Serum Hepcidin in Haemodialysis Patients: Associations with Iron Status and Microinflammation

Transcription

1 The Journal of International Medical Research 2011; 39: Serum Hepcidin in Haemodialysis Patients: Associations with Iron Status and Microinflammation Y XU, XQ DING, JZ ZOU, ZH LIU, SH JIANG AND YM CHEN Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai, China Hepcidin plays a key role in iron homeostasis. This cross-sectional study measured the serum hepcidin levels of 48 maintenance haemodialysis patients and 20 age-matched healthy control subjects using a competitive enzymelinked immunosorbent assay (C-ELISA). Serum hepcidin, interleukin (IL)-6 and high-sensitivity C-reactive protein levels were significantly higher in maintenance haemodialysis patients compared with control subjects. In all patients, there was a positive correlation between serum hepcidin levels and ferritin, transferrin saturation and IL-6, and an inverse correlation between serum hepcidin and unsaturated ironbinding capacity, total iron-binding capacity (TIBC) and transferrin. Linear regression analyses showed that ferritin and TIBC were independently associated with serum hepcidin levels. In conclusion, serum hepcidin levels are associated with iron status and microinflammation (defined as hscrp < 15 mg/l, without clinical manifestation of inflammation) in maintenance haemodialysis patients. The C-ELISA method for measuring serum hepcidin should facilitate the routine measurement of hepcidin in clinical practice. KEY WORDS: HAEMODIALYSIS; ANAEMIA; MICROINFLAMMATION; HEPCIDIN; IRON; HIGH-SENSITIVITY C-REACTIVE PROTEIN Introduction Anaemia is a common complication in maintenance haemodialysis patients and contributes to reduced quality of life. 1 Despite the great success of recombinant human erythropoietin (EPO) in clinical practice for the treatment of anaemia in dialysis patients (chronic kidney disease patients), resistance to this therapy is approximately 10 20%. 2 Iron deficiency is a frequent cause of EPO resistance. 3 An accurate assessment of iron status is important because both anaemia and overtreatment with erythropoiesisstimulating agents (ESA) are associated with poor clinical outcomes. 4 Defining an irondeficient state in maintenance haemodialysis patients is, however, more complex than in the general population and, to date, no reliable marker of iron status has been agreed. 5 Serum ferritin is a surrogate marker for tissue iron stores that continues to gain recognition. The interpretation of serum ferritin levels in isolation is difficult because ferritin also plays a role as an acute-phase reactant and 1961

2 is often elevated irrespective of tissue iron stores in maintenance haemodialysis patients. 6 Moreover, both analytical and intraindividual variability in serum ferritin limits its utility as a test for iron status in maintenance haemodialysis patients. 7 This has led to a search for more reliable markers of iron status in these patients, with serum hepcidin emerging as a promising candidate. 8,9 There are limited data on the concentration and regulation of serum hepcidin in maintenance haemodialysis patients, as well as the contribution of serum hepcidin to the pathogenesis of renal anaemia. This is because an easily applicable technique for measurement has not been available. Recently, a competitive enzyme-linked immunoassay (C-ELISA) for serum hepcidin has been developed which could enable widespread measurement of hepcidin in clinical practice. 10 The present cross-sectional study was conducted to determine serum hepcidin levels in maintenance haemodialysis patients using the C-ELISA method and to investigate associations between serum hepcidin level and markers of microinflammation (i.e. high sensitive C- reactive protein [hscrp] < 15 mg/l and no clinical manifestation of real inflammation) 11 and iron metabolism. Patients and methods STUDY POPULATION This cross-sectional, single-centre study enrolled maintenance haemodialysis patients and age-matched healthy control subjects in January All eligible patients, treated as outpatients in the Blood Purification Centre of Zhongshan Hospital, Fudan University, Shanghai, China, were included in the study. Healthy volunteers were recruited as controls from subjects attending Zhongshan Hospital, Fudan University for routine examination. Males and females aged > 18 years were eligible for inclusion in the study. Inclusion criteria for the maintenance haemodialysis patients were inception of maintenance haemodialysis 6 months (three times per week for 4 5 h per session) preceding the study and a baseline haemoglobin (Hb) level > 10 g/dl. Exclusion criteria were previous treatment with immunosuppressive drugs, clinical signs of acute infection, active inflammatory disease, liver disease, any malignancy, evidence of blood loss (gastrointestinal bleeding, trauma, etc.) and all patients unable or unwilling to participate in the study. Healthy controls had to have no clinical or laboratory evidence of any chronic disease. The study was approved by the Ethical Committee on Human Research of Zhongshan Hospital, Fudan University and was performed in accordance with the Declaration of Helsinki. All patients and control subjects gave written informed consent to participate in the study. STUDY DESIGN This was a cross-sectional, single-centre study conducted in maintenance haemodialysis patients and age-matched healthy control subjects. Maintenance haemodialysis patients were receiving regular haemodialysis treatment three times a week (4 5 h per session) using 500 ml/min of the standard bicarbonate dialysate used at Zhongshan Hospital, Fudan University (Na mmol/l, K mmol/l, Ca mmol/l, Mg mmol/l, Cl mmol/l, HCO mmol/l and glucose mmol/l), and synthetic low-flux polysulphone F6 membranes (Fresenius Kabi, Bad Homburg, Germany). 1962

