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Serum Carboxymethyllysine Predicts Mortality in Hemodialysis Patients
Serum Carboxymethyllysine Predicts Mortality in Hemodialysis Patients Zoltán Wagner, MD, PhD, Márta Molnár, MD, Gergo˝ A. Molnár, MD, Mónika Tamaskó, MD, Boglárka Laczy, MD, László Wagner, MD, PhD, Botond Csiky, MD, PhD, August Heidland, MD, DHc, Judit Nagy, MD, DSc, and István Wittmann, MD, PhD ● Background: Hemodialysis patients show markedly elevated serum levels of advanced glycation end products (AGEs). AGEs have been implicated in the pathogenesis of vascular damage and are regarded as a class of uremic toxins. However, to date, serum AGE level could not be identified as an independent predictor of mortality. The aim of the present study is to test whether serum level of the AGE carboxymethyllysine (CML) predicts all-cause or cardiovascular mortality in hemodialysis patients. Methods: Serum total CML concentration was measured by means of enzyme-linked immunosorbent assay in 154 patients receiving long-term hemodialysis. Patients were divided into groups with serum CML levels less and greater than the median (23.8 ng/mg protein). All-cause and cardiovascular mortality were registered during a follow-up of 51 months. The relationship between serum CML level and mortality was tested by using Kaplan-Meier and Cox regression analyses. Results: In the group with low serum CML levels, 38% of patients died during the follow-up period; 23% had a cardiovascular cause of death. However, in the group with high CML levels, 58% died (P < 0.01) and 36% had a cardiovascular cause of death (P < 0.05). The following parameters proved to be independent risk factors of all-cause mortality: age (hazard ratio, 1.056; P < 0.001), preexisting vascular disease (hazard ratio, 2.53; P < 0.05), smoking (hazard ratio, 3.03; P < 0.005), high serum CML level (hazard ratio, 1.776; P < 0.05), and C-reactive protein level (hazard ratio, 1.017; P < 0.001). Conclusion: The AGE CML may contribute to increased mortality in patients with uremia. Am J Kidney Dis 47: 294-300. © 2005 by the National Kidney Foundation, Inc. INDEX WORDS: Advanced glycation end products; carboxymethyllysine; hemodialysis (HD); mortality; uremia.
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ARKEDLY ELEVATED serum levels of advanced glycation end products (AGEs) have been shown in patients with end-stage renal
From the Second Department of Medicine and Nephrological Center, University of Pécs; Fresenius Medical Care, Dialysis Center Pécs, Pécs; Fresenius Medical Care, Dialysis Center Szigetvár, Szigetvár, Hungary; and Department of Internal Medicine, University of Würzburg, Würzburg, Germany. Received August 16, 2005; accepted in revised form October 5, 2005. Originally published online as doi:10.1053/j.ajkd.2005.10.010 on December 27, 2005. Support: This work was supported by the Hungarian national grants: ETT 562/2003 of the Ministry of Health to J.N. and OTKA T-043788 of the Hungarian Academy of Sciences to I.W.; furthermore, by a grant (P5 research grant) from Fresenius Medical Care to M.M., the János Bolyai Research Scholarship of the Hungarian Academy of Sciences to L.W. and by a grant of the “International Exchange Programme for Young Nephrologists” (Fresenius Medical Care, Bad Homburg, Germany, Dr. Jörg Vienken) to Z.W. Potential conflicts of interest: None. Address reprint requests to Zoltán Wagner, MD, PhD, Second Department of Medicine, University of Pécs, Pacsirta u. 1, H-7624 Pécs, Hungary. E-mail: zoltan.wagner@ aok.pte.hu © 2005 by the National Kidney Foundation, Inc. 0272-6386/05/4702-0010$30.00/0 doi:10.1053/j.ajkd.2005.10.010 294
disease (ESRD) by several studies in recent years.1-3 It has been supposed that AGEs act as uremic toxins contributing to the extremely high cardiovascular risk in patients with ESRD.4,5 This concept was supported by numerous studies exploring the role of AGEs in the pathogenesis of atherosclerosis.6,7 In vitro investigations showed that AGEs induce the secretion of cytokines, growth factors, and adhesion molecules responsible for vascular smooth muscle cell proliferation and mononuclear cell chemotaxis, activation, and transendothelial migration. They impair vasodilatory effects by quenching nitric oxide and increase vasoconstriction by inducing endothelin 1 production. In addition, AGEs exert procoagulant activity by increasing tissue-factor level and downregulating thrombomodulin expression.6,7 Despite increasing indirect evidence of AGErelated toxicity, only minimal clinical investigations supported the link between circulating AGEs and the development of cardiovascular disease. Until now, serum AGE levels could not be identified as independent risk factors of cardiovascular morbidity and mortality in patients with ESRD.8-11 However, findings of Schwedler et al9 indicating that serum level of the AGE carboxymethyllysine (CML) is not predictive of mortality may be biased by the better nutritional status (greater albumin,
American Journal of Kidney Diseases, Vol 47, No 2 (February), 2006: pp 294-300
CARBOXYMETHYLLYSINE AND MORTALITY IN HEMODIALYSIS
creatinine, and blood urea nitrogen levels) of patients with high (greater than the median) serum CML levels. In the present study, we investigate whether serum CML level predicts cardiovascular and overall mortality in 154 maintenance hemodialysis patients followed up during 51 months. METHODS
Subjects One hundred fifty-four hemodialysis patients with ESRD (119 patients without diabetes, 35 patients with diabetes) from the Fresenius Medical Care Dialysis Center at the Medical Faculty, University of Pécs, Hungary, were examined and followed up during 51 months from April 2000 until July 2004. Mean time on dialysis therapy was 42.3 months (range, 1 to 211 months) at the start of the study. During follow-up, dialysis vintage reached or exceeded 9 months in all patients. One hundred fifteen patients (75%) were treated with F5-high-performance steam (HPS) (Fresenius Medical Care, Bad Homburg, Germany), F6-HPS (Fresenius Medical Care), and F8-HPS low-flux polysulfone membranes (Fresenius Medical Care), and 39 patients (25%), with F50-S (Fresenius Medical Care), F60-S (Fresenius Medical Care), and HF80-S high-flux polysulfone membranes (Fresenius Medical Care). Twenty-three patients (15%) had an indwelling (tunneled) venous catheter, and 4 patients (3%) had a temporary catheter as their vascular access for hemodialysis at the start of the study. Most patients received medications commonly used in patients with ESRD, such as phosphate and potassium binders; iron; erythropoietin; vitamin B, C, and D supplements; furosemide; and antihypertensive drugs. The presence of vascular disease was assessed by reviewing clinical charts. Patients with angina pectoris, defined by positive stress test results or pathological findings from coronary angiography, as well as patients with a history of myocardial infarction and those who had undergone percutaneous transluminal coronary angioplasty or coronary artery bypass grafting were classified as having coronary heart disease. History of myocardial infarction is defined as the presence of chest pain, confirmatory electrocardiograms, and enzyme courses. Peripheral vascular disease was diagnosed by the presence of claudication and diminished pulses or history of lower limb arterial reconstruction. Cerebrovascular disease is defined as a history of stroke, transient ischemic attack, or carotid artery surgery or significant stenosis on carotid artery duplex scanning. At baseline, 100 patients (65%) had at least 1 of the 3 defined vascular diseases and are referred to as patients with preexisting cardiovascular disease (CVD). Previous habitual or current smoking was found in 47 patients (31%). On examination at baseline, no clinical signs of acute infection were found. At initial investigations, 65 healthy subjects served as controls. Healthy subjects did not participate in the follow-up study. All participants gave their informed consent before entering the study, and the local Ethics Committee of the University of Pécs approved the study protocol.
