di ab etes re sea rch and c lini ca l pr ac tic e 1 3 9 (2 0 18 ) 4 3–51

Contents available at ScienceDirect

Diabetes Researchand Clinical Practicejournal homepage: www.elsevier.com/locat e/dia bre s

Serum cystatin C level is associated with carotid
arterial wall elasticity in subjects with type 2
diabetes mellitus: A potential marker of early-stage
atherosclerosis
Rei Kaneko a, Shojiro Sawada a,*, Ai Tokita a, Rieko Honkura a, Noriko Tamura a,
Shinjiro Kodama a, Tomohito Izumi a, Kei Takahashi a, Kenji Uno a, Junta Imai a, Tetsuya
Yamada a, Yukiya Miyachi b, Hideyuki Hasegawa c, Hiroshi Kanai d, Yasushi Ishigaki e,
Hideki Katagiri a,*a

Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, Japan
Medical Systems Research and Development Center R&D Management Headquarters, FUJIFILM Corporation, Kaisei, Japanc
Graduate School of Science and Engineering, University of Toyama, Toyama, Japand
Department of Electronic Engineering, Tohoku University Graduate School of Medicine, Sendai, Japane
Department of Internal Medicine, Division of Diabetes and Endocrinology, Iwate Medical University Hospital, Morioka, Japanb

A R T I C L E I N F O

A B S T R A C T

Article history:

Aims: Detection of early-stage atherosclerosis in type 2 diabetes mellitus (T2DM) patients is

Received 1 August 2017

important for preventing cardiovascular disease. A phased tracking method for evaluating

Received in revised form

arterial wall elasticity sensitively detects early-stage atherosclerosis. However, biochemical

29 January 2018

markers for early-stage atherosclerosis have yet to be established.

Accepted 1 February 2018

Methods: This cross-sectional study enrolled 180 T2DM patients, who were classified as not

Available online 15 February 2018

having atherosclerosis according to the carotid intima-media thickness (IMT) criteria. We
measured serum cystatin C, the estimated glomerular filtration rate (eGFR) and urinary

Keywords:
Early-stage atherosclerosis
Cystatin C
Arterial wall elasticity
Type 2 diabetes mellitus

albumin-to-creatinine ratio (ACR), and analyzed the associations between these markers
and arterial wall elasticity (Eh), IMT and the cardio-ankle velocity index.
Results: Multiple linear regression analyses revealed that cystatin C was significantly associated with Eh, while neither eGFR nor ACR showed an association. Furthermore, among
the examined atherosclerotic markers, Eh was most reliably associated with cystatin C.
Additionally, the association between cystatin C and Eh disappeared in the low elasticity
subgroup, which included subjects in whom no atherosclerotic changes had yet been initiated.
Conclusions: In T2DM patients without apparent arterial wall thickening, cystatin C isstrongly and independently associated with arterial wall elasticity, which reflects thedegree of subclinical atherosclerosis. Thus, cystatin C is a potentially useful marker of
early-stage atherosclerosis.
Ó 2018 Elsevier B.V. All rights reserved.

* Corresponding authors at: Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan.
E-mail addresses: ssawada@med.tohoku.ac.jp (S. Sawada), katagiri@med.tohoku.ac.jp (H. Katagiri).
https://doi.org/10.1016/j.diabres.2018.02.003
0168-8227/Ó 2018 Elsevier B.V. All rights reserved.

44

1.

