Reference Intervals for Serum Cystatin C and Factors Influencing Cystatin C Levels Other than Renal Function in the Elderly
Zhao W (2014) Reference Intervals for Serum Cystatin C and Factors Influencing Cystatin C Levels Other than Renal Function in
the Elderly. PLoS ONE 9(1): e86066. doi:10.1371/journal.pone.0086066
Reference Intervals for Serum Cystatin C and Factors Influencing Cystatin C Levels Other than Renal Function in the Elderly
Lu Wei 0
Xiaoshuang Ye 0
Xiaohua Pei 0
Jianqing Wu 0
Weihong Zhao 0
Antonio Carlos Seguro, University of Sao Paulo School of Medicine, Brazil
0 Department of Geriatrics, the First Affiliated Hospital of Nanjing Medical University , Nanjing, Jiangsu , China
Objective: The present study aimed to establish reference intervals for serum cystatin C (Scys-C) stratified by stages of chronic kidney disease, explore factors influencing Scys-C and compare the performance of Scys-C with serum creatinine (Scr) in the young and elderly. Methods: A total of 800 participants, 516 young (,60 years) and 284 old ($60 years) subjects were included in this study. Scys-C and Scr were assayed by the partical-enhanced immunoturbidimetry method and enzymatic method respectively. 95% reference interval was adopted to evaluate reference intervals. Influencing factors were characterized by multivariate linear regression analysis. Relationship between reference glomerular filtration rate (rGFR) and Scys-C or Scr was determined by correlation coefficient. Results: Reference intervals for Scys-C were calculated to be 0.71-1.38 mg/L, 0.83-1.67 mg/L, 1.02-2.61 mg/L, 1.324.48 mg/L, 1.95-6.11 mg/L in the aged in CKD G1, G2, G3a, G3b and G4-5 stages, respectively. Body mass index(BMI), nephritis, kidney neoplasm and hypertension were demonstrated as factors affecting Scys-C in the elderly while gender, nephritis and kidney neoplasm were clarified as influencing factors in the young group. Scr levels were affected by more factors, such as body surface area and hematological disease. Correlation coefficient between rGFR and Scys-C or Scr showed that serum Scys-C was superior to Scr, especially in the subjects with mildly decreased renal function (20.593 vs. 20.520). Conclusions: Factors other than renal function influenced Scys-C when applying to evaluate glomerular filtration rate (GFR), such as BMI, nephritis, kidney neoplasm and hypertension, and Scys-C had higher correlation with GFR than Scr in the elderly.
Funding: This work was supported by the Innovation of Science and Technology Achievement Transformation Fund of Jiangsu Province BL2012066, the National
Natural Science Foundation of China H0511-810705, and the grants from the Major State Basic Research Development Program of China 2013CB530803, a Project
Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions JX10231801. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Chronic kidney disease (CKD) and end-stage renal disease
(ESRD) in particular are major health problems worldwide with
dramatically increasing incidence and prevalence . Several
studies were conducted among the elderly and showed a markedly
high prevalence . In China, the incidence of CKD is 10.8%,
that is to say, there are at least 100 million patients with CKD .
The evaluation of glomerular filtration rate (GFR) is very
important to diagnosis of CKD. Thus, assessing GFR accurately
in early stage of CKD is essential for clinician to achieve explicit
diagnosis and take reasonable therapies.
As we know, GFR can be determined by measuring the
clearance of exogenous substances or evaluated by the serum or
urinary concentration of endogenous substances. Serum creatinine
(Scr) has been used to assess renal function as a traditional
endogenous substance for many years, but several factors other
than renal function have been certified to affect Scr level, for
instance muscle mass, age, gender and malnutrition etc . Inulin
clearance or nuclear medicine techniques such as 99Tc DTPA or
51Cr EDTA, which is considered as the golden standard
measurement of GFR, is expensive, cumbersome and invasive
. Thus, a new, convenient and relatively accurate endogenous
substance is needed to evaluate renal function for clinical
Serum cystatin C (Scys-C) recently was proposed as a promising
alternative marker of GFR owing to better specificity and
sensitivity for detecting mildly decreased GFR. However, several
studies have reported that Scys-C was superior to Scr as a marker
of GFR [18,19], in contrast, some studies didnt show this
advantage [4,20]. The existence of factors other than renal
function influencing Scys-C may lead to this discrepancy especially
Serum cystatin C (mg/L)
Serum creatinine (mmol/L)
rGFR (ml/min/1.73 m2)
BMI: Body mass index, BSA: Body surface area, rGFR: Reference glomerular filtration rate measured by the 99mTc-DTPA renal dynamic imaging.