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Blood Sampling and Laboratory Analyses At the start of the study, predialysis blood samples were drawn before heparin administration. Measurements of albumin, 2-microglobulin, blood urea nitrogen (BUN), creatinine, C-reactive protein (CRP), hemoglobin A1c, total cholesterol, and triglycerides were performed on fresh samples. For CML measurement, serum samples were stored at ⫺70°C until analyzed. CRP was measured by means of nephelometric immunoassay. Determination of albumin level was performed by using the bromocresol green method. Body weight, height, and volume of 24-hour collected urine also were recorded, and body mass index (BMI) was calculated.
CML Measurement Serum CML concentrations were measured in triplicate by means of competitive enzyme-linked immunosorbent assay developed by Roche Diagnostics (Penzberg, Germany), as described earlier.12 Briefly, serum samples were hydrolyzed with proteinase K and diluted 20-fold before CML determination. To estimate absolute CML concentration, N-(carboxymethyl-)amino-caproic acid (Alteon Inc, New York, NY) served as standard. Digested and diluted serum samples and CML standards were incubated with peroxidase-conjugated monoclonal antibody against CML for 1 hour in 96-well plates preincubated with AGE-bovine serum albumin (100 ng/well). Color reaction was induced with 2,2=-azino-di-3-ethylbenzthiazoline-sulfonic acid (Roche Diagnostics) and measured in a microtiter reader (Multiskan Ascent, Labsystems, Helsinki, Finland) at 405 nm (reference, 603 nm); all steps were performed at room temperature. Considering that the majority of circulating CML is protein bound,13 results are expressed as serum CML content in nanograms per milligram of protein.
Follow-Up Study All hemodialysis patients enrolled in April 2000 were followed up until the date of death, censoring for transplantation, or study completion (July 2004). Cause of death was recorded and classified as mortality caused by cardiovascular (myocardial infarction, heart failure, or stroke) and noncardiovascular events (infection, malignancy, and unknown causes). During the median follow-up of 40 months (range, 1 to 51 months), 17 patients were censored for transplantation. Data for patients who underwent transplantation were included in statistical analyses only until the date of transplantation.
Statistical Analyses Results are expressed as mean ⫾ SD. Normality of variables was analyzed by means of Kolmogorov-Smirnov test using Lilliefors significance correction. Most variables were non-normally distributed, except for serum creatinine level. Therefore, in the case of serum creatinine values, Student t-test was used. Otherwise, comparisons of 2 independent samples were performed by using nonparametric Mann-Whitney U test. Comparisons between 2 groups for nominal variables were carried out with chi-square test. Bivariate correlation was analyzed by using Spearman test. Survival was examined by means of Kaplan-Meier analysis
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using log-rank test and a univariate Cox proportional hazards model followed by multivariate Cox regression analysis. Variables supposed to have influence on survival (presence of diabetes mellitus, male sex, smoking. and residual urine volume) and those with P less than 0.05 in univariate analysis were entered in the multivariate model.
RESULTS
Mean serum CML content was 5-fold greater in hemodialysis patients compared with controls (Table 1). All hemodialysis patients had serum CML content greater than 8.8 ng/mg protein, the 95th percentile of levels in healthy subjects. Hemodialysis patients were significantly older and had greater serum creatinine and CRP levels and lower albumin levels and body weights than controls (Table 1). Sex was distributed equally between both groups. There were no significant differences in means of serum CML content between men and women in hemodialysis patients and healthy controls (data not shown). Patients with diabetes mellitus (n ⫽ 35) had a mean serum CML content (22.5 ⫾ 8.3 ng/mg protein) similar to that of patients without diabetes (25.5 ⫾ 10.0 ng/mg protein). No association was found between serum CML and hemoglobin A1c levels in the group of patients with diabetes. Serum CML content was not significantly greater in patients with preexisting CVD (25.8 ⫾ 10.2 ng/mg protein; n ⫽ 100) than those without vascular disease (23.0 ⫾ 8.3 ng/mg protein). Serum CML content was related inversely to residual urine volume ( ⫽ ⫺0.279; P ⬍ 0.001) and correlated significantly with 2-microglobulin level ( ⫽ 0.196; P ⬍ 0.05) and time on Table 1. Characteristics of Hemodialysis Patients and Controls
Characteristics
Age (y) Men/women (%) Body weight (kg) Creatinine (mg/dL) Albumin (g/dL) CRP (mg/dL) Serum CML content (ng/mg protein)
Patients (n ⫽ 154)
Controls (n ⫽ 65)
59.4 ⫾ 15.8 46/54 64.2 ⫾ 13.8 9.3 ⫾ 2.5 3.8 ⫾ 0.3 1.8 ⫾ 2.6
41.8 ⫾ 14.0* 55/45 73.2 ⫾ 13.5* 1.1 ⫾ 0.2* 4.7 ⫾ 0.2* 0.2 ⫾ 0.2*
24.8 ⫾ 9.7
5.0 ⫾ 1.8*
NOTE. Data expressed as mean ⫾ SD. To convert serum creatinine in mg/dL to mol/L, multiply by 88.4; albumin in g/dL to g/L, multiply by 10. *P ⬍ 0.001.