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1Introduction

Type 2 diabetes mellitus (T2DM) is associated with atherosclerosis, which leads to cardiovascular disease and increased
mortality [1]. However, it is difficult to detect atherosclerosis
at its early stages. Arterial stenosis often develops without
symptoms in T2DM patients [2], subsequently leading to cardiovascular events. Therefore, development of strategies foreasily detecting early-stage atherosclerosis, such as identifying reliable biochemical markers, is particularly importantin those with T2DM.
Measurement of carotid intima-media thickness (IMT) is
widely used for atherosclerosis screening in clinical practice
but this examination detects morphological changes ofatherosclerosis. For detecting early-stage atherosclerosis
before the appearance of such morphological changes, wedeveloped a novel non-invasive ultrasonic phased tracking
method, which evaluates deformity at multiple sites in arterial walls during one heartbeat [3–6]. Since the arterial wall
deforms less easily at harder sites during a single heartbeat,this innovative phased tracking method has enabled us toquantitatively analyze arterial wall elasticity. The results
obtained with this method correspond to pathological findings in the extracted human iliac artery [5]. In addition, we
reported that phased tracking arterial wall elasticity, but not
IMT, pulse wave velocity (PWV) or the plaque score, is associated with an increasing number of coronary risk factors suchas diabetes, hypertension, hyperlipidemia and current smoking, in T2DM patients with IMT <1.1 mm [7], who were classified as not having atherosclerosis according to the IMT
criteria [8]. The elasticity in the arterial wall is also associatedwith visceral fat mass in subjects with obesity [9] and anincreasing number of coronary risk factors in T2DM patients
who have not developed apparent cardiovascular disease[10]. Furthermore, arterial wall elasticity is reportedly
increased in smokers, as compared with non-smokers [11],and in subjects with the metabolic syndrome, as comparedwith those without metabolic syndrome [12], among subjects
with IMT <1.1 mm. Collectively, these findings indicate that
arterial wall elasticity is useful for detecting early-stage
atherosclerosis, i.e. that developing before IMT increases
detectably. Therefore, in order to search for biochemicalmarkers of early-stage atherosclerosis, we sought serum
markers which have a strong association with arterial wall
elasticity values.
In the present study, we focused on serum cystatin C. Cystatin C, a 13-kD endogenous cysteine proteinase inhibitor, isconsidered to be a more reliable marker for glomerular filtration rates than the estimated glomerular filtration rate (eGFR)[13]. Moreover, cystatin C was reported to be associated withplaque morphology using multi-detector computed tomography [14] and coronary angiography [15], even in patients without established chronic kidney disease. Furthermore, Wanget al. demonstrated cystatin C, but not eGFR, to be associatedwith the presence of cardiovascular disease in subjects withmild renal impairment [16]. Similar findings, indicating thatcystatin C is superior to eGFR for predicting cardiovascular
events, were also reported in subjects with diabetes [17] aswell as those with [18] and without [19] cardiovascular dis-ease. Thus, cystatin C is not simply regarded as a marker of
impaired kidney function but also as one which reflects cardiovascular disease due to advanced atherosclerosis. However, the association of cystatin C and early-stage
atherosclerosis remains unclear.
Herein, to assess whether the serum cystatin C level islinked to, and useful for detecting, early-stage atherosclerosis, we enrolled T2DM patients who had IMT of less than
1.1 mm, the cut-off value for a plaque lesion, and analyzed
the associations of cystatin C with examinations for assessing
atherosclerosis, such as Eh (an indicator of arterial wall elasticity) as well as IMT and the cardio-ankle vascular index(CAVI). We also compared the utility of cystatin C as an
early-stage atherosclerosis marker with those of other renalmarkers, such as eGFR and urinary albumin excretion.

2.

Materials and methods

2.1.

Study subjects

This was a cross-sectional study consisting of 180 subjects
with T2DM. The subjects were recruited from patients with
T2DM admitted to the Department of Metabolism and Diabetes, Tohoku University Hospital for glycemic control during
the period from January 2012 to December 2015. T2DM wasdefined according to the World Health Organization (WHO)criteria, or treatment for diabetes mellitus. The exclusion criteria for the enrollment in this study were pregnancy, thyroid
disease, tumor and severe inflammation, because these conditions are known to affect cystatin C levels. Subjects withatrial fibrillation were also excluded because the intima
media complex (IMC) during one heart beat was used for calculating Eh. Since this study aimed to evaluate renal markers
in early-stage atherosclerosis, subjects with IMT 1.1 mm,
were also excluded. The study protocol was approved by the
Tohoku University Institutional Review Board, in accordancewith the World Medical Association Declaration of Helsinki.
Written informed consent was obtained from each patient.
We used the following criteria for cardiovascular risk factors. Hyperlipidemia was defined as total cholesterol  5.7
mmol/l (220 mg/dl) and/or triglycerides 1.7 mmol/l (150mg/dl), based on the definition proposed by the Japan
Atherosclerosis Society, or taking anti-hyperlipidemic drugs.
The subjects whose systolic blood pressure 140 mmHg
and/or diastolic blood pressure 90 mmHg, based on the definition proposed by the Japanese Society of Hypertension, or
taking anti-hypertensive drugs were defined as having hypertension. The subjects who currently smoked were classified
as current smokers.

2.2.

Measurement of Eh

Eh in the carotid artery was evaluated using the phased
tracking method. The arteries were scanned with ultrasonicdiagnostic equipment (FUJIFILM FAZONE M) and a linear typeprobe with a frequency range of 5–10 MHz (FUJIFILM FZT
L10-5 probe) in the supine position. By B-mode ultrasound,
the IMC was longitudinally scanned at two points on the
far wall of the common carotid artery, 1 cm and 3 cm prox-

