Tissue level of advanced glycation end products is an independent determinant of highsensitivity creactive protein levels in haemodialysis patients

Nephrology 16 (2011) 299–303
Tissue level of advanced glycation end products is an independent
determinant of high-sensitivity C-reactive protein levels in
haemodialysis patientsnep_1419299.303
MAKIO NAGANO,1 KEI FUKAMI,1 SHO-ICHI YAMAGISHI,2 KAZUKO SAKAI,1 YUSUKE KAIDA,1TAKAFUMI MATSUMOTO,1 TAKUMA HAZAMA,1 MASAHIRO TANAKA,1 SEIJI UEDA1 and SEIYA OKUDA1 1Division of Nephrology, Department of Medicine, and 2Department of Pathophysiology and Therapeutics of Diabetic Complications, Kurume University Schoolof Medicine, Kurume, Japan KEY WORDS:
ABSTRACT:
advanced glycation end products,atherosclerosis, haemodialysis, inflammation.
C-reactive protein (CRP) level predicts future cardiovascular events in
patients on haemodialysis (HD). Advanced glycation end products (AGE)
Correspondence:
play a role in cardiovascular disease (CVD) in HD patients. However, which
variables including tissue AGE levels are independently associated with CRP
Department of Medicine, Kurume University remains unknown. Therefore, whether tissue AGE and CRP levels were
School of Medicine, 67 Asahi-machi, Kurume, correlated with atherosclerosis in HD patients was examined.
Fukuoka 830-0011, Japan. Email: [email protected] Fifty-four HD patients underwent determinations of blood chem-
istries and tissue AGE. Tissue AGE levels were evaluated by measuring skin
Accepted for publication 25 October 2010.
autofluorescence. Pulsatility index (PI) in the carotid artery was measured
Accepted manuscript online 3 November 2010.
using a Doppler ultrasonography.
Results:
Univariate analyses showed that age, white blood cells, serum
albumin (inversely), alkaline phosphatase (inversely), tartrate-resistant acid
phosphatase 5b (TRAP5b) (inversely) and skin AGE levels were significantly
SUMMARY AT A GLANCE
correlated with high-sensitivity CRP (hsCRP). Multiple stepwise regression
analysis revealed that serum albumin, TRAP5b and skin AGE levels were
independent determinants of hsCRP. Further, PI was highest among HD
products (AGE) level and high-sensitivity patients with high skin AGE and high hsCRP levels.
C-reactive protein (hsCRP) in haemodialysis The present study suggests that tissue AGE level is one of the
patients. In addition, tissue AGE and hsCRP independent determinants of hsCRP in HD patients. Tissue AGE and hsCRP
levels could in concert contribute to the levels may be correlated with each other, which could in concert contribute
to the progression of atherosclerosis in these subjects.
Cardiovascular disease (CVD) is a major cause of morbidity Reducing sugars can react non-enzymatically with the and high mortality rates in patients on haemodialysis (HD).1,2 amino groups of proteins to initiate a complex series of Recently, chronic inflammation is shown to be involved in rearrangements and dehydrations, and then to produce a accelerated atherosclerosis, which could lead to increased class of irreversibly cross-linked, fluorescent moieties termed risks of CVD in these high-risk patients.3 Indeed, several advanced glycation end products (AGE).10–12 Recently, the epidemiological studies have shown that an inflammatory formation and/or accumulation of AGE have been known to biomarker, C-reactive protein (CRP) level, is elevated progress in a normal aging process, and at an accelerated rate in patients with HD, which predicts future cardiovascular under diabetes or chronic kidney disease, thus playing a role events and death.4 Although various metabolic and haemo- in the development and progression of a CVD in these dynamic derangements are supposed to cause chronic subjects.13–18 Because AGE are reported to elicit oxidative inflammation in HD patients,5–7 which factors are indepen- stress generation and vascular inflammation in both cell dently associated with CRP level is not fully understood, culture and animal models, it is conceivable that accumula- because these variables are correlated with each other and tion of AGE is one of the causative factors for the elevation of 2011 The AuthorsNephrology 2011 Asian Pacific Society of Nephrology Many AGE form molecular cross-links and fluorescence in vivo.19,20 Therefore, tissue AGE levels could be evaluated non- invasively by measuring skin autofluorescence (AF). Indeed, where Ce/Ce is post-serum/pre-serum urea nitrogen ratio, t is dialy- skin AF value has been shown to be associated with vascular sis time, DBW/BW is the ratio of the ultrafiltrate volume removed complications in diabetes and predict cardiovascular mortal- from the post-dialysis weight and ln is a natural logarithmic (ln) ity in patients with HD.19,20 In this study, we investigated which anthropometric and metabolic variables, including Informed consent was obtained from all patients, and the study tissue AGE levels which were evaluated quantitatively by protocol was approved by the Institutional Ethics Committee of measuring skin AF with an AGE reader, were independently Kurume University School of Medicine.