Fig 1. Cumulative incidence of death from all causes during 51 months of follow-up in relation to high (greater than the median) or low (less than the median) serum CML content (P < 0.01).
dialysis therapy ( ⫽ 0.158; P ⬍ 0.05). 2Microglobulin level also was related inversely to residual urine volume ( ⫽ ⫺0.394; P ⬍ 0.001) and correlated significantly with time on dialysis therapy ( ⫽ 0.309; P ⬍ 0.001). Residual urine volume was related inversely to time on dialysis therapy ( ⫽ ⫺0.416; P ⬍ 0.001). No relationship was found between serum CML levels and age, CRP level, albumin level, BUN level, creatinine level, and BMI in hemodialysis patients. During the follow-up period of 51 months, 74 patients (48%) died. Sixty-two percent had a cardiovascular and 38% had a noncardiovascular cause of death. Calculated mean survival time per patient was 37 months at the beginning of the observation period. Survival was decreased significantly by preexisting CVD (33 versus 46 months; P ⬍ 0.001), presence of diabetes mellitus (34 versus 38 months; P ⬍ 0.05), and inflammation (CRP ⱖ 1 mg/dL; 33 versus 41 months; P ⬍ 0.05), as well as high (greater than the median, 23.8 ng/mg protein) serum CML content (34 versus 40 months; P ⬍ 0.01). Median CML level was used as a cutoff value for creating 2 groups with high and low serum CML content, with the same number of cases. Patients with serum CML content greater than the median had greater all-cause (0.58 versus 0.38; P ⬍ 0.01; Fig 1) and cardiovascular mortality rates (0.36 versus 0.23; P ⬍ 0.05; Fig 2) than patients with low serum CML content. Ten pa-
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analysis, whereas the effect of serum CML content was not significant. DISCUSSION
Fig 2. Cumulative incidence of death from CVD during 51 months of follow-up in relation to high (greater than the median) or low (less than the median) serum CML content (P < 0.05).
tients (13%) underwent kidney transplantation in the low-CML group, and 7 patients (9%), in the high-CML group. Baseline characteristics of patients with high (greater than the median) and low (less than the median) serum CML content are listed in Table 2. No significant differences were seen in age; sex; BMI; serum albumin, BUN, creatinine, CRP, and lipid levels; membrane type; vascular access; smoking habits; preexisting CVD; and presence of diabetes mellitus between groups. In the high-CML group, time on dialysis therapy and 2-microglobulin level were significantly greater, whereas residual urine volume was less than in the low-CML group. Univariate Cox regression analysis showed that age; presence of diabetes mellitus; preexisting CVD; serum creatinine, serum albumin, and serum CRP levels; and high serum CML content were associated with all-cause mortality (Table 3). In the multivariate model, age, preexisting CVD, serum CRP level, serum albumin level, high (greater than the median) serum CML content, and smoking proved to be independent predictors of all-cause mortality (Table 3). Hazard rate ratio of all-cause death was 1.78 (95% confidence interval, 1.05 to 3.01) in the high-CML group (P ⬍ 0.05), adjusted for the variables of interest (Fig 3). Cardiovascular mortality was predicted by age, preexisting CVD, and serum albumin level, estimated by means of multivariate Cox regression
AGEs have been implicated in the pathogenesis of vascular damage. Immunohistochemical studies showed AGE accumulation in atherosclerotic lesions of patients with and without diabetes,14 as well as those with ESRD.15 Furthermore, in hemodialysis patients, left ventricular hypertrophy was associated with elevated serum levels of the AGE CML10 and pentosidine,10,16 and a significant correlation between plasma pentosidine level and aorta calcification index was shown.17 Nevertheless, to date, circulating AGEs were considered inadequate parameters for the demonstration of AGE-induced damage with reTable 2. Characteristics of Hemodialysis Patients With Serum CML Content Less and Greater Than the Median
Characteristics
Age (y) Men/women (%) BMI (kg/m2) Serum albumin (g/dL) Serum creatinine (mg/dL) BUN (mg/dL) CRP (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) High-flux membrane (%) Catheter as a vascular access (%) Smoking (%) Diabetes mellitus (%) Preexisting CVD (%) Time on dialysis (mo) Residual urine volume (mL/d) 2-Microglobulin (mg/dL) Serum CML content (ng/ mg protein)
Serum CML ⱕ Median (n ⫽ 77)
Serum CML ⬎ Median (n ⫽ 77)
58.3 ⫾ 15.5 47/53 24.3 ⫾ 4.8 3.8 ⫾ 0.4 9.0 ⫾ 2.4 59 ⫾ 18 1.7 ⫾ 2.5 174 ⫾ 39 177 ⫾ 106 27.3
60.6 ⫾ 16.1 46/54 23.5 ⫾ 3.8 3.8 ⫾ 0.3 9.7 ⫾ 2.6 62 ⫾ 18 1.9 ⫾ 2.6 170 ⫾ 43 168 ⫾ 97 23.4
18.2 27.3 26.0 58.4 34.8 ⫾ 33.4
16.9 33.8 19.5 71.4 49.7 ⫾ 41.3*
749 ⫾ 627 1.9 ⫾ 1.1
382 ⫾ 453† 2.5 ⫾ 1.5‡
17.8 ⫾ 4.1
31.8 ⫾ 8.5†
NOTE. Data expressed as mean ⫾ SD. To convert serum albumin in g/dL to g/L, multiply by 10; creatinine in mg/dL to mol/L, multiply by 88.4; BUN in mg/dL to mmol/L, multiply by 0.357; total cholesterol in mg/dL to mmol/L, multiply by 0.0259; triglycerides in mg/dL to mmol/L, multiply by 0.0113. *P ⬍ 0.05. †P ⬍ 0.001. ‡P ⬍ 0.01.
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WAGNER ET AL Table 3. Univariate and Multivariate Analyses of the Effect of Variables on Survival by Cox Proportional Hazards Model Univariate Analysis
Variable
Hazard Rate Ratio
95% Confidence Interval
Age (/y increase) Preexisting CVD Serum CRP (/mg/dL increase) High CML Serum albumin (/g/dL increase) Serum creatinine (/mg/dL increase) Diabetes mellitus Smoking Male sex Oligoanuria
1.043 4.235 1.010 1.868 0.910 0.998 1.724 0.857 1.391 0.916
1.024-1.061 2.172-8.257 1.003-1.016 1.170-2.982 0.856-0.967 0.997-1.000 1.053-2.822 0.517-1.420 0.880-2.197 0.579-1.449
Multivariate Analysis
Probability
Hazard Rate Ratio
95% Confidence Interval
Probability
⬍0.001 ⬍0.001 ⬍0.005 ⬍0.01 ⬍0.005 ⬍0.05 ⬍0.05 NS NS NS
1.056 2.533 1.017 1.776 0.917 1.000 1.284 3.03 1.413 0.574
1.026-1.088 1.217-5.272 1.008-1.025 1.051-3.001 0.850-0.989 0.999-1.002 0.739-2.232 1.483-6.191 0.806-2.476 0.320-1.031
⬍0.001 ⬍0.05 ⬍0.001 ⬍0.05 ⬍0.05 NS NS ⬍0.005 NS NS
NOTE. High serum CML content indicates level greater than the median, and oligoanuria indicates urine volume less than 500 mL/d. Abbreviation: NS, not significant.
spect to outcome in patients with ESRD.18 Busch et al11 reported that elevated AGE levels were not related to greater risk for cardiovascular events in a heterogeneous group consisting of patients with chronic renal failure (n ⫽ 99), patients undergoing maintenance hemodialysis treatment (n ⫽ 84), and renal transplant recipients (n ⫽ 49). Suliman et al8 found no association between plasma pentosidine content and
Fig 3. Cumulative hazard of all-cause mortality in relation to high (greater than the median) or low (less than the median) serum CML content (multivariate Cox proportional hazards model, P < 0.05), adjusted for age, sex, smoking, serum CRP level, serum albumin level, serum creatinine level, preexisting CVD, presence of diabetes mellitus, and oligoanuria.