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1

imal to the bifurcation. The portion of the IMC including plaques was excluded. The plaque was defined as a focal raised
lesion, using an IMT cut-off value of 1.1 mm [8]. The quadrature demodulated signals of the IMC during 8 heartbeats
were obtained with a high frame rate of 200 Hz. From these8 heartbeats, we selected one heartbeat which yielded themost precise image. The sampling interval of the quadrature
demodulated signal and the pitch of scan lines were 0.035
and 0.12 mm, respectively. The quadrature demodulated signals were saved as raw data to a personal computer. A single
well-trained sonographer (N.T.), who was unaware of theclinical characteristics of the subjects, analyzed the raw data
to introduce Eh based on the principal described previously[20] (Supplementary Fig. 1). Multiple points were preset fromthe luminal surface to the adventitia along each beam.
Then, the displacement of each point during one heartbeat
was obtained by applying the phased tracking method [3–6]. Er, which is the elastic modulus in the radial directionof each of the sampled points, was calculated using the following equation: Er = DP/e, where e = Dh/h0, and Dh is the
maximum decrease in the thickness of the arterial wall during one heartbeat. DP is the pulse pressure, defined as thedifference between systolic and diastolic brachial blood pressure. The h0 and r0 variables are the initial thickness andradius of the vessel at end-diastole, respectively. Eh, whichis the elastic modulus in the circumferential direction was
calculated using the following equation: Eh = (3/8)  (2r0/h0+ 1)  Er. The intraobserver coefficient of variation for Eh
was 7.0%.
Due to the absence of normative data for Eh, we referred to
the Eh values from the data of a healthy population fromanother cohort. The Sendai knowledge cluster initiative organized by the Ministry of Education, Culture, Sports, Scienceand Technology, Japan, performed population-based healthcare examinations including the determination of Eh in the
carotid artery. After excluding hypertension, hyperlipidemia,
and diabetes mellitus, 156 cases were defined as healthy subjects. Their mean age was 47.7 ± 10.3 years and Eh was 228.3 ±72.0 kPa (unpublished data).

2.3.

Measurement of IMT

IMT of the common carotid artery was measured employing
the same scanning image, as that mentioned above for themeasurement of Eh. IMT was measured at a point on the far
wall of the common carotid artery, in a 1 cm segment proximal to the bifurcation in a region free of plaque [21]. The
mean value of the bilateral measurements was used for the
analysis. The intraobserver coefficient of variation for IMT
was 8.3%.

2.4.

45

s/Pd)PWV2} + b, where Ps is systolic blood pressure, Pd is diastolic blood pressure, PWV is pulse wave velocity, DP is Ps Pd,
q is blood density, and a and b are both constant.

2.5.

Biochemical markers of renal function

The eGFR was calculated using the formula recommended by
the Japanese Society of Nephrology [23], derived from theModification of Diet in Renal Disease (MDRD) study group.
The urinary albumin excretion is presented as the albuminto-creatinine ratio (ACR; mg/g creatinine). Cystatin C wasassayed using colloidal gold immunoassay (Nescaute GC cystatin C; Alfresa Pharma, Osaka, Japan).

2.6.

Statistical analysis

Continuous variables were expressed as means ± S.D. or
medians (inter-quantile range (IQR)). The difference between
2 groups was determined using the unpaired t-test for continuous variables and Fisher’s exact test for non-continuous
variables. Data were logarithmically transformed before statistical analysis, if they were not normally distributed. Pearson correlation was used for correlation analysis. Multiple
linear regression analysis was performed to evaluate theindependent parameters that were found to be significantly
related to the objective variable. The independent parameters
tested were age, male gender, systolic blood pressure, hyperlipidemia, HbA1c, BMI, duration of diabetes, treatment withRAS inhibitors, calcium channel blockers, other antihypertension
agents, statins,
fibrates,other antihyperlipidemic agents, insulin, GLP-1 receptor agonists andother anti-diabetic agents, eGFR, cystatin C and log ACR.
Among these variables, no extreme collinearities were recognized. Receiver-operating characteristic (ROC) curve for cystatin C was generated to determine the optimal cut-off
value for high elasticity of the arterial wall. We computed
the sensitivity and specificity for high elasticity of the arterial
wall using this cut-off value for cystatin C. As for sample size
calculation, the correlation coefficient of cystatin C and Eh
was estimated 0.25 from our preliminary study. We calculated
that 164 patients would be needed at a significance level a = 0.
05, with power of 90%. The number of required patients wasthus set at 180. A level of p < 0.05 was considered to indicate
a statistically significant association. Analyses were carriedout with JMP 11.2 Pro statistical analysis software (SAS Institute, Cavy, NC, USA).

3.

Results

3.1.

Subject characteristics

Measurement of CAVICAVI was measured using a pulse wave analytical instrument(VaSera; Fukuda Denshi, Tokyo, Japan) as described previously[22]. After a 10 min rest, cuffs were applied to the bilateral
upper arms and ankles to detect each of the pulse waves.
Electrocardiograph, phonocardiograph, and blood pressures
were measured. CAVI was calculated using a conventional
PWV measurement with the formula: CAVI = a{(2q/DP)  ln(PAll 180 diabetic subjects enrolled in this study had IMT ofless than 1.1 mm, the cut-off value for detecting atherosclerosis with this examination. The clinical characteristics ofthese subjects are shown in Table 1. Overall, 56.7% of thesubjects were male, mean age was 52.0 ± 15.6 years, the diabetes duration was 8.0 (3.0–15.0) years, BMI was 27.3 ± 6.2 kg/
m2, 64.4% of the subjects had hypertension, 70.0% had
hyperlipidemia.

46

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1

Table 1 – Subject characteristics.