Pulsatility index (PI) in the carotid artery, a surrogate marker of associated with CRP in HD patients. We further examined atherosclerosis, was evaluated by high-resolution ultrasonography whether tissue AGE and CRP levels were correlated with with a 7.5 MHz linear probe (GE Yokogawa Medical Systems, Tokyo, Japan). Common carotid PI was measured as a reflection of imped-ance to flow distal to the point of sampling.23 PI was automaticallycalculated and indicated as ((peak systolic velocity) - (end diastolic Tissue AGE levels were evaluated quantitatively by measuring Patients
skin AF with an AGE reader according to the supplier’s recommen-dations (DiagOptics BV, Groningen, the Netherlands).20 Fifty-four consecutive outpatients on HD (32 men and 22 women;mean age 54.2 1 14.0 years) underwent a complete history andphysical examination, determinations of blood chemistries, anthro- Statistical analysis
pometric and metabolic variables. Mean duration of HD was7.9 1 5.1 years. Patients were dialyzed for 5 h with high-flux dialyz- Results are presented as mean 1 standard deviation (SD). The medi- ers three times a week. Nine patients had diabetes mellitus (DM).
cations for hypertension and dyslipidaemia (renin–angiotensin Thirteen patients had angiographically proven CVD and/or a history system (RAS) inhibitors and statins) and the presence or absence of of coronary heart disease. The remainders had no history of CVD.
DM were coded as dummy variables. Because of their skewed dis- Forty-one patients received inhibitors of the renin–angiotensin tributions, a natural logarithmic (ln) transformation was performed system, and only two patients received statins for the treatment of for hsCRP. Univariate analysis was performed for determinants of dyslipidaemia. We excluded any patients with inflammatory, neo- hsCRP. To determine independent determinants of hsCRP and tissue plastic disorders and those who had a recent (<3 months) acute AGE, multiple stepwise regression analysis was performed. PI levels coronary syndrome, stroke and any acute infection.
stratified by tissue AGE and hsCRP levels were compared usingunpaired Student’s t-test. Statistical significance was defined asP < 0.05. All statistical analyses were performed with SPSS software(SPSS, Chicago, IL, USA).
Data collection
The medical history was ascertained by a questionnaire. Height and weight were measured, and body mass index (BMI) (kg/m2)was calculated as an index of presence or absence of obesity. Blood Demographic data are shown in Table 1. Univariate analyses pressure (BP) was measured in the sitting position using an showed that age (b = 0.298, P = 0.03), white blood cells upright standard sphygmomanometer. Vigorous physical acti- (WBC) (b = 0.294, P = 0.03), serum albumin (b = -0.506, vity and smoking were avoided for at least 30 min before BP P < 0.001), alkaline phosphatase (ALP) (b = -0.275, P = 0.04), TRAP5b (b = -0.412, P = 0.002) and skin AGE levels Blood was drawn from arteriovenous shunt just before starting HD for determinations of lipids (total cholesterol, low-density lipo- (b = 0.378, P = 0.005) were significantly correlated with protein (LDL) cholesterol and triglycerides), haemoglobin, haemo- serum levels of hsCRP (Table 2). Because these significant globin A1c (HbA1c), albumin, blood urea nitrogen (BUN), creatinine parameters could be closely correlated with each other, mul- (Cr), uric acid, Ca, P and Fe. Intact parathyroid hormone (PTH) was tiple regression analysis was performed. Multiple stepwise evaluated by an immunoradiometric assay (IRMA, Allegro Intact regression analysis revealed that serum albumin (b = -0.431, PTH; Nichols Institute, San Juan Capistrano, CA, USA). Tartrate- P < 0.001), TRAP5b (b = -0.390, P < 0.001) and skin AGE resistant acid phosphatase 5b (TRAP5b) activity, a bone resorption levels (b = 0.308, P = 0.004) were independent determinants marker unaffected by renal function, was measured by a novel of hsCRP (Table 2). We also found that the presence of DM fragment-absorbed immunocapture enzymatic assay (FAICEA) as (b = 0.385, P = 0.002) and hsCRP (b = 0.379, P = 0.002) described previously.21 High-sensitivity CRP (hsCRP) was measured were independently associated with skin AGE levels in our with nephelometry (N-Latex, CRPII; Dade Behring, Tokyo, Japan).
