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Dissecting Inflammation in ESRD: Do Cytokines and C-Reactive Protein Have a Complementary Prognostic Value for Mortality in Dialysis Patients?

Carmine Zoccali^*,, Giovanni Tripepi and Francesca Mallamaci^*,

^* Nephrology, Hypertension & Renal Transplantation Unit, Ospedali Riuniti, Reggio Calabria, and CNR-IBIM Clinical Epidemiology of Renal Diseases and Hypertension, Calabria, Italy

Address correspondence to: Prof. Carmine Zoccali, Nephrology, Hypertension & Renal Transplantation Unit & CNR-IBIM, Clinical Epidemiology of Renal Diseases and Hypertension, c/o Ki Point Gransial SRL, Via Filippini n. 85, 89100 Reggio Calabria, Italy. Phone: +39-0965-397010; Fax: +39-0965-26879; E-mail: carmine.zoccali{at}tin.it

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Because atherogenesis represents a type of chronic inflammation that involves multiple elements of the inflammatory-immune response, the simultaneous prediction power for death of C-reactive protein (CRP) and proinflammatory cytokines (IL-1, IL-6, IL-18, and TNF-) was tested in a cohort of 217 patients with ESRD. During the follow-up period (average 41 mo), 112 patients died. In an analysis that was adjusted for other risk factors, the relative risks for death of patients who were exposed to high levels of one, two, three, and four or more inflammation biomarkers were 1.48, 1.64, 2.76, and 3.05 times higher, respectively, than that of patients in the reference category (no inflammation). In this model, the explained variation in mortality that was attributable to overall inflammation burden (+9.1%) was marginally higher (P = 0.06) than that provided by IL-6 alone (+6.1%). In an alternative analysis based on the Bayesian approach (receiver operating characteristic curves analysis), the prediction power of the combined inflammatory burden was identical to that provided by the sole IL-6 (0.59 ± 0.04 versus 0.59 ± 0.04). IL-6 captures almost entirely the prediction power of the overall inflammation burden in patients with ESRD. IL-6 seems to be an almost ideal indicator of the severity of inflammation. The use of this biomarker can be recommended in clinical studies that aim to better the understanding of inflammation or to modify it in this population.

	Introduction

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Substantial evidence has been accrued that inflammation is a major factor in the high mortality of ESRD, and a variety of experimental and epidemiologic studies coherently indicate that cytokines and other inflammatory proteins are not only conducive to cardiovascular damage in experimental models but also predictive of cardiovascular events in patients with ESRD. We recently reported that IL-6, a key player in the acute-phase response, is the strongest predictor of mortality among inflammation markers and that C-reactive protein (CRP) seems to be the most suitable marker to grade the inflammation status in clinical practice on cost-effectiveness grounds (1). Whereas the predictive power of individual inflammatory proteins now is well established, the important question of whether the overall inflammation burden that is estimated by the combined measurement of these molecules provides additional prognostic information in comparison with the measurement of individual cytokines and acute-phase reactants has not been studied. The issue is relevant because the major cytokines—IL-1, IL-6, IL-18, TNF-, and CRP, the pentraxin that is synthesized by the liver under the stimulus of these cytokines—have pleiotropic and redundant actions at least in part reflecting complementary aspects of the inflammatory-immune process (2,3).

In the present study, we therefore tested the prediction power for all-cause mortality of overall inflammation burden as estimated by the measurement of plasma concentration of CRP and of IL-1, IL-6, IL-18, and TNF- in a cohort of patients with ESRD. The results show that IL-6 captures almost entirely the prognostic power for death of the inflammatory process in patients with ESRD.

	Materials and Methods

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Protocol
The protocol conformed to the ethical guidelines of our institution, and informed consent was obtained from each participant. For hemodialysis (HD) patients, all studies were performed during a midweek nondialysis day; for continuous ambulatory peritoneal dialysis (CAPD) patients at empty abdomen, studies were performed between 8:00 a.m. and 1:00 p.m.

Study Cohort
A total of 217 patients with ESRD (120 men and 97 women) who had been on regular dialysis treatment for at least 6 mo (median duration 43 mo; interquartile range 20 to 101 mo) without history of congestive heart failure and without intercurrent inflammatory illnesses were considered eligible for the study. The main demographic and clinical characteristics of the study population are detailed in Table 1. The prevalence of diabetes in this cohort was 15% (33 of 217 patients).

Follow-Up
After the initial assessment, patients were followed up for an average of 41 mo (range 0.8 to 70.0 mo). During the follow-up, fatal cardiovascular events (myocardial infarction, electrocardiogram-documented arrhythmia, heart failure, stroke, and other thrombotic events except for arteriovenous fistula thromboses) and death were recorded accurately. Each death was reviewed and assigned an underlying cause by a panel of five physicians. As a part of the review process, all available medical information about each death was collected. This information always included study and hospitalization records. In the case of an out-of-hospital death, family members were interviewed by telephone for better ascertainment of the circumstances surrounding death.