**P,0.01 compared with the young.
in the elderly, although these factors are not completely clear. In
addition, aging has becoming a serious social problem worldwide
, and failed physiological functions and pathologic
abnormalities in the elderly probably lead to the different performance of
Scr and Scys-C in evaluation of renal function between the young
and elderly. But, few studies focused on the comparison between
these two groups. Thus, further detailed studies are needed to
evaluate the performance of Scys-C compared with Scr, especially
in the elderly.
Therefore, the present study aimed to establish reference
intervals for Scys-C in subjects stratified by age and stages of
CKD and identified factors other than renal function influencing
Scys-C. Besides, the performance of Scys-C and Scr as a GFR
marker was compared in subjects stratified by age and early or
advanced stage of CKD.
Subjects and measurements
Totally 800 participants, 516 young (age,60 ys) and 284 old
subjects (age$60 ys), who were outpatients or inpatients of our
hospital from December 2009 to March 2013 were included. The
basic therapies of these participants were anti-hypertensive drugs,
oral hypoglycemic drugs, corticosteroid etc. Reference glomerular
filtration rate (rGFR) was measured by the 99mTc-DTPA renal
dynamic imaging on a single photon emission computed
tomography (Siemens E.CAM, Siemens Co., Ltd, Germany) ,
and Scys-C concentration was assayed by the partical-enhanced
immunoturbidimetry method (Beijing Leadman Biomedical Co.,
Coronary heart disease
**P,0.01 compared with the young.
Ltd, China) with a reference range of 0.601.55 mg/L.
Meanwhile, Scr levels were analyzed using enzymatic method on
Shanghai kehua Dongling Diagnostic Products with a reference
range of 44136 mmol/L. Both two markers were examined by an
Olympus AU5400 autoanalyzer (Olympus Co., Ltd, Japan). All
participants provided their written informed consent to participate
in this study and Nanjing Medical University Ethics committee
approved this study.
Included subjects were divided into ten groups stratified by age
(,60 ys and $60 ys) and stages of CKD according to decreased
degrees of GFR (G1, G2, G3a, G3b and G45 stages). According
to the KDIGO clinical practice guideline for evaluation and
management of CKD, it further acknowledges the importance of
dividing stage 3 based on data supporting different outcomes and
risk profiles in categories G3a (GFR 4559 ml/min/1.73 m2) and
G3b (GFR 3044 ml/min/1.73 m2) [8,9]. Thus, we divided the
stage 3 into G3a and G3b stages to calculate reference intervals
separately. Since Scys-C concentrations were not normally
distributed, the natural logarithm of Scys-C value which was
normally distributed after transformed was used for this analysis.
Reference intervals for Scys-C were calculated by 95% reference
interval. Single-factor ANOVA analysis was used to determine the
statistical significance of differences between groups stratified by
CKD stages. Simultaneously, the difference between the young
and elderly was evaluated using t-test.
Influencing factors and correlation coefficients
Multivariate linear regression analysis was performed to analyze
influencing factors other than renal function on Scys-C
concentrations. Scys-C and rGFR levels were ln-transformed. BMI, body
surface area (BSA) and age were expressed as the initial data.
Gender, nephritis, kidney neoplasm, hematological disease,
hypertension and diabetes mellitus (DM) were used as binary
factors. The same analysis was performed on Scr.
Correlation coefficients were assessed using Spearmans rank
correlation. We calculated R values between rGFR and Scys-C or
Scr in early stages and advanced stages respectively.
Statistical analysis was performed using SPSS 17.0 for
Windows. Data was presented as median.
Mann-WhitneyWilcoxon test and Chi-square test were adopted to determine
Reference interval Mean
Reference interval Mean
*P,0.05 compared with the young.
the statistical difference between the young and elderly. Two-tailed
P value of ,0.05 was considered to be statistically significant.
Characteristics of the included subjects were presented in
Table 1. The rGFR of the elderly was significantly lower than that
of the young, whereas, age, BMI, and the levels of Scys-C and Scr
in the elderly were higher than those in the young subjects. The
totally constituent ratio of hematological disease, hypertension,
coronary heart disease and DM was statistically different between
the young and elderly (Table 2), however, the constituent ratio of
all basic diseases had no age-related significant difference in the
G1 and G2 stages.
According to age and stages of CKD, all subjects were divided
to ten groups, and the corresponding reference intervals, means
and medians of Scys-C of each group were shown in Table 3.