3 SAMPLE COLLECTION AND LABORATORY MEASUREMENTS Peripheral blood samples were collected midweek from maintenance haemodialysis patients, after an overnight fast, immediately before and after a single session of haemodialysis. The samples were put into pyrogen-free tubes containing clot activator (without anticoagulant) and serum was harvested, aliquoted and stored at 70 C until analysis. For healthy control subjects, blood was also drawn from a peripheral vein after an overnight fast. Serum samples were prepared for the evaluation of hepcidin and interleukin (IL)-6. Serum hepcidin and IL-6 concentrations were measured using the commercially available human hepcidin C- ELISA kit and human IL-6 ELISA kit (Cusabio Biotech, Wuhan, China) according to the manufacturer s instructions. Serum levels of albumin, prealbumin, creatinine, iron, ferritin, transferrin and hscrp, and blood urea nitrogen, total ironbinding capacity (TIBC), unsaturated ironbinding capacity (UIBC), Hb and white blood cell count (WBC) were determined by automated procedures carried out at the Department of Clinical Chemistry, Zhongshan Hospital, Fudan University. Percentage transferrin saturation (TSAT) was calculated as the ratio of serum iron levels to TIBC. The ESA resistance index (ERI) was calculated as the ratio of the weekly EPO dose to Hb per unit weight. 12 The single-pool urea clearance index was used to represent weekly dialysis dose and was calculated as K t /V, where K is the clearance of urea by the dialyser, t is the dialysis time and V is the volume of distribution of urea. STATISTICAL ANALYSES Statistical analyses were carried out using the SPSS statistical package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows. Data are reported as mean ± SD for normally distributed (parametric) continuous variables or as median (interquartile range) for non-normally distributed (nonparametric) values. Comparisons between the maintenance haemodialysis patients and controls were assessed with Student s unpaired t-test, Mann Whitney U-test, Wilcoxon s signed-rank test or the χ 2 test, as appropriate. Spearman s rank correlation coefficient and linear regression were used to determine the relationship between hepcidin levels and clinical and laboratory parameters. A value of P < 0.05 was considered to be statistically significant. Results The study was conducted in 48 maintenance haemodialysis patients (27 males, 21 females; mean ± SD age 58.9 ± 14.3 years) and 20 age-matched healthy control subjects (11 males, nine female; mean ± SD age 56.5 ± 12.7 years). There was no significant difference in age or gender between the maintenance haemodialysis patients and healthy control subjects. Serum hepcidin, IL- 6 and hscrp levels were significantly higher in pre- and posthaemodialysis patients compared with healthy controls (P < 0.01, P < 0.05 and P < 0.01, respectively; Table 1). No significant difference was observed between pre- and post-haemodialysis patients. The clinical and laboratory characteristics of the maintenance haemodialysis patients and univariate analysis of their association with serum hepcidin level are shown in Table 2. There were significant positive correlations between serum levels of hepcidin and ferritin (r = 0.892, P < 0.001), TSAT (r = 0.289, P = 0.049) and IL-6 (r = 0.439, P = 0.002), and inverse correlations between serum levels of hepcidin and UIBC (r = 0.570, P < 0.001), TIBC (r = 0.552, P < 0.001) and transferrin 1963