Laboratory Measurements
Blood sampling was performed after an overnight fast between 8:00 a.m. and 10:00 a.m. always during a midweek nondialysis day for HD patients and at empty abdomen for CAPD patients. After 20 to 30 min of quiet resting in semirecumbent position, samples were taken into chilled EDTA Vacutainers, placed immediately on ice, and centrifuged within 30 min at –4°C, and the plasma was stored at –80°C before assay. Serum lipids, albumin, calcium, phosphate, and hemoglobin measurements were made using standard methods in the routine clinical laboratory. The plasma concentrations of asymmetric dimethylarginine and homocysteine were determined as reported elsewhere (4,5). Serum CRP was measured by using a commercially available kit (immunonephelometric method, lower limit of detection 3.5 mg/L; Behring, Scoppito, L’Aquila, Italy). Serum levels of IL-6, IL-1, IL-18, and TNF- were measured by ELISA with the use of Quantikine High Sensitivity kits (intra-assay coefficient of variation for these substances 1.6 to 10%; interassay coefficient of variation 3.3 to 10.2%; R&D Systems, Minneapolis, MN).

Statistical Analyses
Data are reported as mean ± SD (normally distributed data), median, and interquartile range (nonnormally distributed data) or as percentage frequency, and comparisons among groups were made by a P for trend. The combined prognostic power of inflammatory proteins (inflammatory burden) for all-cause mortality was analyzed by a score that was calculated as the sum of five biomarkers (CRP, IL-1, IL-6, IL-18, and TNF-), each defined in categorical terms (1 = third tertile of the corresponding data distribution; 0 = others two tertiles). We used the third tertile of each biomarker because in a previous article, this functional form of variable provided adequate risk stratification in the same dialysis cohort (1). We constructed six separate Cox models on the basis of either the inflammation score or inflammation biomarkers considered individually. These models included all covariates that were associated to all-cause death with P < 0.10 at univariate Cox regression analysis as well as variables that resulted to be associated with the inflammatory score with P < 0.10 at univariate analysis (Table 1). The treatment modality (HD/CAPD) was always introduced into these Cox models. The Cox models then were compared by using the –2 log likelihood test (6). Furthermore, we performed an alternative analysis by the Bayesian approach (i.e., by comparing the areas under the receiver operating characteristic [ROC] curves of the combined inflammatory load with that of IL-6 in the prediction of death [7]).

Hazard ratios and their 95% confidence intervals were calculated with the use of the estimated regression coefficients and their standard errors in the Cox regression analysis. All calculations were made using a standard statistical package (SPSS for Windows Version 9.0.1; SPSS, Chicago, IL).

	Results

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The cutoff values of serum CRP, IL-1, IL-6, IL-18, and TNF- that identified the third tertile of the relative data distribution were 12.9 mg/L, 0.87 pg/ml, 8.5 pg/ml, 676 pg/ml, and 9.0 pg/ml, respectively. In the aggregate, 187 (86%) of 217 patients had at least one biomarker within the corresponding third tertile (one biomarker in 74 cases; two biomarkers in 65 cases; three biomarkers in 32 cases, and four or more biomarkers in 16 cases), whereas a minority of patients had all cytokines within the first two tertiles of the relative data distribution (n = 30; 14%). As shown in Table 1, the number of altered (third tertile) inflammatory biomarkers was related directly to age and inversely to serum albumin and tended to be inversely associated with serum cholesterol (P = 0.07) and homocysteine (P = 0.10). As expected, the average plasma concentration of each biomarker rose in a stepwise manner from the reference group (no inflammation) to the group that showed four or more biomarkers in the corresponding third tertile.

Overall Inflammatory Burden and Mortality: –2 Log-Likelihood Statistics
During the follow-up period, 112 patients died, 65 of them (58% of total deaths) of cardiovascular causes. The independent prognostic value of overall inflammation burden for all-cause mortality was analyzed in a multiple Cox regression analysis that adjusted for a series of traditional and nontraditional risk factors. In this analysis, the hazard ratios of all-cause mortality increased in a dose-response manner according to the number of altered (third tertile) biomarkers so that the relative risks of patients who were exposed to one, two, three, and four or more increased levels of these inflammatory proteins were 1.48, 1.64, 2.76, and 3.05 times higher, respectively, than in those in the reference category (Table 2). However, the explained variation in mortality that was attributable to overall inflammatory burden was marginally (P = 0.06) higher than that provided by IL-6 (9.1 versus 6.1%; P = 0.06), which by far resulted to be the biomarker that showed the highest (P < 0.01) prognostic power for death (CRP +3.0%; IL-18 +2.9%; IL-1 +0.5%; and TNF- +0.5%).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
An inflammation score that was composed of CRP, IL-6, IL-1, IL-18, and TNF- predicts death no better than the sole IL-6 in patients with ESRD. This finding indicates that IL-6 captures almost entirely the high risk for inflammation in ESRD and further highlights the importance of high levels of this cytokine in patients who are on long-term dialysis.