Male gender (%)
Age (years)
Duration of diabetes (years)
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Fasting plasma glucose (mg/dl)
HbA1c (%)
Total cholesterol (mg/dl)
Triglyceride (mg/dl)
HDL cholesterol (mg/dl)
LDL cholesterol (mg/dl)eGFR (ml/min/1.73 m2)
Cystatin C (mg/l)
ACR (mg/g creatinine)
Hypertension (%)
Hyperlipidemia (%)
Current smokers (%)
Eh (kPa)
IMT (mm)CAVI
RAS inhibitors (%)
Calcium channel blockers (%)
Other agents for hypertension (%)
Statins (%)
Fibrates (%)
Other agents for hyperlipidemia (%)
Insulin (%)
GLP-1 receptor agonists (%)
Other agents for diabetes (%)

All subjects (n = 180)

Low Eh (n = 90)

High Eh (n = 90)p

56.7
52.0 ± 15.6
8.0 (3.0–15.0)
27.3 ± 6.2
118.3 ± 15.7
73.5 ± 11.5
151.8 ± 54.1
10.0 ± 2.0
182.8 ± 41.6
131.5 (93.0–175.3)
43.6 ± 12.6
108.5 ± 35.6
85.9 ± 29.5
0.94 ± 0.30
15.6 (6.3–52.3)
64.4
70.0
35.8
259.1 ± 82.1
0.70 ± 0.16
7.9 ± 1.6
55.6
33.3
16.1
45.6
3.9
12.2
57.2
7.2
80.0

50.0
48.0 ± 15.1
6.0 (2.0–13.0)
27.9 ± 6.0
114.8 ± 13.4
74.0 ± 9.8
157.0 ± 58.5
10.3 ± 2.1
188.6 ± 41.8
132.5 (94.8–193.3)
43.7 ± 13.4
115.0 ± 34.7
92.0 ± 26.8
0.89 ± 0.21
15.7 (6.4–38.0)
54.4
68.9
38.6
197.0 ± 31.2
0.69 ± 0.17
7.5 ± 1.6
52.2
23.3
8.9
41.1
2.2
8.9
52.2
5.6
80.0

63.3
55.4 ± 15.3
10.0 (4.0–20.0)
26.7 ± 6.3
121.8 ± 17.0
72.9 ± 13.0
146.3 ± 48.9
9.7 ± 1.9
177.0 ± 40.8
131.5 (90.5–160.3)
43.4 ± 11.8
102.3 ± 35.5
79.9 ± 30.9
1.00 ± 0.37
12.3 (6.3–54.7)
70.0
71.1
33.0
321.3 ± 69.2
0.70 ± 0.15
8.4 ± 1.6
58.9
43.3
23.3
50.0
5.6
15.6
62.2
8.9
80.0

0.10
<0.01
<0.01
0.21
<0.01
0.53
0.17
<0.05
0.06
0.91
0.89
<0.05
<0.01
<0.05
0.97
0.16
0.87
0.53
<0.01
0.81
<0.01
0.45
<0.01
<0.05
0.29
0.44
0.25
0.23
0.57
1.00

Data are presented as means ± SD and or medians (inter-quantile range (IQR)) for continuous variables and % for categorical variables. p value
for the comparison between low and high Eh group.

3.2.

Associations of Eh with cardiovascular risk factors

We first assessed the associations of Eh with cardiovascularrisk factors. Eh was significantly associated with age (r =0.35, p < 0.01), BMI (r = 0.17, p = <0.05), systolic blood pressure (r = 0.35, p < 0.01), HbA1c (r = 0.19, p < 0.05), eGFR (r =0.36, p < 0.01) and serum cystatin C (r = 0.42, p < 0.01)(Table 2). We then focused on the associations of Eh with renalmarkers such as eGFR, cystatin C and ACR levels. Eh was significantly associated with eGFR (r = 0.36, p < 0.01) and cystatin C (r = 0.42, p < 0.01), but not with ACR (r = 0.05, p = 0.48)(Supplementary Fig. 2A–C). Next, the subjects were divided

Table 2 – Correlations of Eh with cardiovascular risk factors
in all subjects.

Age (years)
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Fasting blood glucose (mg/dl)
HbA1c (%)
eGFR (ml/min/1.73 m2)
Cystatin C (mg/l)
log ACR (mg/g creatinine)
Hyperlipidemia

r

p

0.35
0.17
0.35
0.02
0.12
0.19
0.36
0.42
0.05
0.02

<0.01
<0.05
<0.01
0.79
0.10
<0.05
<0.01
<0.01
0.48
0.80

into two groups by eGFR = 60 mL/min/1.73 m2, cystatin C = 0.
95 mg/l and ACR = 30 mg/g creatinine. Subjects with eGFR
<60 had significantly higher Eh than those with eGFR 60,and subjects with cystatin C 0.95 had significantly higher
Eh than those with cystatin C <0.95. In contrast, subjects with
ACR 30 had Eh values similar to those with ACR <30 (Supplementary Fig. 3A–C).
Next, to identify the independent variables affecting Eh, we
performed multiple regression analysis. Several clinicalparameters, including age, gender, systolic blood pressure,
hyperlipidemia, body mass index, HbA1c, duration of diabetes, use of medications, and each of the renal markers were
included in the regression models. Cystatin C, eGFR and ACR
were employed in models 1, 2 and 3, respectively. This analysis revealed that cystatin C (b = 0.29, p < 0.01), but neither
eGFR (b = 0.15, p = 0.09) nor ACR (b = 0.01, p = 0.88), wasindependently associated with Eh in T2DM patients with
IMT <1.1 mm (Table 3). Thus, in T2DM subjects with IMT ofless than 1.1 mm, serum cystatin C is strongly and independently associated with arterial wall elasticity which reflects
the degree of atherosclerosis in its early stages.