survival in a cohort of 191 patients with ESRD before starting dialysis therapy. However, in that study, AGEs measured in patients with ESRD before starting dialysis therapy may not be representative of AGE levels when already on dialysis therapy because serum AGE levels are influenced by the hemodialysis treatment itself.19,20 Schwedler et al9 indicated that high serum AGE concentration was linked to better survival in 312 stable hemodialysis patients who were followed up over 32 months. It is well established that malnutrition is linked to increased mortality in hemodialysis patients.21 Thus, in the study of Schwedler et al,9 greater albumin, creatinine, and BUN levels (ie, better nutritional status) may explain better survival in patients with high serum AGE levels. Furthermore, in the study of Schwedler et al,9 patients with high CML levels had a lower incidence of preexisting vascular disease, which also contributed to better survival in that patient group. In accordance with previous studies,3,22 we found markedly elevated serum CML content in patients undergoing hemodialysis treatment compared with healthy subjects, with no difference between patients with and without diabetes. More importantly, serum CML content predicted overall mortality in our 154 hemodialysis patients after a follow-up of 51 months. In the multivariate Cox regression model, serum CML content turned out to be an independent risk factor for mortality. Patients with high CML levels had albumin, BUN,
CARBOXYMETHYLLYSINE AND MORTALITY IN HEMODIALYSIS
and creatinine levels similar to those of patients with low CML levels; thus, it may be concluded that nutritional status was similar in both groups, although more reliable estimation of nutritional status was not carried out in our study. Effects of age and preexisting CVD on allcause mortality in our study were similar to previous findings.8,9,23 Conversely, the effect of inflammation was not significant in the study of Suliman et al,8 whereas CRP level showed a stronger relation to mortality (hazard ratios, 1.13 and 1.16, respectively) in the latter 2 studies9,23 compared with our data (hazard ratio, 1.017). The prevalence of preexisting CVD was relatively high in our patients, which can be explained by the high prevalence of CVD in the Hungarian general population. The cardiovascular mortality rate in Hungary is approximately 2 times greater than in western European countries.24 Increased cardiovascular mortality was noticed in the high-CML group with Kaplan-Meier analysis. Nevertheless, with multivariate Cox regression analysis, serum CML content did not prove to be an independent predictor of cardiovascular mortality. In agreement with previous studies, both serum 2-microglobulin25 and serum AGE19 levels were related inversely to residual diuresis in our study, which may explain the correlation of CML level with that of the middle-molecular-weight marker 2-microglobulin. This finding is not necessarily inconsistent with the fact that CML circulates mainly in high-molecular-weight protein-bound form because residual diuresis can affect serum CML concentration through modulation of the level of middle- and low-molecularweight AGE precursors. The significant correlation between serum CML level and time on dialysis therapy may be a consequence of the inverse relation between time on dialysis therapy and residual urine volume. In summary, our study shows that in addition to such well-established risk factors as age, preexisting CVD, smoking, and inflammation, circulating AGEs also may contribute to increased mortality in hemodialysis patients. This finding is in accordance with the current hypothesis suggesting that AGEs contribute to the toxicity of uremia.
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ACKNOWLEDGMENT The authors thank Dr Reinhard Schinzel for invaluable comments to this manuscript and Dr Rosemarie KientschEngel (Roche Diagnostics, Penzberg, Germany) for providing the CML enzyme-linked immunosorbent assay kit.
REFERENCES 1. Makita Z, Radoff S, Rayfield EJ, et al: Advanced glycosylation end products in patients with diabetic nephropathy. N Engl J Med 325:836-842, 1991 2. Dawnay A, Millar DJ: The pathogenesis and consequences of AGE formation in uraemia and its treatment. Cell Mol Biol 44:1081-1094, 1998 3. Schinzel R, Münch G, Heidland A, Sebekova K: Advanced glycation end products in end-stage renal disease and their removal. Nephron 87:295-303, 2001 4. Ritz E, Deppisch R, Nawroth PP: Toxicity of uraemia— Does it come of AGE? Nephrol Dial Transplant 9:1-2, 1994 5. Vlassara H: Serum advanced glycosylation end products: A new class of uremic toxins? Blood Purif 12:54-59, 1994 6. Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP: AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc Res 37:586-600, 1998 7. Raj DSC, Choudhury D, Welbourne TC, Levi M: Advanced glycation end products: A nephrologist’s perspective. Am J Kidney Dis 35:365-380, 2000 8. Suliman ME, Heimbürger O, Bárány P, et al: Plasma pentosidine is associated with inflammation and malnutrition in end-stage renal disease patients starting on dialysis therapy. J Am Soc Nephrol 14:1614-1622, 2003 9. Schwedler SB, Metzger T, Schinzel R, Wanner C: Advanced glycation end products and mortality in hemodialysis patients. Kidney Int 62:301-310, 2002 10. Stein G, Busch M, Müller A, et al: Are advanced glycation end products cardiovascular risk factors in patients with CRF? Am J Kidney Dis 41:S52-S56, 2003 (suppl 1) 11. Busch M, Franke S, Müller A, et al: Potential cardiovascular risk factors in chronic kidney disease: AGEs, total homocysteine and metabolites, and the C-reactive protein. Kidney Int 66:338-347, 2004 12. Wagner Z, Wittmann I, Mazák I, et al: N-(carboxymethyl)lysine levels in type 2 diabetic patients: Role of renal function. Am J Kidney Dis 38:785-791, 2001 13. Gerdemann A, Lemke HD, Nothdurft A, et al: Lowmolecular but not high-molecular advanced glycation end products (AGEs) are removed by high-flux dialysis. Clin Nephrol 54:276-283, 2000 14. Massy ZA: Importance of homocysteine, lipoprotein(a) and non-classical cardiovascular risk factors (fibrinogen and advanced glycation end-products) for atherogenesis in uraemic patients. Nephrol Dial Transplant 15:S81-S91, 2000 (suppl 5) 15. Sakata N, Imanaga Y, Meng J, et al: Increased advanced glycation end products in atherosclerotic lesions of patients with end-stage renal disease. Atherosclerosis 142:6777, 1999 16. Zocalli C, Mallamaci F, Asahia K, et al: Pentosidine, carotid atherosclerosis and alterations in left ventricular
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geometry in hemodialysis patients. J Nephrol 14:293-298, 2001 17. Kitauchi T, Yoshida K, Yoneda T, et al: Association between pentosidine and arteriosclerosis in patients receiving hemodialysis. Clin Exp Nephrol 8:48-53, 2004 18. Locatelli F, Canaud B, Eckardt K, Stenvinkel P, Wanner C, Zocalli C: Oxidative stress in end-stage renal disease: An emerging threat to patient outcome. Nephrol Dial Transplant 18:1272-1280, 2003 19. Jadoul M, Ueda Y, Yasuda Y, et al: Influence of hemodialysis membrane type on pentosidine plasma level, a marker of “carbonyl stress.” Kidney Int 53:2487-2492, 1999 20. Gerdemann A, Wagner Z, Solf A, et al: Plasma levels of advanced glycation end products during haemodialysis, haemodiafiltration and haemofiltration: Potential importance of dialysate quality. Nephrol Dial Transplant 17:1045-1049, 2002 21. Pupim LB, Caglar K, Hakim RM, Shyr Y, Ikizler TA:
