Interleukin-18 is a strong predictor of hospitalization in haemodialysis patients

Chih-Kang Chiang1,2,3, Shih-Ping Hsu1, Mei-Fen Pai1, Yu-Sen Peng1, Tai-I Ho1, Shing-Hwa Liu3, Kuan-Yu Hung1,2 and Tun-Jun Tsai2

1 Department of Internal Medicine, Far Eastern Memorial Hospital, 2 National Taiwan University Hospital, Taipei, Taiwan and 3 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan

Correspondence and offprint requests to: Dr Kuan-Yu Hung, Department of Internal Medicine, Far Eastern Memorial Hospital, No. 21, Sect. 2, Nan-Ya South Road, Pan Chiao, Taipei 220, Taiwan, ROC. Email: d820612{at}ha.mc.ntu.edu.tw



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Morbidity and mortality rates are high among patients with end-stage renal disease (ESRD), and recent evidence suggests that this may be linked to inflammation. Current research has also demonstrated the crucial involvement of interleukin-18 (IL-18) in inflammation. In agreement, the activity of IL-18 has been markedly up-regulated in ESRD patients. However, it has not been established whether elevated plasma IL-18 predicts outcome in haemodialysis (HD) patients.

Methods. To determine whether plasma IL-18 predicts overall hospitalization, we studied 184 ESRD patients (62% males, 58.5±1.0 years of age) undergoing maintenance HD treatment. The patients were followed for 12 months and were stratified by the tertiles of plasma IL-18 levels. Classic factors, such as age, body mass index, duration of HD, nutritional and inflammatory parameters, co-morbidity, dialysis adequacy, and lipid status were entered into a Cox regression model to predict hospitalization. The Kaplan–Meier method was used to analyse the cumulative proportion of hospitalization-free events.

Results. Significantly different hospitalization days and frequencies (P<0.05) were observed when patients were divided according to tertiles of plasma IL-18 levels. Patients were stratified according to IL-18 tertiles and analysed separately according to the hospitalization-free period. In the Kaplan–Meier model, the upper tertile of IL-18 had the highest probability of a hospitalization event during the entire follow-up period (P log rank = 0.027). In the Cox proportional hazard model, the relative risk for first hospital admission for each increase in Ln IL-18 (pg/ml) concentration was associated with a 1.709 (95% CI, 1.114 to 2.620; P = 0.014) increase in the risk for future hospitalization events.

Conclusions. The present study demonstrated a strong predictive value of elevated IL-18 levels for poor outcome in HD patients.

Keywords: hospitalization; haemodialysis; interleukin-18



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The annual morbidity and mortality rate in dialysis patients remains high despite marked improvements in dialysis technology and patient care. Dialysis patients show serological evidence of an activated inflammatory response [1,2], as clearly indicated by increased circulating levels of non-specific markers of inflammation and pro-inflammatory cytokines including interleukin-18 (IL-18) [3,4]. Originally identified as interferon-{gamma}-inducing factor in Kupffer's cells and macrophages, IL-18 plays a central role in the inflammatory cascade [5]. IL-18 levels have been positively correlated with C-reactive protein (CRP), which itself is a marker of the hepatic acute-phase response [6]. Consistent findings support a predictive role of CRP in the cardiovascular risk profile of healthy individuals and of patients with end-stage renal disease (ESRD) [2,6]. In dialysis patients, albumin is known as an important predictor of mortality [7]. The relationship between IL-18 and malnutrition has not been clarified in dialysis patients. Recently, elevated concentrations of IL-18 were shown to be a strong predictor of cardiovascular mortality in stable and unstable angina patients. This correlation was independent of prevalent vascular disease, smoking, and traditional risk factors, and was stronger than, but additive to, the predictive power of CRP [8].

Based on these observations, we aimed to evaluate whether baseline plasma levels of IL-18 can serve as a useful and relatively convenient screening device for risk of death and hospitalization, two crucial and untoward patient outcomes, in haemodialysis (HD) patients. Special attention was focused on the predictive power of IL-18 as it compares to traditional markers of co-morbidity.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
In September 2002, we recruited a group of 184 chronic stable and ambulatory adult HD patients from a total of 223 patients at the Far Eastern Memorial Hospital (FEMH), Taiwan. Excluded from this study were patients on dialysis for <3 months, and those with recent infection, cardiovascular events, hospitalization, surgery, or malignancy. We prospectively obtained the annual hospitalization indices as well as demographic and laboratory data of all 184 patients by recording the medical chart information after the FEMH Institutional Review Board had approved exemption from written consent. HD was given 4 h/day for three times a week in the majority of patients. HD was applied using single-use hollow-fibre dialysers equipped with modified cellulose-based membranes in more than 90% of these patients. The dialysate had a standard ionic composition that included a bicarbonate-based buffer in all cases. Although smoking may have considerable impact on inflammatory mediators, the prevalence of heavy smoking (20 or more cigarettes per day) was low (<3%) in our patients. Patient characteristics, including age, sex, biological and haematological data and primary renal aetiology are listed in Table 1.