Reference intervals for Scys-C were calculated to be
0.561.27 mg/L, 0.721.51 mg/L, 0.982.61 mg/L, 1.574.01 mg/L,
1.8010.49 mg/L in the young group and 0.711.38 mg/L, 0.83
1.67 mg/L, 1.022.61 mg/L, 1.324.48 mg/L, 1.956.11 mg/L
in the elderly group in G1, G2, G3a, G3b and G4-5 stages,
respectively. Scys-C levels of the elderly were significantly higher
than those of the young with normal renal function (P = 0.001),
while the rGFR values were not significantly different between
above-mentioned two groups (P = 0.121), which suggested that age
was a factor influencing Scys-C in the patients with normal GFR.
BMI, Body mass index, BSA, Body surface area, rGFR, Reference glomerular filtration rate, Scys-C, serum cystatin C, Scr, serum creatinine.
The one-way ANOVA analysis and pairwise comparisons revealed
statistically significant difference of Scys-C between G1, G2, G3a,
G3b and G4-5 stages in both two groups, thus to the clinician,
Scys-C could be used to evaluate the decreased degree of GFR.
Table 4 showed the results of multivariate linear regression
analysis in all, the young and the elderly subjects. In terms of all
included subjects, gender, nephritis and kidney neoplasm were
significantly associated with serum concentrations of Scys-C
besides the age (r = 20.062, 0.155 and 20.072, respectively). In
the young group, nephritis was independently positively associated
with Scys-C concentrations (r = 0.146) while the female gender and
patients with kidney neoplasm had lower Scys-C concentrations
after adjustment for rGFR (r = 20.094 and 20.063, respectively),
whereas, BMI, hematological disease, hypertension and DM did
not have significantly correlation with Scys-C (P$0.05).
Compared with the young group, Scys-C was influenced by more
factors in the elderly. After adjustment for rGFR, kidney neoplasm
were negatively associated with Scys-C (r = 20.064) while BMI,
nephritis and hypertension were all significantly positively
associated with it (r = 0.020, 0.138 and 0.064, respectively), and
gender, hematological disease and DM did not have effect on
Scys-C in the elderly(P$0.05). As expected, Scr levels were
influenced by more factors, such as BSA, hematological disease
and the influencing power of the same factors was stronger than
Scys-C after adjustment for rGFR.
Figure 1. Relationship between rGFR and serum CysC or Scr stratified by age and progression of CKD (Scys-C, serum cystatin C, Scr,
serum creatinine, rGFR, reference glomerular filtration rate).
The correlation coefficients of Scys-C were consistently higher
than those of Scr in subjects stratified by age, and the young had
better correlation with rGFR than the elderly especially in the
early stage of CKD, but there was not significant difference in
correlation coefficients between Scys-C and Scr. Negative
relationship between rGFR and the Scys-C or Scr was shown
and Scys-C had greater correlation and accuracy than Scr (Table 5
and Figure 1).
Total rGFR$60 ml/min/1.73 m2
rGFR,60 ml/min/1.73 m2
,60 y rGFR$60 ml/min/1.73 m2
$60 y rGFR$60 ml/min/1.73 m2
rGFR,60 ml/min/1.73 m2
rGFR,60 ml/min/1.73 m2
Scr has been widely used to assess renal function for a while
though it can not accurately predict renal function because of its
poor sensitivity, especially in the early stage of CKD. Recently,
several studies have clarified that Scys-C is superior to Scr in
evaluation of GFR, thus Scys-C was proposed as an alternative
parameter to evaluate renal function. But, few studies
concentrated on reference intervals for Scys-C to evaluate progression of
CKD and factors other than renal function influencing Scys-C,
and fewer studies explored the difference between the young and
elderly. Therefore, the main objectives of our study were to
establish reference intervals for Scys-C and analyze factors other
than renal function influencing Scys-C.
Reference intervals were established by 95% reference interval
using the natural logarithm of Scys-C values and difference among
groups was evaluated by t-test and one-way ANOVA analysis. In
the early stage, defined as rGFR$60 ml/min/1.73 m2, reference
intervals of the elderly were significantly higher than those of the
young, but no significant difference of rGFR levels and the
constituent ratio of all basic diseases were found in G1 stage
between the two groups. Hence, age may be a factor influencing
Scys-C levels in patients with normal renal function. Besides, the
force of age would decrease along with the reduced GFR, then
decreased renal function would be the main influencing factor
taking place of age when GFR decreased below 60 ml/min/
1.73 m2. The one-way ANOVA analyses was adopted to examine
the difference between five stage groups, and the results showed
significant difference. These results may provide a reference to
clinician that Scys-C can be used as a parameter to assess the
progression of CKD, and provide evidence that the elderly have
higher Scys-C levels than the young when the renal function is
normal or mildly-decreased.