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Uremic malnutrition is a predictor of death independent of inflammatory status. Kidney Int 66:2054-2060, 2004 22. Degenhardt T, Grass L, Reddy S, Thorpe S, Diamandis E, Baynes JW: The serum concentration of N-(carboxymethyl)lysine is increased in uremia. Kidney Int 52:10641067, 1997 23. Zimmermann J, Herrlinger S, Pruy A, Metzger T, Wanner C: Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int 55:648-658, 1999 24. World Health Organization Regional Office for Europe: The health situation in the European region, in Nosikov A, Jardel JP (eds): The European Health Report 2002. Copenhagen, WHO, 2002, pp 7-64 25. Catizone L, Cocchi R, Fusaroli M, Zucchelli P: Relationship between plasma beta 2-microglobulin and residual diuresis in continuous ambulatory peritoneal dialysis and hemodialysis patients. Perit Dial Int 13:S523-S526, 1993 (suppl 2)
M
ARKEDLY ELEVATED serum levels of advanced glycation end products (AGEs) have been shown in patients with end-stage renal
From the Second Department of Medicine and Nephrological Center, University of Pécs; Fresenius Medical Care, Dialysis Center Pécs, Pécs; Fresenius Medical Care, Dialysis Center Szigetvár, Szigetvár, Hungary; and Department of Internal Medicine, University of Würzburg, Würzburg, Germany. Received August 16, 2005; accepted in revised form October 5, 2005. Originally published online as doi:10.1053/j.ajkd.2005.10.010 on December 27, 2005. Support: This work was supported by the Hungarian national grants: ETT 562/2003 of the Ministry of Health to J.N. and OTKA T-043788 of the Hungarian Academy of Sciences to I.W.; furthermore, by a grant (P5 research grant) from Fresenius Medical Care to M.M., the János Bolyai Research Scholarship of the Hungarian Academy of Sciences to L.W. and by a grant of the “International Exchange Programme for Young Nephrologists” (Fresenius Medical Care, Bad Homburg, Germany, Dr. Jörg Vienken) to Z.W. Potential conflicts of interest: None. Address reprint requests to Zoltán Wagner, MD, PhD, Second Department of Medicine, University of Pécs, Pacsirta u. 1, H-7624 Pécs, Hungary. E-mail: zoltan.wagner@ aok.pte.hu © 2005 by the National Kidney Foundation, Inc. 0272-6386/05/4702-0010$30.00/0 doi:10.1053/j.ajkd.2005.10.010 294
disease (ESRD) by several studies in recent years.1-3 It has been supposed that AGEs act as uremic toxins contributing to the extremely high cardiovascular risk in patients with ESRD.4,5 This concept was supported by numerous studies exploring the role of AGEs in the pathogenesis of atherosclerosis.6,7 In vitro investigations showed that AGEs induce the secretion of cytokines, growth factors, and adhesion molecules responsible for vascular smooth muscle cell proliferation and mononuclear cell chemotaxis, activation, and transendothelial migration. They impair vasodilatory effects by quenching nitric oxide and increase vasoconstriction by inducing endothelin 1 production. In addition, AGEs exert procoagulant activity by increasing tissue-factor level and downregulating thrombomodulin expression.6,7 Despite increasing indirect evidence of AGErelated toxicity, only minimal clinical investigations supported the link between circulating AGEs and the development of cardiovascular disease. Until now, serum AGE levels could not be identified as independent risk factors of cardiovascular morbidity and mortality in patients with ESRD.8-11 However, findings of Schwedler et al9 indicating that serum level of the AGE carboxymethyllysine (CML) is not predictive of mortality may be biased by the better nutritional status (greater albumin,
American Journal of Kidney Diseases, Vol 47, No 2 (February), 2006: pp 294-300
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creatinine, and blood urea nitrogen levels) of patients with high (greater than the median) serum CML levels. In the present study, we investigate whether serum CML level predicts cardiovascular and overall mortality in 154 maintenance hemodialysis patients followed up during 51 months. METHODS
Subjects One hundred fifty-four hemodialysis patients with ESRD (119 patients without diabetes, 35 patients with diabetes) from the Fresenius Medical Care Dialysis Center at the Medical Faculty, University of Pécs, Hungary, were examined and followed up during 51 months from April 2000 until July 2004. Mean time on dialysis therapy was 42.3 months (range, 1 to 211 months) at the start of the study. During follow-up, dialysis vintage reached or exceeded 9 months in all patients. One hundred fifteen patients (75%) were treated with F5-high-performance steam (HPS) (Fresenius Medical Care, Bad Homburg, Germany), F6-HPS (Fresenius Medical Care), and F8-HPS low-flux polysulfone membranes (Fresenius Medical Care), and 39 patients (25%), with F50-S (Fresenius Medical Care), F60-S (Fresenius Medical Care), and HF80-S high-flux polysulfone membranes (Fresenius Medical Care). Twenty-three patients (15%) had an indwelling (tunneled) venous catheter, and 4 patients (3%) had a temporary catheter as their vascular access for hemodialysis at the start of the study. Most patients received medications commonly used in patients with ESRD, such as phosphate and potassium binders; iron; erythropoietin; vitamin B, C, and D supplements; furosemide; and antihypertensive drugs. The presence of vascular disease was assessed by reviewing clinical charts. Patients with angina pectoris, defined by positive stress test results or pathological findings from coronary angiography, as well as patients with a history of myocardial infarction and those who had undergone percutaneous transluminal coronary angioplasty or coronary artery bypass grafting were classified as having coronary heart disease. History of myocardial infarction is defined as the presence of chest pain, confirmatory electrocardiograms, and enzyme courses. Peripheral vascular disease was diagnosed by the presence of claudication and diminished pulses or history of lower limb arterial reconstruction. Cerebrovascular disease is defined as a history of stroke, transient ischemic attack, or carotid artery surgery or significant stenosis on carotid artery duplex scanning. At baseline, 100 patients (65%) had at least 1 of the 3 defined vascular diseases and are referred to as patients with preexisting cardiovascular disease (CVD). Previous habitual or current smoking was found in 47 patients (31%). On examination at baseline, no clinical signs of acute infection were found. At initial investigations, 65 healthy subjects served as controls. Healthy subjects did not participate in the follow-up study. All participants gave their informed consent before entering the study, and the local Ethics Committee of the University of Pécs approved the study protocol.