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Table 1. Baseline characteristics of patients according to tertiles of IL-18a

 
Hospitalization
Hospitalization data from over a 12-month period were prospectively obtained on all 184 HD patients by recording all hospitalization events. Hospitalization was defined as any hospital admission that included at least one overnight stay. The admission day was counted as one full hospitalization day, but the discharge day was not. Therefore, the minimum hospitalization per admission was 1 day. Exclusion criteria included scheduled operations for problems such as hyperparathyroid patients receiving parathyroidectomy or access-related issues. For the 20 patients who died during the prospective 12-month follow-up, absolute hospitalization days and frequency of hospitalization were calculated. There were 103 hospital admission events in 75 patients. Hospitalization events included 33.3% cardiovascular events, 5.3% cerebral vascular events, 1.3% peripheral arterial occlusion disease, 33.3% infection-related events and 28% for other events, including seven events of gastrointestinal origin, five dialysis-related events, four newly-diagnosed malignancies, three events of respiratory origin and two unspecific haematuria events.

We applied three methods to assess the 12-month prospective hospitalizations in terms of clinical outcomes. Annual hospitalization days (H1), expressed as days per patient-year, was the sum of all hospitalization days of a given patient during that same period. Annual hospitalization frequency (H2), expressed as times per patient-year, was calculated from the total number of hospital admissions during the 12-month prospective period, regardless of the length of each admission. The number of days at risk from study onset to the first hospitalization event for each individual per week was assessed in a survival model (H3). Accordingly, risk time for each individual was defined as weeks from study entry until the first hospitalization, a censoring event or when a study anniversary day occurred.

Biochemical parameters and interleukin-18
Non-fasting blood samples were drawn from the arterial end of the vascular access just before starting HD. The samples were collected in Vacutainer tubes containing ethylenediamine tetra-acetic acid. They were then centrifuged at 2000 g for 5 min, aliquoted, and kept frozen at –80°C until assayed. Blood urea nitrogen, creatinine, albumin, total cholesterol, triglyceride and other biochemistry parameters were measured by standard laboratory techniques with an automatic analyser. The protein catabolic rate (PCR) in HD patients was calculated using validated equations [9] and normalized (nPCR) to actual body weight. Dialysis clearance of urea was expressed as weekly Kt/Vurea according to Daugirdas [10].

A single plasma IL-18 measurement was made at study onset and was assayed by a commercially available immunoenzymatic method (Colorimetric Sandwich ELISA; Boehringer Mannheim, Mannheim, Germany). The ELISA kit methods were followed according to manufacturer instructions. The detection limit for IL-18 was 37.8 pg/ml. The interassay and intraassay variation of IL-18 were 12.9 and 3.3%, respectively.

Statistical analysis
Results were expressed as means±SEM and were compared using one-way analysis of variance and the Bonferroni test. Since the cytokine data did not show a normal distribution, differences between groups were examined by nonparametric analysis with the Mann–Whitney U-tests. Hospitalization days and frequency were analysed by nonparametric analysis with the Kruskal–Wallis test. Correlation coefficients were calculated with Spearman rank analysis. Survival analyses were made with Kaplan–Meier and Cox regression analyses. For survival analysis, patients were divided according to tertiles based on the plasma levels of IL-18. Differences were considered significant at the P<0.05 level. All statistical calculations were performed with SPSS 10.0 for Windows.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Characteristics of the study population
A total of 184 adult HD patients (85 males and 99 females), with a mean duration of 54.8±3.9 months on HD, were enrolled for analysis. Patient characteristics, including age, sex, biological and haematological data are listed in Table 1. Patient ages ranged from 19.6 to 85.8 years (58.5±1.0 years). Primary renal disease was present in diabetes (DM) (39%), in chronic glomerulonephritis (33%) and in hypertension (14%). The median IL-18 level was 482.7 pg/ml (33.3th/66.7th levels, 383.2/601.3). Table 1 also provides patient characteristics according to tertiles of IL-18. From these, there were no differences in age, gender, percentage of diabetic patients, biochemical results, haematocrit, adequacy of HD and nPCR, although there were significantly higher levels of triglycerides, leucocyte and platelet counts, and CRP in the upper tertile.