Some studies have investigated that many factors other than
renal function may influence Scys-C, and more studies are needed
for verification [10,11,12]. Multivariate linear regression analysis
was conducted in three groups, all subjects, the young and the
elderly. Female gender was associated with lower Scys-C in young
subjects (P,0.001), but in the elderly, it was not an influencing
factor (P = 0.836). Some studies revealed that Scys-C was
influenced by gender, while Yashiro et al. reported that Scys-C
levels were not gender-related [10,11,12]. Decreased secretion of
sex hormone in the elderly may contribute the difference between
these two groups. Stevens et al. reported that percent change of
Scys-C was higher than the value of Scr according to the change in
BMI (mean 27.7 kg/m2), and speculated an association between
fat mass and Scys-C . But, in this study, BMI (mean 22.84 kg/
m2) was not associated with Scys-C in the young group, similarly
with the results examined in patients from Japan, with a mean
BMI of 22.9 kg/m2 . Meanwhile, the mean of BMI in the
elderly was 23.47 kg/m2 and was significantly different from this
of the young group, which may explain statistically significant
effect of BMI on Scys-C in the old group. Race and the prevalence
of obesity may contribute to the inconsistent conclusions regarding
the association with BMI.
In both young and elderly groups, kidney neoplasm was
negatively associated with Scys-C concentrations. A review
showed that tumors have been suggested to influence Scys-C
production, although this is still widely debated . In the
present study, included subjects with kidney neoplasia may have a
relatively increased renal function, which result in the negatively
association between kidney neoplasm and Scys-C. Hence, more
studies are needed to clarify whether kidney neoplasm is an
influencing factor on Scys-C.
Nephritis acted as a positively correlative factor in both two
groups. The application of corticosteroid, which is essential drug in
the treatment of some kinds of nephritis may influence Scys-C
concentrations. The hypothesis that increased Scys-C was
associated with corticosteroid doses had been demonstrated in
asthmatics, adult renal transplant patients, and patients with
nephritis [12,14]. Besides, the patients with nephritis were in the
inflammatory status, and inflammation acted as an influencing
factor has been clarified by many studies, which found that Scys-C
was associated with many inflammatory markers like IL-6, TNF-a
but not with C-reactive protein (CRP) .
Hypertension, as a common chronic disease and classical
cardiovascular risk factor in the elderly, was positively associated
with Scys-C concentrations in the elderly. Some studies have
reported that the prevalence of an elevated Scys-C in the general
population was found to be high and was associated with the
presence of classical cardiovascular risk factors such as DM,
hypertension [12,16,17]. But, DM was not an influencing factor in
our study. This converse results may due to the diabetes sample
size and its low constituent ratio, and LA Stevens et al.
demonstrated that the association of higher Scys-C with diabetes
may, in part, also reflect the association with fat mass, which
inferred that low fat mass of included subjects may be another
reason leading to this variance . Given the high and increasing
prevalence of hypertension and DM, these are important
consideration to clinician when using Scys-C to evaluate renal
function, especially in the elderly.
Compared with Scys-C, Scr levels were affected by more factors
other than renal function, such as BSA, hematological disease.
Besides, the influencing power of the same factors was stronger on
Scr, especially in the elderly. This result suggested that considering
to the influencing factors other than renal function, Scys-C was
better than Scr in the evaluation of renal function.
The superior of Scys-C was more than in the terms of less
influencing factors, and the advantage on the correlation with
rGFR was clarified by correlation coefficient. We calculated the
correlation coefficient in early and advanced stages of CKD. The
performance of Scys-C was surpassing than Scr in both the young
and the elderly. But the advantage was not apparent in the old
subjects with normal to mildly-scathing renal function, and
correlation between Scys-C and rGFR of the old subjects was
worse than that of the young subjects though statistically
significant difference was not detected in both groups. Some
factors such as age and chronic underlying diseases which are
incidental in the elderly may result in this disadvantage. The
majority of compositions demonstrated that Scys-C was superior
than Scr in terms of correlation coefficient with rGFR while little
of these demonstrated if statistically significant difference existed,
but a part of those got opposite results [4,18,19,20].
Factors other than renal function influenced Scys-C when
applying to evaluate glomerular filtration rate, such as BMI,
nephritis, kidney neoplasm and hypertension, and Scys-C had
higher correlation with GFR than Scr in the elderly. But, the
difference was existed between the young and the elderly, so
clinician needs to consider the difference and the influencing
factors when applying Scys-C to evaluate renal function in the
elderly. In addition, the influencing factors also should be
considered when using Scys-C to creat equations to estimate GFR.
We thank Jianfeng Ma and Chengjing Yan for laboratory measurements,
and Lihua Bao and Zhaoqiang Xu for GFR measurement.
Conceived and designed the experiments: LW XHP WHZ JQW.
Performed the experiments: LW XSY. Analyzed the data: LW. Wrote
the paper: LW WHZ.
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