Decoy Receptor 3, a Novel Inflammatory Marker, and Mortality in Hemodialysis Patients

Study Design

This prospective cohort study was carried out at four dialysis centers in the Taipei metropolitan area. Study participants were recruited from November 1 to December 31, 2004. Initially, all patients (n=402) undergoing hemodialysis were screened, and 350 patients aged >20 years who had been on hemodialysis for >6 months were included. Exclusion criteria were dialysis for <12 h/wk (n=4); inadequacy of dialysis defined as Kt/V urea <1.2 (n=5); and conditions of malignancy (n=3), infectious disease, or sepsis (n=5), or hepatobiliary disease (n=17). Finally, 316 clinically stable patients (150 men and 166 women; mean age of 59 years) were followed up until June 30, 2009. All of the patients were subjected to a standard bicarbonate dialysis session. Hemodialysis was performed three times weekly using single-use dialyzers with a membrane surface area of 1.6−1.7 m². The median duration of hemodialysis before study entry was 71 months (interquartile range, 34−120). The study protocol was approved by the institutional review board of each affiliated hospital. Informed consent was obtained from all participants, and our study complies with the Declaration of Helsinki.

In all patients, a thorough medical history was taken at the time of study enrollment. Presence of CVD was defined as a medical history and clinical findings of congestive heart failure and coronary artery, cerebrovascular, and/or peripheral vascular disease. No major modifications were made in dialysis treatments or schedules during the follow-up period. The primary outcome measures were cardiovascular and all-cause mortality from the time of inclusion in the study. Cardiovascular mortality included fatal myocardial infarction, stroke, congestive heart failure, arrhythmia, complicated peripheral vascular disease, and sudden death. The overall mortality category comprised death due to cardiovascular events, infection, sepsis, malignancy, gastrointestinal bleeding, chronic obstructive lung disease, or cachexia. Each medical chart was reviewed and a trained physician who was blinded to the DcR3 levels independently assigned the cause of death on the basis of all of the available clinical information.

Laboratory Measurements

All blood samples were drawn from patients who had fasted overnight at the start of a mid-week dialysis session, and heparin was then administered. Plasma and serum were separated and kept frozen at –70°C when not analyzed immediately. Serum levels of DcR3 (Biovendor, Modrice, Czech Republic), ICAM-1 and VCAM-1 (BioSource, Camarillo, CA), and IL-6 (R&D Systems, Minneapolis, MN) were measured using commercially available ELISA kits according to the manufacturer’s instructions. Intra-assay and interassay coefficients of variance for DcR3 were 4.8% and 4.3% at 0.50 ng/ml and 1.5% and 1.6% at 3.50 ng/ml, respectively. Intra-assay and inter-assay coefficients of variance for other concentrations were <5%. Albumin, urea, creatinine, calcium, phosphate, iron, and total iron-binding capacity in serum were determined using commercial kits by a Hitachi 7600 autoanalyzer (Roche Modular; Hitachi Ltd, Tokyo, Japan). Transferrin saturation was calculated as the serum iron concentration/total iron-binding capacity × 100. Serum ferritin and intact parathyroid hormone levels were determined with a RIA (Incstar, Stillwater, MN). Serum high-sensitivity C-reactive protein (hs-CRP) levels were measured using an immunoturbidimetric assay and rate nephelometry (IMMAGE; Beckman Coulter, Galway, Ireland). The adequacy of dialysis was estimated by measuring mid-week urea clearance (Kt/V urea) using the standard method (13). BP was measured and recorded by an automated sphygmomanometer. Predialysis BP was measured in the nonaccess arm after a 5-minute rest while the patient was seated with both feet on the floor before placement of a dialysis needle.

Statistical Analyses

All variables were expressed as percentages for categorical data and as means ± SD or medians and interquartile ranges for continuous data with or without a normal distribution, respectively. Potential differences among the three patient groups for each baseline serum DcR3 tertile were assessed by ANOVA, the Kruskal–Wallis test, or the chi-squared test, as appropriate. A Pearson correlation analysis was used to assess the associations of DcR3 with malnutrition-inflammation parameters. Multivariate regression analyses were used to assess independent predictors of serum DcR3 levels. To assess the predictive accuracy of the DcR3 for mortality, we used the time-dependent receiver operating characteristic (ROC) curve for censored data and the area under the ROC curve (AUC) as the criterion (14). An AUC of 0.5 indicates no predictive ability, whereas a value of 1 represents perfect predictive ability. Using the C statistic with stepwise addition of VCAM-1, albumin, IL-6, and DcR3 to traditional cardiovascular risk factors, the incremental change in AUC was assessed for mortality prediction at 48 months of the study.

The Kaplan–Meier method was used to describe survival curves. During the follow-up, 14 patients received a kidney transplant, 4 were shifted to peritoneal dialysis, and 33 were transferred to other dialysis units. Censoring occurred at the time of kidney transplantation, peritoneal dialysis, and withdrawal from the study, or on June 30, 2009. Cox proportional hazards regression analysis was used to examine the association of baseline variables with cardiovascular and all-cause mortality. The univariate and multivariate Cox regression analyses are presented as hazard ratios (HRs) and 95% confidence intervals (95% CIs). Adjustments for age and sex were initially performed to calculate adjusted HRs. The multivariate regression analysis was further adjusted for age and sex, and covariates served as potential confounders of the association between DcR3 concentration and cardiovascular or all-cause death. The potential confounding factors were smoking status, diabetes, prior CVD, body mass index, total cholesterol, systolic BP, dialysis vintage, urea Kt/V, hemoglobin, serum albumin, IL-6, and VCAM-1. Furthermore, the significance of linear trends across the DcR3 tertiles was tested by assigning each patient the median of the tertile and modeling this value as a continuous variable. The Akaike Information Criterion (AIC) (15) was used for calculating the prediction gain by DcR3 when this receptor was added to a multivariate Cox regression model adjusted for traditional cardiovascular risk factors. The prediction gain was then calculated by adding VCAM-1, albumin, hs-CRP, or IL-6 into the same regression model, respectively. P<0.05 was considered statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences (version 16.0; SPSS Inc, Chicago, IL).