Overlapping and Complementary Role of Inflammatory Proteins and Atherosclerosis
Mainly because of the intellectual drive by Ross (3), atherosclerosis now is seen as an inflammatory disease, and substantial evidence has been accrued that the inflammation is a major contributor to arterial disease in patients with ESRD (1,8). Proinflammatory and anti-inflammatory cytokines are crucial elements in such process because the balance and the interplay of these proteins control the direction, amplitude, and duration of inflammation as well as tissue remodeling. Cytokines and acute-phase response proteins intervene at various levels in the atherosclerosis process. Thus, CRP seems to be implicated in endothelial dysfunction, foam cell formation, and inhibition of progenitor cell survival and differentiation (2). IL-6, a ubiquitous cytokine that is fundamental for leukocyte and endothelial cell activation, is highly expressed at the shoulder of the atherosclerotic plaque, where it may induce plaque instability by driving the local synthesis of matrix metalloproteinases, monocyte chemotactic protein-1, and TNF- (9). Furthermore, IL-6 stimulates fibrinogen synthesis in the liver via IL-6–responsive sequences in the promoter region of the fibrinogen gene, thereby enhancing the risk for thrombosis (10). IL-1 (11) and IL-18 (12) play a relevant role in the inflammatory component of atherosclerosis in experimental models. TNF-, besides being involved in plaque instability, is implicated in myocardial dysfunction and remodeling after an acute ischemic insult (13). The disparate interferences of cytokines and CRP with atherothrombosis suggest that the combined measurement of these biomarkers may provide prognostic information that is superior to what can be derived by individual biomarkers.

Individual and Combined Prediction Power of Inflammation Biomarkers in ESRD
In patients who have ESRD without clinically apparent inflammatory complications, independent of exposure to dialysis membranes and/or contaminated dialysis fluid, plasma levels of IL-6, IL-1, and TNF- and of CRP are raised substantially (8). The detrimental effect of inflammation in ESRD seems particularly prominent in the cardiovascular system because high CRP and high cytokine levels signal a high risk for mortality and incident cardiovascular complications (1,14–19). We previously showed that the plasma concentrations of IL-6, CRP, and other cytokines are mutually interrelated in dialysis patients without overt inflammatory processes (1). The hypothesis that the overall inflammation burden that is estimated by the combined measurement of cytokines and CRP has not been examined so far in patients with ESRD. To address this problem, we constructed a composite score based on the categorization of each inflammation marker into tertiles. We did so because this categorization allowed adequate risk stratification in our study cohort (1). Somewhat unexpected, this analysis showed that the combined use of these biomarkers provides very small, if any, gain in prediction power for death in comparison with that provided by the sole IL-6. The "inflammation score" was expected to capture the prognostic power of nonoverlapping or partially overlapping pathogenetic pathways that lead to arterial injury better than IL-6 or other individual cytokines. In other words, we hypothesized that this score reflects several critical aspects of the offending potential of inflammation, from endothelial dysfunction and plaque formation to plaque instability and atherothrombosis. Contrary to our expectations, the sole measurement of IL-6 was almost as informative as the composite inflammation score. IL-6 perhaps is the most investigated cytokine in human diseases (20). This protein has a very marked pleiotropy, being involved not only in inflammation but also in the regulation of several fundamental organ functions. Apart from the action on multipotent hematopoietic cell progenitors and on B cells or from its stimulatory role for the synthesis of other cytokines and acute-phase reactants in the liver, IL-6 affects myocardial cell growth, stimulates thermogenesis, suppresses the thyroid axis, and induces growth hormone secretion (20). This large variety of effects makes IL-6 unique among proinflammatory cytokines and indicates that it represents not only a potential agent of organ damage in pathophysiologic conditions but also a potent marker of the overall severity of the inflammation process. Our observation that IL-6 captures almost entirely the predictive power of inflammation in ESRD is in keeping with findings in the general population, in which IL-6 emerged as the sole inflammation marker to predict progression of atherosclerosis in an analysis that considers also CRP, adhesion molecules, and E-selectin (21). The marked pleiotropy of IL-6 makes it the best inflammation marker in ESRD, its prediction power being almost equal to what can be derived from simultaneous measurements of several other cytokines and CRP.

	Conclusion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
IL-6 captures almost entirely the prediction power of the overall inflammation burden in patients with ESRD. IL-6 seems to be an almost ideal indicator of the severity of inflammation. The use of this biomarker can be recommended in clinical studies that aim to better our understanding of inflammation or to modify it in this population.

	References

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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