Tissue level of advanced glycation end products is an independent determinant of highsensitivity creactive protein levels in haemodialysis patients
Tissue level of advanced glycation end products is an independent
determinant of high-sensitivity C-reactive protein levels in
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
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)
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.
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
.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
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-
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).
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
< 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-
< 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
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 (R
2 = 0.311,
Nephrology 2011 Asian Paciﬁc Society of Nephrology
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
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
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
Univariate and multiple stepwise regression analysis for the correlates of log hsCRP
Bolded text indicates statistically signiﬁcant values. b, standardized regression coefﬁcients. r
2 = 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
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
. 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
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
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.
18. Kilhovd BK, Juutilainen A, Lehto S et al
. Increased serum levels
of advanced glycation endproducts predict total, cardiovascular
1. Rayner HC, Pisoni RL, Bommer J et al
. Mortality and
and coronary mortality in women with type 2 diabetes: a
hospitalization in haemodialysis patients in five European
population-based 18 year follow-up study. Diabetologia
countries: results from the dialysis outcomes and practice patterns
study (DOPPS). Nephrol. Dial. Transplant.
2004; 19: 108–20.
19. Lutgers HL, Graaff R, Links TP et al
. Skin autofluorescence as a
2. Ganesh SK, Hulbert-Shearon T, Port FK, Eagle K, Stack AG.
noninvasive marker of vascular damage in patients with type 2
Mortality differences by dialysis modality among incident ESRD
diabetes. Diabetes Care
2006; 29: 2654–9.
patients with and without coronary artery disease. J. Am. Soc.
20. Meerwaldt R, Hartog JW, Graaff R et al
. Skin autofluorescence, a
measure of cumulative metabolic stress and advanced glycation
3. Bevc S, Sabic S, Hojs R. Atherosclerosis in hemodialysis patients –
end products, predicts mortality in hemodialysis patients. J. Am.
the role of microinflammation. Ren. Fail.
2008; 30: 1012–16.
2005; 16: 3687–93.
4. Zimmermann J, Herrlinger S, Pruy A, Metzger T, Wanner C.
21. Yamada S, Inaba M, Kurajoh M et al
. Utility of serum tartrate-
Inflammation enhances cardiovascular risk and mortality in
resistant acid phosphatase (TRACP5b) as a bone resorption marker
hemodialysis patients. Kidney Int.
1999; 55: 648–58.
in patients with chronic kidney disease: independence from renal
5. Piroddi M, Depunzio I, Calabrese V et al
. Oxidatively-modified and
dysfunction. Clin. Endocrinol. (Oxf.)
2008; 69: 189–96.
glycated proteins as candidate pro-inflammatory toxins in uremia
22. Daugirdas JT. Linear estimates of variable-volume, single-pool
and dialysis patients. Amino Acids
2007; 32: 573–92.
Kt/V: an analysis of error. Am. J. Kidney Dis.
1993; 22: 267–70.
6. Suliman ME, Heimburger O, Barany P et al
. Plasma pentosidine is
23. Gosling RG, King DH. Arterial assessment by Doppler-shift
associated with inflammation and malnutrition in end-stage renal
ultrasound. Proc. R. Soc. Med.
1974; 67: 447–9.
disease patients starting on dialysis therapy. J. Am. Soc. Nephrol.
24. Matsumoto T, Tsurumoto T, Baba H et al
. Measurement of
advanced glycation endproducts in skin of patients with
7. Yu TM, Chen YH, Hsu JY et al
. Systemic inflammation is associated
rheumatoid arthritis, osteoarthritis, and dialysis-related
with pulmonary hypertension in patients undergoing
spondyloarthropathy using non-invasive methods. Rheumatol. Int.
haemodialysis. Nephrol. Dial. Transplant.
2009; 24: 1946–51.
8. Krane V, Winkler K, Drechsler C, Lilienthal J, Marz W, Wanner C.
25. Arsov S, Graaff R, Morariu AM et al
. Does hepatitis C increase the
Association of LDL cholesterol and inflammation with
accumulation of advanced glycation end products in haemodialysis
cardiovascular events and mortality in hemodialysis patients with
patients? Nephrol. Dial. Transplant.
type 2 diabetes mellitus. Am. J. Kidney Dis.