3.3.

Associations of cystatin C with Eh, IMT, and CAVITo determine which of the atherosclerosis examinations, Eh,
IMT or CAVI, is most strongly associated with cystatin C, we
next analyzed the results employing a different approach.

47

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1

Table 3 – Multiple regression analysis of Eh associations with cardiovascular risk factors in all subjects.
Model 1aAge (years)
Male gender
Systolic blood pressure (mmHg)
Hyperlipidemia
Body mass index (kg/m2)
HbA1c (%)
Duration of diabetes (years)
RAS inhibitors
Calcium channel blockers
Other agents for hypertension
Statins
Fibrates
Other agents for hyperlipidemia
Insulin
GLP-1 receptor agonists
Other agents for diabetes
Cystatin C (mg/l)
eGFR (ml/min/1.73 m2)
log ACR (mg/g creatinine)

Model 2bModel 3c

b

p

b

p

b

p

0.21
0.10
0.33
0.01
0.24
0.12
0.02
0.14
0.05
0.00
0.02
0.06
0.05
0.10
0.22
0.05
0.29
–
–

<0.05
0.15
<0.01
0.95
<0.01
0.09
0.82
0.07
0.55
0.98
0.79
0.39
0.48
0.16
<0.01
0.52
<0.01
–
–

0.20
0.12
0.33
0.02
0.20
0.15
0.02
0.14
0.04
0.05
0.02
0.08
0.07
0.13
0.21
0.03
–
0.15
–

<0.05
0.10
<0.01
0.85
<0.05
<0.05
0.84
0.09
0.62
0.51
0.85
0.25
0.34
0.10
<0.01
0.71
–
0.09
–

0.27
0.14
0.34
0.00
0.19
0.16
0.03
0.12
0.05
0.07
0.01
0.10
0.06
0.11
0.21
0.01
–
–
0.01

<0.01
0.05
<0.01
0.97
<0.05
<0.05
0.71
0.13
0.60
0.41
0.90
0.15
0.40
0.14
<0.01
0.87
–
–
0.88

Multiple regression analysis was used to examine the relationships of Eh (objective variable) with age, male gender, systolic blood pressure,
hyperlipidemia, body mass index, HbA1c, duration of diabetes, RAS inhibitors, calcium channel blockers, other agents for hypertension,
statins, fibrates, other agents for hyperlipidemia, insulin, GLP-1 receptor agonists, other agents for diabetes, and each renal marker(explanatory variables).
aR2 = 0.43.
bR2 = 0.38.
c
R2 = 0.37.

First, simple correlation analysis revealed that Eh (r = 0.42, p <0.01) and CAVI (r = 0.26, p < 0.01) were significantly associatedwith cystatin C, while IMT was not (Supplementary Fig. 4A–C).
In addition, multiple regression analysis showed Eh alone tobe associated with cystatin C (b = 0.29, p < 0.01) independentlyof age, gender, systolic blood pressure, hyperlipidemia, bodymass index, HbA1c, duration of diabetes, use of medications,while neither IMT (b = 0.04, p = 0.66) nor CAVI (b = 0.05, p =0.45) showed association (Table 4). These results indicate thatcystatin C is strongly associated with arterial wall elasticity,i.e. Eh, which is the most sensitive examination of the three
for early-stage atherosclerosis. Collectively, these observations suggest the serum cystatin C level to be a highly sensitive marker for early-stage atherosclerosis.

3.4.