The other chemistries were measured at a commercially availablelaboratory as described previously (Wako Pure Chemical Industries, Further, we found that hsCRP was significantly correlated Osaka, Japan). HD adequacy was evaluated by a single-pool frac- with PI (b = 0.396, P = 0.003) and that hsCRP was one of the tional clearance of body water for urea (Kt/V).22 Single-pool Kt/V independent determinants of PI in our patients (R2 = 0.311, Nephrology 2011 Asian Pacific Society of Nephrology Table 1 Clinical characteristics of the patients
each other, both of which could in concert contribute to theprogression of atherosclerosis in patients with HD.
In this study, we evaluated tissue AGE levels by measuring skin AF because: (i) measurement of skin AF with a com- mercially available AGE reader is a non-invasive, reliable and time-saving method to estimate tissue accumulation of AGE;3 (ii) skin AF values are reported to significantly corre- late with skin AGE levels evaluated by high-performance liquid chromatography (HPLC) in both diabetic and non- diabetic subjects;20 and (iii) skin AF was positively associated with the severity of vascular complications in patients with both type 1 and type 2 diabetes mellitus and a predictor of future cardiovascular events and death in patients with HD.19,20 Skin AF levels in our HD patients were 2.84 1 0.71, whose value was 1.6-fold higher than that in age- and sex- matched Japanese controls.24 The results were consistent with the previous findings of Arsov et al., who showed that skin AF values were increased by approximately 1.4-fold in HD patients compared with controls.25 These observations further support the concept that measurement of skin AF with an AGE reader is a reliable diagnostic tool to evaluate tissue AGE accumulation in HD patients.
This study was cross-sectional one and thus could not assess the questions of whether elevation of tissue AGE level was a cause or consequence of chronic inflammation. However, we have previously shown that AGE could induce CRP produc- tion by hepatoma cells through oxidative stress and inflam- matory reactions.26 Further, inflammation is shown to cause Values are shown as mean 1 standard deviation. AGE, advanced glycation end oxidative stress generation, which could lead to promotion of products; ALP, alkaline phosphatase; a.u., arbitrary units; BUN, blood urea the formation and accumulation of AGE.16 These observations nitrogen; HbA1c, haemoglobin A1c; HD, haemodialysis; hsCRP, high-sensitivity suggest that decreased renal clearance of AGE in HD patients C-reactive protein; LDL, low-density lipoprotein; PI, pulsatility index; PTH, par- could contribute to enhanced tissue accumulation of AGE and athyroid hormone; RAS, renin–angiotensin system; TRAP5b, tartrate-resistant subsequently evoke inflammatory reactions, which may in turn further stimulate the formation and accumulation ofAGE. In other words, AGE accumulation and inflammatory Then, in order to examine the relationships among skin reactions may be correlated with each other in patients with AGE, hsCRP and carotid PI, we divided the study participants HD, which could account for the positive correlations into four groups according to their AGE (low AGE, Յ2.84; between skin AF and hsCRP levels in our subjects. Tan et al.27 high AGE, >2.85) and hsCRP (low hsCRP, log Յ2.42; high reported that serum AGE levels were independently associ- hsCRP, log >2.43) levels. As shown in Figure 1, carotid PI was ated with CRP in patients with type 2 diabetes as well.