Association between IL-18 with age, lipid values and prognostic factors in HD patients
IL-18 concentrations were positively and significantly correlated with leucocyte counts and CRP, which acted as acute inflammation reactants (Table 2). Plasma IL-18 levels were also positively correlated with cholesterol and triglyceride, which together are considered the major risk factors for cardiovascular disease. Plasma IL-18 did not correlate with age, a major predictor of morbidity and mortality in HD patients. There were no correlations between plasma IL-18 and nutritional parameters, such as albumin or nPCR in HD patients. IL-18 levels were negatively correlated with dialysis adequacy, such as Kt/Vurea (r =–0.162; P<0.05). Patients with diabetes had significantly higher IL-18 levels than non-diabetes patients (676.0±59.8 vs 516.5±37.6 pg/ml, P = 0.019).


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Table 2. IL-18 associations with age, lipid values and prognostic factors in HD patients

 
IL-18 concentrations and future hospitalization events
As shown in Figure 1, median plasma concentrations of IL-18 at baseline were significantly higher among patients who had experienced at least one hospitalization than those who had not (535.8 vs 429.5 pg/ml, P = 0.009); however, these levels were not higher in patients who had died (574.7 vs 479.1 pg/ml, P = 0.108). The results from the Kaplan–Meier survival analysis for overall admission rates are shown in Figure 2. We found that the highest hospitalization rates were observed among patients in the upper tertile of IL-18 levels (P log rank = 0.027). The Cox proportional hazard model was applied to the overall study population to adjust for event-free periods according to age, HD duration, body mass index (BMI), co-morbidity (DM, hypertension and hepatitis), CRP, lipid status, nutritional state (albumin level and nPCR) and dialysis clearance (Kt/Vurea). While age, Ln CRP, DM, albumin levels and Ln IL-18 were associated with admission in the univariate analysis, only age, albumin and Ln IL-18 were independently associated with hospital admission in multivariate analysis (Table 3). The relative risk (RR) for the first hospital admission for each pg/ml increase in the Ln IL-18 concentrations was 1.709 (95% CI, 1.114 to 2.620; P = 0.014). Because other pathophysiological events can lead to either cardiovascular or infectious causes of hospital admission, we performed a separate subgroup analysis. Cox regression analysis showed that plasma IL-18 concentrations acted as a predictor not only of future hospital admissions for cardiovascular causes (RR: 1.863, 95% CI, 1.045 to 3.319; P = 0.035) but also for infectious events (RR: 2.042, 95% CI, 1.085 to 3.844; P = 0.027). We also found in this cohort that a total of 19 (10.8%) patients died, which was caused by seven (36.8%) cardiovascular events, seven (36.8%) infection-related events and five (26.4%) events based on other aetiologies. However, plasma IL-18 levels did not predict future mortality in HD patients (data not shown), even though a higher trend of plasma IL-18 levels was observed.



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Fig. 1. Distribution (box plot representation) of IL-18 according to follow-up hospitalization, expressed by medians (429.5 vs 535.8 pg/ml, P = 0.009).

 


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Fig. 2. Kaplan–Meier curve for hospitalization according to tertiles of IL-18 (T1–T3). The log-rank test was computed for increasing tertiles, P = 0.027.

 

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Table 3. Independent predictors of hospitalization after 12 months follow-up using the Cox regression model with 184 HD patients

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In support of increasing experimental evidence implicating a role for inflammation and immune reaction in ESRD, a wide range of circulating markers of inflammation have been shown to predict overall morbidity and mortality in dialysis patients [11]. Because IL-18 is an important inflammatory marker, its role in the morbidity and mortality of ESRD is worthy of research. In this study, we demonstrated that elevated plasma IL-18 levels predicted outcomes in HD patients. The predictive power of IL-18 persisted even after adjusting for the effects of age, BMI, HD duration, co-morbidity (DM, hypertension and hepatitis), CRP, lipid status, nutritional state (albumin level and nPCR) and dialysis adequacy (Kt/Vurea). To the best of our knowledge, this is the first demonstration that IL-18 is an important predictor of patient outcome in an ESRD population.