Subgroup analysis based on low and high EhFinally, we investigated whether cystatin C is associated withearly-stage atherosclerosis, i.e., whether the cystatin C association with Eh values is less significant in subjects who have
not yet developed atherosclerosis. All 180 diabetic subjects
with IMT of less than 1.1 mm were divided into two groups
using the median Eh value (243 kPa) as the cut-off. In the
low Eh group, mean Eh was 197.0 ± 31.2 kPa (Table 1). In comparison to the Eh values of the aforementioned healthy population (228.3 ± 72.0 kPa), we assumed that there would be
essentially no atherosclerosis, even in the early stage, in subjects in the low Eh group. In contrast, subjects in the high Ehgroup (321.3 ± 69.2 kPa) would be likely to have early-stage
atherosclerosis not yet detectable as IMT change but withhigher than average Eh. Consistent with this assumption,age, duration of diabetes, systolic blood pressure, cystatin C
and CAVI values were significantly higher in the high Eh group(Table 1). Eh showed a weak correlation with eGFR in the low
Eh group (r = 0.21, p < 0.05), while Eh showed a modest correlation with eGFR in the high Eh group (r = 0.39, p < 0.01) (Supplementary Fig. 2D, G). Interestingly, Eh showed a strong
correlation with cystatin C in the high Eh group (r = 0.56, p <0.01), while no significant difference was detected in the low
Eh group (r = 0.05, p = 0.67). (Supplementary Fig. 2E, H). The
subjects with eGFR <60 had significantly higher Eh than thosewith eGFR 60 in the high Eh group (Supplementary Fig. 3G).
Additionally, subjects with cystatin C 0.95 had significantly
higher Eh than those with cystatin C <0.95 in the high Eh group(Supplementary Fig. 3H). However, there were no differencesin Eh between any two groups according to the renal markers
in the low Eh group (Supplementary Fig. 3D–F). In addition,
multiple regression analysis demonstrated cystatin C to be
associated with Eh in the high Eh group independently ofage, gender, systolic blood pressure, hyperlipidemia, bodymass index and HbA1c, duration of diabetes and use of medications (b = 0.51, p < 0.01), while no significant association
was observed in the low Eh group (Table 5). This subgroup
analysis showed the association between cystatin C and Eh
to be even stronger in the high Eh group than in the low Ehgroup. Thus, cystatin C was not associated with arterial wall
elasticity in subjects in whom no atherosclerotic changes
had yet been initiated (IMT <1.1 mm and Eh <243 kPa). In contrast, cystatin C very sensitively reflects the degree ofatherosclerotic change in the early stage, as indicated byincreased arterial elasticity (IMT <1.1 mm and Eh 243 kPa).
Finally, the ROC curve for cystatin C was generated to deter-

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d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1

Table 4 – Multiple regression analysis of Eh, IMT and CAVI associations with cardiovascular risk factors.
Eha

Age (years)
Male gender
Systolic blood pressure (mmHg)
Hyperlipidemia
Body mass index (kg/m2)
HbA1c (%)
Duration of diabetes (years)
RAS inhibitors
Calcium channel blockers
Other agents for hypertension
Statins
Fibrates
Other agents for hyperlipidemia
Insulin
GLP-1 receptor agonists
Other agents for diabetes
Cystatin C (mg/l)

IMTb

CAVIc

b

p

b

p

b

p

0.21
0.10
0.33
0.01
0.24
0.12
0.02
0.14
0.05
0.00
0.02
0.06
0.05
0.10
0.22
0.05
0.29

<0.05
0.15
<0.01
0.95
<0.01
0.09
0.82
0.07
0.55
0.98
0.79
0.39
0.48
0.16
<0.01
0.52
<0.01

0.56
0.15
0.19
0.11
0.05
0.15
0.07
0.07
0.08
0.06
0.02
0.03
0.03
0.02
0.03
0.05
0.04

<0.01
<0.05
<0.05
0.27
0.59
<0.05
0.40
0.39
0.38
0.43
0.85
0.64
0.70
0.83
0.66
0.54
0.66

0.54
0.19
0.02
0.02
0.19
0.06
0.18
0.10
0.08
0.14
0.08
0.03
0.03
0.15
0.04
0.17
0.05

<0.01
<0.01
0.76
0.83
<0.01
0.29
<0.05
0.15
0.27
<0.05
0.29
0.58
0.62
<0.05
0.54
<0.01
0.45

Multiple regression analysis was used to examine the relationships of Eh, IMT, and CAVI (objective variables) with age, male gender, systolicblood pressure, hyperlipidemia, body mass index, HbA1c, duration of diabetes, RAS inhibitors, calcium channel blockers, other agents for
hypertension, statins, fibrates, other agents for hyperlipidemia, insulin, GLP-1 receptor agonists, other agents for diabetes, and cystatin C(explanatory variables).
aR2 = 0.43.
bR2 = 0.33.
c
R2 = 0.58.

Table 5 – Multiple regression analysis of Eh associations with cardiovascular risk factors, in low and high Eh subgroups.
Low Eh (n = 90)aAge (years)
Male gender
Systolic blood pressure (mmHg)
Hyperlipidemia
Body mass index (kg/m2)
HbA1c (%)
Duration of diabetes (years)
RAS inhibitors
Calcium channel blockers
Other agents for hypertension
Statins
Fibrates
Other agents for hyperlipidemia
Insulin
GLP-1 receptor agonists
Other agents for diabetes
Cystatin C (mg/l)

High Eh (n = 90)b

b

p

b

p

0.29
0.10
0.25
0.21
0.29
0.03
0.26
0.16
0.09
0.04
0.21
0.14
0.33
0.18
0.45
0.07
0.03

<0.05
0.36
<0.05
0.14
<0.05
0.79
0.07
0.23
0.50
0.73
0.15
0.17
<0.01
0.15
<0.01
0.57
0.84

0.07
0.04
0.37
0.07
0.29
0.17
0.03
0.05
0.04
0.02
0.08
0.00
0.11
0.08
0.15
0.09
0.51