found to be highest among HD patients with high skin AGE In the present study, PI, a surrogate marker of atheroscle- rosis, which reflects the degree of vascular resistance,25,28 ishighest among HD patients with high skin AGE and high DISCUSSION
hsCRP levels. Nakatou et al. have demonstrated that the PI issignificantly correlated with the atherosclerosis risk score The salient findings of this study are: (i) tissue AGE levels and the existence of cerebral infarction in diabetic patients.28 evaluated by skin AF with a specific AGE reader is one of the There is a growing body of evidence that AGE could elicit independent determinants of hsCRP levels in HD patients; vascular inflammation and thrombogenesis, playing a central (ii) besides the presence of DM, hsCRP is a sole determinant role in atherosclerosis. Further, recently, CRP has been of skin AGE levels; and (iii) carotid PI, a surrogate marker of shown to evoke endothelial cell damage, pro-inflammatory atherosclerosis, is highest among HD patients with high reactions and smooth muscle cell proliferation, thereby being tissue AGE and high hsCRP levels. Therefore, our present involved in the pathogenesis of CVD as well. An enhanced study suggests that: (i) tissue accumulation of AGE might be positive feedback loop between tissue AGE accumulation one of the causative factors for the elevation of hsCRP in HD and CRP induction as described above could in concert patients; and (ii) tissue AGE and CRP are correlated with contribute to the development and progression of atheroscle- 2011 The AuthorsNephrology 2011 Asian Pacific Society of Nephrology Table 2 Univariate and multiple stepwise regression analysis for the correlates of log hsCRP
<0.001
<0.001
<0.001
Bolded text indicates statistically significant values. b, standardized regression coefficients. r2 = 0.495. ALP, alkaline phosphatase; BP, blood pressure; BUN, bloodurea nitrogen; CVD, cardiovascular disease; DM, diabetes mellitus; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; PTH, parathyroidhormone; TRAP5b, tartrate-resistant acid phosphatase 5b; SE, standard error; WBC, white blood cell.
subject population and ethnicity could account for the In this study, low serum albumin and TRAP5b levels were also independently correlated with hsCRP. There is accumu- lating evidence to show that malnutrition is involved ininflammation and atherosclerosis in chronic kidney disease patients.30 Further, chronic inflammation is shown to impair bone turnover in HD patients.31 The findings suggest that lowserum albumin and low TRAP5b levels could reflect malnu- trition and impaired bone turnover in our patients, respec-tively, which could explain the link between high hsCRP and The study was a cross-sectional non-intervention one.
Therefore, it did not elucidate the causative relationships between tissue AGE and hsCRP levels. However, as described above, AGE are reported to stimulate CRP production by the liver in vitro.28 So, it is conceivable that tissue AGE accumula- tion could be one of the causative factors for the elevation ofhsCRP in HD patients. Tissue AGE accumulation and hsCRP elevation may act in concert to promote atherosclerosis inthese patients. Longitudinal and/or interventional studies are Fig. 1 Relationships among skin AGE, hsCRP and carotid PI in HD patients.
AGE, advanced glycation end products; hsCRP, high-sensitivity C-reactive
needed to clarify whether suppression of AGE formation could decrease tissue AGE and hsCRP levels and subsequentlyreduce the risk of future cardiovascular events in HD patients.
In this study, we measured tissue AGE levels by evaluating rosis in HD patients. However, it should be noted that Mulder skin AF with an AGE reader. However, we have to say that et al. reported that skin AF values were elevated in patients there are some limitations of this method: (i) not only tissue with stable coronary artery disease, but not correlated with AGE, but also other fluorescent contents of skin are measured CRP levels.29 We did not know the exact reasons for the with this method; (ii) some AGE are not fluorescent; and (iii) discrepant results between ours and theirs. The difference of the measurement could be influenced by skin abnormalities.32 Nephrology 2011 Asian Pacific Society of Nephrology ACKNOWLEDGEMENT
16. Yamagishi S. Advanced glycation end products and receptor- oxidative stress system in diabetic vascular complications. Ther. Dr Fukami is supported by a Grant-in-Aid for Scientific Apher. Dial. 2009; 13: 534–9.
Research (C) from the Ministry of Education, Culture, 17. Kilhovd BK, Juutilainen A, Lehto S et al. High serum levels of Sports, Science and Technology (Tokyo, Japan) (no.
advanced glycation end products predict increased coronary heart disease mortality in nondiabetic women but not in nondiabeticmen: a population-based 18-year follow-up study. Arterioscler.
Thromb. Vasc. Biol. 2005; 25: 815–20.
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2011 The AuthorsNephrology 2011 Asian Pacific Society of Nephrology

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