4 TABLE 1: Levels of serum hepcidin, interleukin (IL)-6 and high sensitive C-reactive protein (hscrp) in maintenance haemodialysis patients (n = 48) and healthy control subjects (n = 20) Hepcidin (ng/ml) IL-6 (pg/ml) hscrp (mg/l) Control subjects 90.4 ( ) 43.9 ( ) 0.9 ( ) Prehaemodialysis ( )** 48.0 ( )* 3.6 ( )** Posthaemodialysis ( )** 58.7 ( )* 3.7 ( )** Data presented as median (interquartile range). *P < 0.05, **P < 0.01 versus controls; Mann Whitney U-test for prehaemodialysis and posthaemodialysis patients versus controls; Wilcoxon signed rank test for prehaemodialysis versus posthaemodialysis patients. (r = 0.532, P < 0.001). No correlations were observed between serum hepcidin and the various other clinical and laboratory parameters measured, including weekly EPO dose, ERI, Hb and hscrp (Table 2). Linear regression analysis showed serum ferritin and TIBC to be independently associated with serum hepcidin levels (Table 3). Discussion Anaemia in maintenance haemodialysis patients is a multifactorial condition and its clinical management remains challenging. The interactions between iron metabolism, EPO deficiency and chronic inflammation are difficult to dissect and new markers are urgently needed to optimize treatment approaches. 5 Hepcidin was originally described as an antimicrobial peptide produced by the liver, 9 but its main biological role is in the regulation of body iron homeostasis through interactions with ferroportin. 13,14 Hepcidin might, therefore, potentially be used as a clinical marker to optimize treatment approaches in several systemic iron disorders. It suppresses iron absorption from the intestine and stimulates iron retention in cells expressing ferroportin, notably macrophages and hepatocytes. 15 Hepcidin levels are also regulated in response to iron, erythropoietic demand, hypoxia and inflammatory signals. 14 Iron administration upregulates hepcidin, thereby providing a feedback mechanism to limit further iron absorption, whereas anaemia and hypoxia inhibit hepcidin, increasing iron availability for erythropoiesis. Hepcidin is also induced by inflammation, which is believed to be part of the host defence mechanism to fight infection and cancer by limiting iron availability. 16,17 5,18 20 In agreement with previous data, the present study demonstrated that serum hepcidin levels were higher in maintenance haemodialysis patients than in healthy control subjects, using a novel C-ELISA method. The observation that there was a significant and independent correlation between hepcidin and ferritin levels suggests that it plays a major role in regulating iron homeostasis in this patient group. 17 Hepcidin levels are likely to be higher in chronic kidney disease patients due to limited hepcidin excretion, tissue iron overload and inflammation. 16 Patients undergoing continuous dialysis are in a chronic inflammatory state. The effects of inflammation on the synthesis of hepcidin are well understood and are mediated, at least in part, by IL-6 through induction and binding of signal transducer and activator of transcription 3 (STAT 3) to the hepcidin gene promoter. 21 There was a significant positive correlation between serum IL-6 and serum hepcidin levels in the 1964

5 TABLE 2: Univariate analysis of the association between serum hepcidin level and relevant clinical and laboratory parameters in maintenance haemodialysis patients (n = 48) Maintenance Spearman s Clinical and laboratory haemodialysis correlation Statistical parameter patients coefficient (r) significance Age, years 58.9 ± NS Dialysis vintage, months 50 ( ) NS K t /V single pool 1.29 ( ) NS EPO dose, units/week ( ) NS ERI, units/kg per week per g Hb 1.25 ( ) NS Serum albumin, g/l 40.3 ± NS Prealbumin, g/l 0.36 ± NS SCr, µmol/l ± NS BUN, mmol/l 27.3 ± NS UIBC, µmol/l 28 (24 35) P < TIBC, µmol/l 40 (36 46) P < Serum iron, µmol/l 11.4 ( ) NS Transferrin, g/l 2.02 ( ) P < Ferritin, ng/ml ( ) P < TSAT, % 27.5 ( ) P = Hb, g/l ± NS WBC, 10 9 /l 6.4 ± NS hscrp, mg/l 3.6 ( ) NS IL-6, pg/ml 48.0 ( ) P = Data presented as mean ± SD or median (interquartile range). Dialysis vintage, length of time on dialysis in months to years; NS, not statistically significant (P > 0.05); K t /V, single pool urea clearance index (used to represent weekly dialysis dose, where K is the clearance of urea by the dialyser, t is the dialysis time, and V is the volume of distribution of urea); EPO, erythropoietin; ERI, ESA (erythropoiesis-stimulating agent) resistance index; SCr, serum creatinine; BUN, blood urea nitrogen; UIBC, unsaturated iron-binding capacity; TIBC, total iron-binding capacity; TSAT, transferrin saturation; Hb, haemoglobin; WBC, white blood cells; hscrp, high sensitive C-reactive protein; IL-6, interleukin-6. present study, which would seem to support this proposed mechanism. In contrast, no relationship was observed between hepcidin and hscrp levels, or between hepcidin levels and EPO (ESA) dose, which is consistent with previous studies. 12 Several factors may explain this lack of relationship, including differences in the half-lives of hscrp and hepcidin. 22,23 Another explanation may be the distinct features of the study population, TABLE 3: Linear regression analysis showing the clinical and laboratory parameters with a significant independent association with serum hepcidin level in maintenance haemodialysis patients Unstandardized Standardized Statistical Independent variable b-coefficient b-coefficient significance Ferritin P < TIBC P = TIBC, total iron-binding capacity. 1965