There are three mechanisms that may explain how elevated IL-18 concentrations predict morbidity in ESRD patients. First, it has been shown that IL-18 mediates cardiovascular causes of death in patients having coronary artery disease, which is a major cause of death in HD patients [12]. Secondly, the finding that recombinant IL-18 injection leads to an increase in atherosclerotic lesions indicates an important role for IL-18 in the atherosclerotic process [13]. Moreover, increased IL-18 expression has been found during the fibrous plaque stage of the atherosclerotic process [14]. Although past findings demonstrating that baseline levels of plasma IL-18 predict patient survival for many months prior to adverse events may seem puzzling, such associations have also been found for CRP (a marker of acute-phase responses) in various groups of ESRD patients [1,2,7]. Similarly, we also found higher Ln CRP levels in patients who had been admitted (–0.714±0.13 vs –1.19±0.11, P = 0.005). As the third mechanism, IL-18 gene expression is stimulated by lipopolysaccharides. The effects of chronic exposure of endotoxin may be discussed as distant triggers of IL-18, which could lead to future hospitalization events in HD patients [15]. We found a significant correlation between the CRP and IL-18 in our patient population. The finding of higher IL-18 levels in HD patients having sub-clinical inflammation and the positive correlation between IL-18 levels and total cholesterol and triglyceride points to a highly probable link between inflammation, atherosclerotic status, and infection in ESRD. In our work, patients with DM, a predictor of poor outcome, had higher IL-18 levels than non-DM patients (676.0±59.8 vs 516.5±37.6 pg/ml, P = 0.019). DM patients also had a higher hospitalization rate as shown by univariate analysis. However, multivariate analysis yielded an independent role for IL-18 that overcame the effect of DM (Table 3). At present, only a single report has shown higher IL-18 levels in patients with type II diabetes [16]. Kumada et al. [17] identified serum IL-18 as a strong independent predictor of future cardiovascular events. In this prospective study of 1229 subjects, one in six patients was diabetic. The present data support these observations.

In recent work, malnutrition, inflammation, and atherosclerotic cardiovascular disease (MIA syndrome) have been linked to higher morbidity and mortality rates in HD patients [18]. As expected, the current study showed that serum albumin levels were also a strong prospective predictor of hospitalization (Table 3). However, there was no correlation between albumin levels and inflammatory parameters, such as IL-18 and CRP (Table 2). To further explore this relationship, we compared serum albumin values between hospitalized or non-hospitalized patients during the study period. We found that mean albumin levels were significantly lower in hospitalized (3.7±0.07 mg/dl) than in hospitalization-free (4.1±0.04 mg/dl) patients (P<0.05). A similar albumen difference was observed between patients who survived (4.0±0.03 mg/dl) and patients who died (3.4±0.14 mg/dl) during the study period (P<0.05). These findings suggest that serum albumin also exerts independent effects on survival of HD patients. Therefore, identification of possible mechanisms of malnutrition-related co-morbidity in HD patients is worthy of further investigation.

Markedly elevated IL-18 levels have been observed in ESRD patients [3,4]. Possible mechanisms explaining IL-18 elevations include impaired cytokine removal, increased synthesis due to various infectious processes, co-morbid conditions such as coronary heart disease [8], and increased body fat mass [19]. In our study, IL-18 was negatively correlated with individual Kt/Vurea (Table 2). This finding indicates that the levels of this cytokine are in some way related to dialysis efficacy. Further studies in patients on maintenance HD will be necessary to establish to what extent IL-18 production and excretion is influenced by the dialysis process. However, we also observed that Kt/Vurea per se did not act as an independent predictor of patient hospitalization (Table 3). It has been reported that dialysis adequacy, indicated by Kt/Vurea, is important for patient survival [20]. The lack of relationship between Kt/Vurea and patient hospitalization may have been due to the relatively better Kt/Vurea of our patients (1.54±0.02, Table 1) than suggested by DOQI recommendations.

There were several limitations in the present study. First, IL-18 values were obtained from one cross-sectional assay. Past studies have shown a fluctuating release of inflammatory markers during the HD process. Therefore, examining the time course of IL-18 changes before, during, and after HD may improve its predictive value. Secondly, we had no data on serum IL-18 values in the healthy general population. Thirdly, an already existing atherosclerotic cardiovascular disease may be a strong factor that influences inflammation, morbidity and mortality. Although we did not conduct a cross-sectional survey showing the burden of existing atherosclerotic lesions, the prevalence of ischaemic heart disease and cerebrovascular accidents were low (2.7 and 11.6%, respectively) in our patients. Fourthly, we did not examine IL-18 binding protein or other cytokines, which are implicated in Th-1 or Th-2 response and which improve the predictive power of a single IL-18 measurement. At present, the assessment of IL-18 is an expensive and time-consuming procedure. Before implementation of IL-18 measurement in clinical settings becomes feasible, it will be necessary to have standardized and reproducible assays, as well as results from consistent prospective studies, especially from the initially healthy population.

In conclusion, we demonstrated that elevated plasma IL-18 acts an independent predictor of the future hospital admissions in patients with HD. This finding highlights the importance of inflammation as an unfavourable prognostic factor in HD patients.



   Acknowledgments
 
The authors thank Ms Chyi-Wen Tzeng and Quei-Hua Huang for data collection. The authors thank Ms Chen-Chih Chang for technical support. This study was supported by Ta-Tung Kidney Foundation and Mrs Hsiu-Chin Lee Kidney Research Fund, Taipei.

Conflict of interest statement. None declared.

Footnotes

In this paper, the contributions of Shing-Hwa Liu and Kuan-Yu Hung were equal.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14.10.03
Accepted in revised form: 23. 4.04





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