0.58
0.70
<0.01
0.63
<0.05
0.10
0.76
0.66
0.73
0.86
0.58
1.00
0.24
0.45
0.10
0.40
<0.01

Multiple regression analysis was used to examine the relationships of Eh (objective variable) with age, male gender, systolic blood pressure,
hyperlipidemia, body mass index, HbA1c, duration of diabetes, RAS inhibitors, calcium channel blockers, other agents for hypertension,
statins, fibrates, other agents for hyperlipidemia, insulin, GLP-1 receptor agonists, other agents for diabetes, and cystatin C (explanatory
variables).
aR2 = 0.39.
b
R2 = 0.55.

mine the optimal cut-off for high elasticity of the arterial wall
(Supplementary Fig. 5). The area under the curve was 0.59,
and the sensitivity and specificity were 0.66 and 0.49, respectively, at cystatin C = 0.86 mg/l.

4.

Discussion

In the present study, to examine whether the serum cystatin
C level is linked to early-stage atherosclerosis, we analyzed

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1

the associations between renal markers and atherosclerosis
markers in T2DM patients with IMT of less than 1.1 mm,
who are classified as not having atherosclerosis according to
the IMT criteria [8]. In these subjects, multiple regression
analyses revealed cystatin C to be strongly and independently
associated with Eh, while neither eGFR nor ACR showed a significant association. In addition, Eh was the more strongly
associated with cystatin C than either IMT or CAVI. Thesefindings demonstrate that serum cystatin C levels reflect thedegrees of atherosclerosis before arterial wall thickening
becomes detectable.
Furthermore, the association between cystatin C and Eh
was more evident in the high Eh subgroup. In contrast, thesignificance of this association disappeared in the low Eh subgroup. Eh values in the low Eh subgroup were significantly
smaller than those in a healthy population. Considering that
all of the enrolled subjects had IMT of less than 1.1 mm, thesubjects in the low Eh subgroup presumably had normal arterial structure and stiffness. Therefore, these findings indicatethat the association between cystatin C and Eh manifests
after atherosclerotic changes have been initiated. Thus, theserum cystatin C level is likely to be useful as a marker for
screening early-stage atherosclerosis, i.e. that after increased
elasticity has been initiated but before arterial wall thickening
becomes apparent. Moreover, in our study, the optimal cut-off
value of cystatin C for predicting high arterial wall elasticity
was 0.86 mg/l. Thus, in clinical practice, considerable attention should be paid to diabetic patients with cystatin C>0.86 mg/l as potentially having early stage-atherosclerosis,
even if thickened carotid intima-media has not been
detected.
T2DM is well known to be associated with atherosclerosis,
which leads to cardiovascular disease and increasing mortality rates [1]. In patients with T2DM especially, arterial stenosis
often develops without symptoms [2]. According to a metaanalysis of prospective studies, 26.1% of asymptomatic
T2DM patients have silent myocardial ischemia based onscreening by myocardial perfusion scintigraphy [24]. Therefore, to reduce the risk of future cardiovascular disease inpatients with T2DM, detection of subclinical atherosclerosis
is particularly important. Conventional angiography, computed tomography and magnetic resonance angiography areoften used to diagnose atherosclerosis in clinical practice.
However, these modalities are useful for detecting advanced,
rather than early-stage, atherosclerosis. Our phased tracking
method, which evaluates the deformities of multiple sites inarterial walls during one heartbeat, is useful for detecting
early-stage atherosclerosis [3–6]. Employing the results
obtained with the phased tracking method, the elastic modulus in the radial direction (Er) and that in the circumferential
direction (Eh) are quantitatively calculated. We and othergroups previously reported these elastic modulus values tobe more reliable and useful for detecting early-stage
atherosclerosis than examining carotid IMT, PWV and the plaque score of the carotid artery, all of which are widely used for
atherosclerosis screening in clinical practice [7,9–12]. Whenconsidering Er and Eh, the latter appears to be a more reliable
marker for arterial wall elasticity, since Eh is less influenced
by the thickness of the arterial wall [3]. Therefore, we measured the elastic modulus, Eh, in the present study. We