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Blood Sampling and Laboratory Analyses At the start of the study, predialysis blood samples were drawn before heparin administration. Measurements of albumin, 2-microglobulin, blood urea nitrogen (BUN), creatinine, C-reactive protein (CRP), hemoglobin A1c, total cholesterol, and triglycerides were performed on fresh samples. For CML measurement, serum samples were stored at ⫺70°C until analyzed. CRP was measured by means of nephelometric immunoassay. Determination of albumin level was performed by using the bromocresol green method. Body weight, height, and volume of 24-hour collected urine also were recorded, and body mass index (BMI) was calculated.
CML Measurement Serum CML concentrations were measured in triplicate by means of competitive enzyme-linked immunosorbent assay developed by Roche Diagnostics (Penzberg, Germany), as described earlier.12 Briefly, serum samples were hydrolyzed with proteinase K and diluted 20-fold before CML determination. To estimate absolute CML concentration, N-(carboxymethyl-)amino-caproic acid (Alteon Inc, New York, NY) served as standard. Digested and diluted serum samples and CML standards were incubated with peroxidase-conjugated monoclonal antibody against CML for 1 hour in 96-well plates preincubated with AGE-bovine serum albumin (100 ng/well). Color reaction was induced with 2,2=-azino-di-3-ethylbenzthiazoline-sulfonic acid (Roche Diagnostics) and measured in a microtiter reader (Multiskan Ascent, Labsystems, Helsinki, Finland) at 405 nm (reference, 603 nm); all steps were performed at room temperature. Considering that the majority of circulating CML is protein bound,13 results are expressed as serum CML content in nanograms per milligram of protein.
Follow-Up Study All hemodialysis patients enrolled in April 2000 were followed up until the date of death, censoring for transplantation, or study completion (July 2004). Cause of death was recorded and classified as mortality caused by cardiovascular (myocardial infarction, heart failure, or stroke) and noncardiovascular events (infection, malignancy, and unknown causes). During the median follow-up of 40 months (range, 1 to 51 months), 17 patients were censored for transplantation. Data for patients who underwent transplantation were included in statistical analyses only until the date of transplantation.
Statistical Analyses Results are expressed as mean ⫾ SD. Normality of variables was analyzed by means of Kolmogorov-Smirnov test using Lilliefors significance correction. Most variables were non-normally distributed, except for serum creatinine level. Therefore, in the case of serum creatinine values, Student t-test was used. Otherwise, comparisons of 2 independent samples were performed by using nonparametric Mann-Whitney U test. Comparisons between 2 groups for nominal variables were carried out with chi-square test. Bivariate correlation was analyzed by using Spearman test. Survival was examined by means of Kaplan-Meier analysis
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using log-rank test and a univariate Cox proportional hazards model followed by multivariate Cox regression analysis. Variables supposed to have influence on survival (presence of diabetes mellitus, male sex, smoking. and residual urine volume) and those with P less than 0.05 in univariate analysis were entered in the multivariate model.
RESULTS
Mean serum CML content was 5-fold greater in hemodialysis patients compared with controls (Table 1). All hemodialysis patients had serum CML content greater than 8.8 ng/mg protein, the 95th percentile of levels in healthy subjects. Hemodialysis patients were significantly older and had greater serum creatinine and CRP levels and lower albumin levels and body weights than controls (Table 1). Sex was distributed equally between both groups. There were no significant differences in means of serum CML content between men and women in hemodialysis patients and healthy controls (data not shown). Patients with diabetes mellitus (n ⫽ 35) had a mean serum CML content (22.5 ⫾ 8.3 ng/mg protein) similar to that of patients without diabetes (25.5 ⫾ 10.0 ng/mg protein). No association was found between serum CML and hemoglobin A1c levels in the group of patients with diabetes. Serum CML content was not significantly greater in patients with preexisting CVD (25.8 ⫾ 10.2 ng/mg protein; n ⫽ 100) than those without vascular disease (23.0 ⫾ 8.3 ng/mg protein). Serum CML content was related inversely to residual urine volume ( ⫽ ⫺0.279; P ⬍ 0.001) and correlated significantly with 2-microglobulin level ( ⫽ 0.196; P ⬍ 0.05) and time on Table 1. Characteristics of Hemodialysis Patients and Controls
Characteristics
Age (y) Men/women (%) Body weight (kg) Creatinine (mg/dL) Albumin (g/dL) CRP (mg/dL) Serum CML content (ng/mg protein)
Patients (n ⫽ 154)
Controls (n ⫽ 65)
59.4 ⫾ 15.8 46/54 64.2 ⫾ 13.8 9.3 ⫾ 2.5 3.8 ⫾ 0.3 1.8 ⫾ 2.6
41.8 ⫾ 14.0* 55/45 73.2 ⫾ 13.5* 1.1 ⫾ 0.2* 4.7 ⫾ 0.2* 0.2 ⫾ 0.2*
24.8 ⫾ 9.7
5.0 ⫾ 1.8*
NOTE. Data expressed as mean ⫾ SD. To convert serum creatinine in mg/dL to mol/L, multiply by 88.4; albumin in g/dL to g/L, multiply by 10. *P ⬍ 0.001.
Fig 1. Cumulative incidence of death from all causes during 51 months of follow-up in relation to high (greater than the median) or low (less than the median) serum CML content (P < 0.01).
dialysis therapy ( ⫽ 0.158; P ⬍ 0.05). 2Microglobulin level also was related inversely to residual urine volume ( ⫽ ⫺0.394; P ⬍ 0.001) and correlated significantly with time on dialysis therapy ( ⫽ 0.309; P ⬍ 0.001). Residual urine volume was related inversely to time on dialysis therapy ( ⫽ ⫺0.416; P ⬍ 0.001). No relationship was found between serum CML levels and age, CRP level, albumin level, BUN level, creatinine level, and BMI in hemodialysis patients. During the follow-up period of 51 months, 74 patients (48%) died. Sixty-two percent had a cardiovascular and 38% had a noncardiovascular cause of death. Calculated mean survival time per patient was 37 months at the beginning of the observation period. Survival was decreased significantly by preexisting CVD (33 versus 46 months; P ⬍ 0.001), presence of diabetes mellitus (34 versus 38 months; P ⬍ 0.05), and inflammation (CRP ⱖ 1 mg/dL; 33 versus 41 months; P ⬍ 0.05), as well as high (greater than the median, 23.8 ng/mg protein) serum CML content (34 versus 40 months; P ⬍ 0.01). Median CML level was used as a cutoff value for creating 2 groups with high and low serum CML content, with the same number of cases. Patients with serum CML content greater than the median had greater all-cause (0.58 versus 0.38; P ⬍ 0.01; Fig 1) and cardiovascular mortality rates (0.36 versus 0.23; P ⬍ 0.05; Fig 2) than patients with low serum CML content. Ten pa-
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analysis, whereas the effect of serum CML content was not significant. DISCUSSION
Fig 2. Cumulative incidence of death from CVD during 51 months of follow-up in relation to high (greater than the median) or low (less than the median) serum CML content (P < 0.05).