6 i.e. stable maintenance haemodialysis patients with little or no dialysis-related inflammation, stable Hb levels and receiving ESAs at baseline. 20 Previous studies have shown that comparison between hepcidin and hscrp may serve as a quick and easy method for identifying the difference between iron deficiency, microinflammation or mixed anaemia. 23 In such a diagnostic scheme, low hscrp and low hepcidin would indicate iron deficiency, high hscrp and high hepcidin would indicate microinflammation, while high hscrp and low hepcidin would indicate a mixture of microinflammation and iron deficiency. 23 Zaritsky et al. 18 reported that hepcidin could be cleared efficiently by haemodialysis as it is a very small molecule. The findings of the present study were unexpected as no significant difference was observed in serum hepcidin levels in a single haemodialysis session. This was consistent with a study by Ashby et al. 24 that showed no reduction following a standard dialysis session. The cause of this variability remains unclear, but might be attributable to differences in the membrane of the dialyser, residual renal function or induction of hepcidin by the haemodialysis procedure. 25 There were several limitations to the present study. First, it was limited by the cross-sectional design, and baseline laboratory abnormalities may have been confounded by the patients current treatment, resulting in difficulty to distinguish a cause and effect relationship. Secondly, the study was limited by the sample size and, finally, as only a single determination of hepcidin was made, any variation that may have occurred over time cannot be taken into account. In conclusion, serum hepcidin levels were associated with iron status and microinflammation in maintenance haemodialysis patients. Availability of the C- ELISA assay for serum hepcidin will facilitate the routine measurement of hepcidin in clinical practice. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 12 May 2011 Accepted subject to revision 23 May 2011 Revised accepted 26 August 2011 Copyright 2011 Field House Publishing LLP References 1 Eleftheriadis T, Liakopoulos V, Antoniadi G, et al: The role of hepcidin in iron homeostasis and anemia in hemodialysis patients. Semin Dial 2009; 22: KDOQI, National Kidney Foundation: KDOQI clinical practice guidelines and clinical practice recommendations for anaemia in chronic kidney disease. Am J Kidney Dis 2006; 47(5 suppl 3): S11 S Lankhorst CE, Wish JB: Anaemia in renal disease: diagnosis and management. Blood Rev 2010; 24: Ford BA, Eby CS, Scott MG, et al: Intraindividual variability in serum hepcidin precludes its use as a marker of iron status in haemodialysis patients. Kidney Int 2010; 78: Weiss G, Theurl I, Eder S, et al: Serum hepcidin concentration in chronic haemodialysis patients: associations and effects of dialysis, iron and erythropoietin therapy. Eur J Clin Invest 2009; 39: Hasuike Y, Nonoguchi H, Tokuyama M, et al: Serum ferritin predicts prognosis in haemodialysis patients: the Nishinomiya study. Clin Exp Nephrol 2010; 14: Ford BA, Coyne DW, Eby CS, et al: Variability of ferritin measurements in chronic kidney disease; implications for iron management. Kidney Int 2009; 75: Hamada Y, Fukagawa M: Is hepcidin the star player in iron metabolism in chronic kidney disease. Kidney Int 2009; 75: Park CH, Valore EU, Waring AJ, et al: Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001; 276: Ganz T, Olbina G, Girelli D, et al: Immunoassay 1966

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