haematocrit, haemoglobin (Hb) concentration,
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haematological indices and red cell distribution width (RDW) were measured by using
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an automatic blood cell counter (Sysmex K1000; Sysmex, Hamburg, Germany). Reticulocyte count was measured by microscopic counting on blood smears after vital
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staining with New methylene blue (reticulocyte stain; Sigma, St Louis, MO, USA). The reticulocyte production index (RPI) was calculated to measure the effective reticulocyte production. Serum iron concentration was determined using a colorimetric method (Iron, Randox Laboratories Ltd., North Ireland, UK), whereas serum ferritin and serum transferrin were measured by immunoturbidimetry (Ferritin, Laboratories Ltd., North Ireland, UK; Transferrin, Laboratories Ltd., North Ireland, UK). Transferrin saturation
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(TS) was calculated by the formula: TS (%) = 70.9 x serum iron concentration (µg/dL) / serum transferrin concentration (mg/dL). Enzyme-linked immunosorbent assays were used for measurement of plasma soluble transferrin receptors (s-TfR) (Human sTfR immunoassay, R&D systems, MN, USA),
IL-6 (human IL-6, Bender MedSystems,
Vienna, Austria) and BMP-2 (BMP-2 human, R & D Systems, Minnesota, EUA). Serum C-reactive protein (CRP) was determined by nephelometry (N high sensitivity
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CRP, Dade Behring). enzymatic
Serum hepcidin concentration was performed by using an
immunoassay
(Hepcidin-25,
EIA
Kit
Extraction-Free,
Peninsula
Laboratories, LLC, San Carlos, USA). For statistical analysis, the Statistical Package for Social Sciences, version 17.0 was
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used. For comparisons between groups, we used the Students t-test whenever the
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parameters presented a Gaussian distribution and the Mann-Whitney U-test in the case of a non-Gaussian distribution. Spearman or Pearson ranks correlations coefficients
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were used to evaluate relationships between sets of data. Multiple regression analysis using the stepwise method was used to determine independent factors affecting BMP-2
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serum concentration. Significance was accepted at p