49

obtained results indicating the serum cystatin C level to bestrongly associated with Eh in T2DM patients with IMT <1.1
mm. This association was independent of age, gender, systolic blood pressure, hyperlipidemia, body mass index,
HbA1c, duration of diabetes, and use of medications. Thus,
the phased tracking method enabled us to show that theserum cystatin C level is associated with atherosclerosis
development at an early stage, i.e. before arterial wall thickening becomes apparent.
Carotid arterial IMT is a well-established surrogate marker for cardiovascular disease. Measuring IMT with ultrasonography is non-invasive, reproducible and easy to
perform, and, therefore, is widely used in clinical practice.
A number of studies have shown that IMT predicts future
myocardial infarction [25,26] and cerebrovascular disease[26,27]. However, employing this procedure, it is not possible
to make a diagnosis of atherosclerosis until the appearanceof arterial wall thickening. Therefore, IMT measurement is
not optimal for sensitively detecting early-stage atherosclerosis. Indeed, the significance of carotid IMT in addition toconventional coronary risk factors for predicting future cardiovascular events remains a matter of debate [28,29]. In
the present study, we enrolled T2DM patients with IMT ofless than 1.1 mm, who are classified as not having
atherosclerosis according to the IMT criteria [8]. Within thisIMT range, there was no significant association between cystatin C and IMT.
Cystatin C may provide a more sensitive and accurate estimation of renal function than eGFR, since cystatin C is lessinfluenced by age and muscle mass [13]. We obtained data
indicating the serum cystatin C level to be strongly associatedwith Eh, a sensitive marker of early-stage atherosclerosis,
while neither eGFR nor ACR was associated with Eh, In addition, Eh was more strongly associated with cystatin C thanwas either IMT or CAVI. Furthermore, multiple regression
analysis revealed a strong relationship between cystatin C
and Eh, especially in the subgroup with relatively high arterial
wall elasticity. One possible explanation of these results isthat renal impairment is associated with atherosclerosis
development starting in the very early stages of both disorders. On the other hand, it is also possible that cystatin C
might itself be directly involved in the development of
atherosclerosis [30]. Cystatin C is an endogenous inhibitorof cysteine protease. Atherosclerosis is an inflammatory disease characterized by remodeling of the extracellular matrixof the arterial walls and cysteine protease induces degradation of the extracellular matrix. The imbalance between cysteine protease and its inhibitor, cystatin C, may result inincreased degradation of extracellular matrix and migrationof monocytes/macrophages into the intima, thus leading to
the development of atherosclerosis. Tissue cystatin C levels
are reportedly reduced in atherosclerotic plaques [31]. In contrast, inflammatory cytokines stimulate cells outside of thevascular walls to secrete cystatin C into the circulation. Thus,
serum cystatin C is considered to be compensatorily upregulated in subjects who have developed atherosclerosis[32]. Although further studies are needed to examine whether
this mechanism also accounts for serum cystatin C levels rising in states of subclinical atherosclerosis, as found in thisstudy, the serum cystatin C level is likely to reflect the degree

50

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 3 9 ( 2 0 1 8 ) 4 3 –5 1of the atherosclerotic states via mechanisms both dependent
on, and independent of, glomerular filtration rates.
This study has limitations. First, the possible causal relationship between cystatin C and Eh could not be clarified
due to the cross-sectional study design. Second, although
we adjusted for well-known confounders such as age, gender,
blood pressure, hyperlipidemia, body mass index, HbA1c,
duration of diabetes, and use of medications, residual confounding, due to factors such as postmenopausal and inflammatory status, may have occurred. Third, our patients wererecruited in a hospital setting after admission for poor glycemic control, such that recruitment bias may exist. In fact, theaverage HbA1c of our patients was 10.0%, which is extremely
high. Moreover, because blood was drawn one day after theadmission, fasting plasma glucose may have improved owing
to dietary treatment while hospitalized. Also, fasting plasma
glucose may not necessarily reflect the conditions of glycemic
control. Fourth, there is little evidence indicating that carotid
arterial wall elasticity determined by the phased tracking
method is a good surrogate marker of cardiovascular diseases, because long-term outcomes have not been observed.
In addition, although we and another group reported the significance of carotid arterial wall elasticity [3–7,9–12], further
accumulation of evidence is needed to establish that Eh is agood surrogate marker of early stage-atherosclerosis. Fifth,
all of the subjects had T2DM and the lack of age- andgender-matched healthy controls limits the generalizabilityof our findings. Further studies, especially larger,
population-based prospective studies, are needed to overcome these limitations.
In conclusion, our results indicate that the serum cystatin
C level is associated with arterial wall elasticity, a sensitive
examination for early-stage atherosclerosis, in subjects classified as not having atherosclerosis as defined by IMT criteria.
Thus, the serum cystatin C level may reflect the degrees ofatherosclerotic changes in the early stages of this disorder.
Measuring the serum cystatin C level appears to be usefulfor detecting subclinical atherosclerosis in subjects with
T2DM.

Funding sources
This work was supported by Grants-in-Aid for Scientific
Research (16 K09773 and 15 K09416) to S. Sawada and Y. Ishigaki, respectively, from Japan Society for the Promotion of
Science – Japan.

Conflicts of interests
The authors declare that they have no competing interests.

Author contributions
R.K. conceived the hypothesis, performed the experiments,
and analyzed the data. S.S. A.T, R.H. and Y.I performed theexperiments. S.K., T.I., K.T., K.U., J.I. and T.Y contributed to
discussions and recruitment of patients. Y.M., H.H. and H.K.
provided technical advice on the ultrasonic equipment. N.T.

provided ultrasonic analysis of arterial wall elasticity. H.K.
directed the project, and prepared the manuscript.

Appendix A. Supplementary material
Supplementary data associated with this article can be found,in the online version, at https://doi.org/10.1016/j.diabres.2018.
02.003.

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