2009; 54: 902–11.
26. Yoshida T, Yamagishi S, Nakamura K et al
. Telmisartan inhibits
9. Cai W, Zhu L, Chen X, Uribarri J, Peppa M. Association of
AGE-induced C-reactive protein production through
advanced glycoxidation end products and inflammation markers
downregulation of the receptor for AGE via peroxisome
with thrombosis of arteriovenous grafts in hemodialysis patients.
proliferator-activated receptor-gamma activation. Diabetologia
Am. J. Nephrol.
2006; 26: 181–5.
10. Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end
27. Tan KC, Chow WS, Tam S, Bucala R, Betteridge J. Association
products in tissue and the biochemical basis of diabetic
between acute-phase reactants and advanced glycation end
complications. N. Engl. J. Med.
1988; 318: 1315–21.
products in type 2 diabetes. Diabetes Care
2004; 27: 223–8.
11. Grandhee SK, Monnier VM. Mechanism of formation of the
28. Nakatou T, Nakata K, Nakamura A, Itoshima T. Carotid
Maillard protein cross-link pentosidine. Glucose, fructose, and
haemodynamic parameters as risk factors for cerebral infarction in
ascorbate as pentosidine precursors. J. Biol. Chem.
Type 2 diabetic patients. Diabet. Med.
2004; 21: 223–9.
29. Mulder DJ, van Haelst PL, Gross S et al
. Skin autofluorescence is
12. Dyer DG, Blackledge JA, Thorpe SR, Baynes JW. Formation of
elevated in patients with stable coronary artery disease and is
pentosidine during nonenzymatic browning of proteins by glucose.
associated with serum levels of neopterin and the soluble receptor
Identification of glucose and other carbohydrates as possible
for advanced glycation end products. Atherosclerosis
precursors of pentosidine in vivo
. J. Biol. Chem.
30. Stenvinkel P, Heimburger O, Paultre F et al
. Strong association
13. Makita Z, Radoff S, Rayfield EJ et al
. Advanced glycosylation end
between malnutrition, inflammation, and atherosclerosis in
products in patients with diabetic nephropathy. N. Engl. J. Med.
chronic renal failure. Kidney Int.
1999; 55: 1899–911.
31. Eleftheriadis T, Kartsios C, Antoniadi G et al
. The impact of chronic
14. Vlassara H. Recent progress in advanced glycation end products
inflammation on bone turnover in hemodialysis patients. Ren. Fail.
and diabetic complications. Diabetes
1997; 46 (Suppl 2): S19–25.
15. Nakamura T, Sato E, Fujiwara N et al
. Positive association of serum
32. Mulder DJ, Water TV, Lutgers HL et al
. Skin autofluorescence, a
levels of advanced glycation end products and high mobility group
novel marker for glycemic and oxidative stress-derived advanced
box-1 with asymmetric dimethylarginine in nondiabetic chronic
glycation endproducts: an overview of current clinical studies,
kidney disease patients. Metabolism
2009; 58: 1624–8.
evidence, and limitations. Diabetes Technol. Ther.
2006; 8: 523–35.
2011 The AuthorsNephrology 2011 Asian Paciﬁc Society of Nephrology
XIX FIGO WORLD CONGRESS OF GYNECOLOGY AND OBSTETRICS INDUSTRY-SPONSORED SYMPOSIA Monday, 5 October 2009 13h00-14h15 Ballroom East (CTICC) Contraception and Beyond: Evidence-based Indications for LNG-IUS 1. Wider Use of Intrauterine Contraception - David Grimes 2. The LNG-IUS in Heavy Menstrual Bleeding: First-line Treatment Based on Comprehensive Clinical Data - Anita Nelson3. S
Das in der letzten Woche vom französischen Pharma-konzern präsentierte Zahlenwerk entsprach weitestge-hend den Erwartungen. Dabei sorgte der Wegfall von wichtigen Einzelpatenten zu Ergebnisbelastungen. Sa-nofi hatte sich jedoch in den vergangenen Jahren auf diesen Fall vorbereitet und die eigene Umsatzbasis gezielt erweitert. Die unter dem Vehikel "Wachstums-plattform" zusam