tients (13%) underwent kidney transplantation in the low-CML group, and 7 patients (9%), in the high-CML group. Baseline characteristics of patients with high (greater than the median) and low (less than the median) serum CML content are listed in Table 2. No significant differences were seen in age; sex; BMI; serum albumin, BUN, creatinine, CRP, and lipid levels; membrane type; vascular access; smoking habits; preexisting CVD; and presence of diabetes mellitus between groups. In the high-CML group, time on dialysis therapy and 2-microglobulin level were significantly greater, whereas residual urine volume was less than in the low-CML group. Univariate Cox regression analysis showed that age; presence of diabetes mellitus; preexisting CVD; serum creatinine, serum albumin, and serum CRP levels; and high serum CML content were associated with all-cause mortality (Table 3). In the multivariate model, age, preexisting CVD, serum CRP level, serum albumin level, high (greater than the median) serum CML content, and smoking proved to be independent predictors of all-cause mortality (Table 3). Hazard rate ratio of all-cause death was 1.78 (95% confidence interval, 1.05 to 3.01) in the high-CML group (P ⬍ 0.05), adjusted for the variables of interest (Fig 3). Cardiovascular mortality was predicted by age, preexisting CVD, and serum albumin level, estimated by means of multivariate Cox regression
AGEs have been implicated in the pathogenesis of vascular damage. Immunohistochemical studies showed AGE accumulation in atherosclerotic lesions of patients with and without diabetes,14 as well as those with ESRD.15 Furthermore, in hemodialysis patients, left ventricular hypertrophy was associated with elevated serum levels of the AGE CML10 and pentosidine,10,16 and a significant correlation between plasma pentosidine level and aorta calcification index was shown.17 Nevertheless, to date, circulating AGEs were considered inadequate parameters for the demonstration of AGE-induced damage with reTable 2. Characteristics of Hemodialysis Patients With Serum CML Content Less and Greater Than the Median
Characteristics
Age (y) Men/women (%) BMI (kg/m2) Serum albumin (g/dL) Serum creatinine (mg/dL) BUN (mg/dL) CRP (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) High-flux membrane (%) Catheter as a vascular access (%) Smoking (%) Diabetes mellitus (%) Preexisting CVD (%) Time on dialysis (mo) Residual urine volume (mL/d) 2-Microglobulin (mg/dL) Serum CML content (ng/ mg protein)
Serum CML ⱕ Median (n ⫽ 77)
Serum CML ⬎ Median (n ⫽ 77)
58.3 ⫾ 15.5 47/53 24.3 ⫾ 4.8 3.8 ⫾ 0.4 9.0 ⫾ 2.4 59 ⫾ 18 1.7 ⫾ 2.5 174 ⫾ 39 177 ⫾ 106 27.3
60.6 ⫾ 16.1 46/54 23.5 ⫾ 3.8 3.8 ⫾ 0.3 9.7 ⫾ 2.6 62 ⫾ 18 1.9 ⫾ 2.6 170 ⫾ 43 168 ⫾ 97 23.4
18.2 27.3 26.0 58.4 34.8 ⫾ 33.4
16.9 33.8 19.5 71.4 49.7 ⫾ 41.3*
749 ⫾ 627 1.9 ⫾ 1.1
382 ⫾ 453† 2.5 ⫾ 1.5‡
17.8 ⫾ 4.1
31.8 ⫾ 8.5†
NOTE. Data expressed as mean ⫾ SD. To convert serum albumin in g/dL to g/L, multiply by 10; creatinine in mg/dL to mol/L, multiply by 88.4; BUN in mg/dL to mmol/L, multiply by 0.357; total cholesterol in mg/dL to mmol/L, multiply by 0.0259; triglycerides in mg/dL to mmol/L, multiply by 0.0113. *P ⬍ 0.05. †P ⬍ 0.001. ‡P ⬍ 0.01.
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WAGNER ET AL Table 3. Univariate and Multivariate Analyses of the Effect of Variables on Survival by Cox Proportional Hazards Model Univariate Analysis
Variable
Hazard Rate Ratio
95% Confidence Interval
Age (/y increase) Preexisting CVD Serum CRP (/mg/dL increase) High CML Serum albumin (/g/dL increase) Serum creatinine (/mg/dL increase) Diabetes mellitus Smoking Male sex Oligoanuria
1.043 4.235 1.010 1.868 0.910 0.998 1.724 0.857 1.391 0.916
1.024-1.061 2.172-8.257 1.003-1.016 1.170-2.982 0.856-0.967 0.997-1.000 1.053-2.822 0.517-1.420 0.880-2.197 0.579-1.449
Multivariate Analysis
Probability
Hazard Rate Ratio
95% Confidence Interval
Probability
⬍0.001 ⬍0.001 ⬍0.005 ⬍0.01 ⬍0.005 ⬍0.05 ⬍0.05 NS NS NS
1.056 2.533 1.017 1.776 0.917 1.000 1.284 3.03 1.413 0.574
1.026-1.088 1.217-5.272 1.008-1.025 1.051-3.001 0.850-0.989 0.999-1.002 0.739-2.232 1.483-6.191 0.806-2.476 0.320-1.031
⬍0.001 ⬍0.05 ⬍0.001 ⬍0.05 ⬍0.05 NS NS ⬍0.005 NS NS
NOTE. High serum CML content indicates level greater than the median, and oligoanuria indicates urine volume less than 500 mL/d. Abbreviation: NS, not significant.
spect to outcome in patients with ESRD.18 Busch et al11 reported that elevated AGE levels were not related to greater risk for cardiovascular events in a heterogeneous group consisting of patients with chronic renal failure (n ⫽ 99), patients undergoing maintenance hemodialysis treatment (n ⫽ 84), and renal transplant recipients (n ⫽ 49). Suliman et al8 found no association between plasma pentosidine content and
Fig 3. Cumulative hazard of all-cause mortality in relation to high (greater than the median) or low (less than the median) serum CML content (multivariate Cox proportional hazards model, P < 0.05), adjusted for age, sex, smoking, serum CRP level, serum albumin level, serum creatinine level, preexisting CVD, presence of diabetes mellitus, and oligoanuria.
survival in a cohort of 191 patients with ESRD before starting dialysis therapy. However, in that study, AGEs measured in patients with ESRD before starting dialysis therapy may not be representative of AGE levels when already on dialysis therapy because serum AGE levels are influenced by the hemodialysis treatment itself.19,20 Schwedler et al9 indicated that high serum AGE concentration was linked to better survival in 312 stable hemodialysis patients who were followed up over 32 months. It is well established that malnutrition is linked to increased mortality in hemodialysis patients.21 Thus, in the study of Schwedler et al,9 greater albumin, creatinine, and BUN levels (ie, better nutritional status) may explain better survival in patients with high serum AGE levels. Furthermore, in the study of Schwedler et al,9 patients with high CML levels had a lower incidence of preexisting vascular disease, which also contributed to better survival in that patient group. In accordance with previous studies,3,22 we found markedly elevated serum CML content in patients undergoing hemodialysis treatment compared with healthy subjects, with no difference between patients with and without diabetes. More importantly, serum CML content predicted overall mortality in our 154 hemodialysis patients after a follow-up of 51 months. In the multivariate Cox regression model, serum CML content turned out to be an independent risk factor for mortality. Patients with high CML levels had albumin, BUN,
CARBOXYMETHYLLYSINE AND MORTALITY IN HEMODIALYSIS
and creatinine levels similar to those of patients with low CML levels; thus, it may be concluded that nutritional status was similar in both groups, although more reliable estimation of nutritional status was not carried out in our study. Effects of age and preexisting CVD on allcause mortality in our study were similar to previous findings.8,9,23 Conversely, the effect of inflammation was not significant in the study of Suliman et al,8 whereas CRP level showed a stronger relation to mortality (hazard ratios, 1.13 and 1.16, respectively) in the latter 2 studies9,23 compared with our data (hazard ratio, 1.017). The prevalence of preexisting CVD was relatively high in our patients, which can be explained by the high prevalence of CVD in the Hungarian general population. The cardiovascular mortality rate in Hungary is approximately 2 times greater than in western European countries.24 Increased cardiovascular mortality was noticed in the high-CML group with Kaplan-Meier analysis. Nevertheless, with multivariate Cox regression analysis, serum CML content did not prove to be an independent predictor of cardiovascular mortality. In agreement with previous studies, both serum 2-microglobulin25 and serum AGE19 levels were related inversely to residual diuresis in our study, which may explain the correlation of CML level with that of the middle-molecular-weight marker 2-microglobulin. This finding is not necessarily inconsistent with the fact that CML circulates mainly in high-molecular-weight protein-bound form because residual diuresis can affect serum CML concentration through modulation of the level of middle- and low-molecularweight AGE precursors. The significant correlation between serum CML level and time on dialysis therapy may be a consequence of the inverse relation between time on dialysis therapy and residual urine volume. In summary, our study shows that in addition to such well-established risk factors as age, preexisting CVD, smoking, and inflammation, circulating AGEs also may contribute to increased mortality in hemodialysis patients. This finding is in accordance with the current hypothesis suggesting that AGEs contribute to the toxicity of uremia.
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ACKNOWLEDGMENT The authors thank Dr Reinhard Schinzel for invaluable comments to this manuscript and Dr Rosemarie KientschEngel (Roche Diagnostics, Penzberg, Germany) for providing the CML enzyme-linked immunosorbent assay kit.
REFERENCES 1. Makita Z, Radoff S, Rayfield EJ, et al: Advanced glycosylation end products in patients with diabetic nephropathy. N Engl J Med 325:836-842, 1991 2. Dawnay A, Millar DJ: The pathogenesis and consequences of AGE formation in uraemia and its treatment. Cell Mol Biol 44:1081-1094, 1998 3. Schinzel R, Münch G, Heidland A, Sebekova K: Advanced glycation end products in end-stage renal disease and their removal. Nephron 87:295-303, 2001 4. Ritz E, Deppisch R, Nawroth PP: Toxicity of uraemia— Does it come of AGE? Nephrol Dial Transplant 9:1-2, 1994 5. Vlassara H: Serum advanced glycosylation end products: A new class of uremic toxins? Blood Purif 12:54-59, 1994 6. Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP: AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc Res 37:586-600, 1998 7. Raj DSC, Choudhury D, Welbourne TC, Levi M: Advanced glycation end products: A nephrologist’s perspective. Am J Kidney Dis 35:365-380, 2000 8. Suliman ME, Heimbürger O, Bárány P, et al: Plasma pentosidine is associated with inflammation and malnutrition in end-stage renal disease patients starting on dialysis therapy. J Am Soc Nephrol 14:1614-1622, 2003 9. Schwedler SB, Metzger T, Schinzel R, Wanner C: Advanced glycation end products and mortality in hemodialysis patients. Kidney Int 62:301-310, 2002 10. Stein G, Busch M, Müller A, et al: Are advanced glycation end products cardiovascular risk factors in patients with CRF? Am J Kidney Dis 41:S52-S56, 2003 (suppl 1) 11. Busch M, Franke S, Müller A, et al: Potential cardiovascular risk factors in chronic kidney disease: AGEs, total homocysteine and metabolites, and the C-reactive protein. Kidney Int 66:338-347, 2004 12. Wagner Z, Wittmann I, Mazák I, et al: N-(carboxymethyl)lysine levels in type 2 diabetic patients: Role of renal function. Am J Kidney Dis 38:785-791, 2001 13. Gerdemann A, Lemke HD, Nothdurft A, et al: Lowmolecular but not high-molecular advanced glycation end products (AGEs) are removed by high-flux dialysis. Clin Nephrol 54:276-283, 2000 14. Massy ZA: Importance of homocysteine, lipoprotein(a) and non-classical cardiovascular risk factors (fibrinogen and advanced glycation end-products) for atherogenesis in uraemic patients. Nephrol Dial Transplant 15:S81-S91, 2000 (suppl 5) 15. Sakata N, Imanaga Y, Meng J, et al: Increased advanced glycation end products in atherosclerotic lesions of patients with end-stage renal disease. Atherosclerosis 142:6777, 1999 16. Zocalli C, Mallamaci F, Asahia K, et al: Pentosidine, carotid atherosclerosis and alterations in left ventricular
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Uremic malnutrition is a predictor of death independent of inflammatory status. Kidney Int 66:2054-2060, 2004 22. Degenhardt T, Grass L, Reddy S, Thorpe S, Diamandis E, Baynes JW: The serum concentration of N-(carboxymethyl)lysine is increased in uremia. Kidney Int 52:10641067, 1997 23. Zimmermann J, Herrlinger S, Pruy A, Metzger T, Wanner C: Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int 55:648-658, 1999 24. World Health Organization Regional Office for Europe: The health situation in the European region, in Nosikov A, Jardel JP (eds): The European Health Report 2002. Copenhagen, WHO, 2002, pp 7-64 25. Catizone L, Cocchi R, Fusaroli M, Zucchelli P: Relationship between plasma beta 2-microglobulin and residual diuresis in continuous ambulatory peritoneal dialysis and hemodialysis patients. Perit Dial Int 13:S523-S526, 1993 (suppl 2)