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ORIGINAL RESEARCH
published: 17 May 2021
doi: 10.3389/fmed.2021.672000
Frontiers in Medicine | www.frontiersin.org 1May 2021 | Volume 8 | Article 672000
Edited by:
Rujun Gong,
University of Toledo Medical Center,
United States
Reviewed by:
Rong Zhou,
Yangpu Hospital, Tongji
University, China
Bohan Chen,
University of Toledo, United States
*Correspondence:
Shan Mou
shan_mou@shsmu.edu.cn
Weiming Zhang
weimingzh1965@163.com
Specialty section:
This article was submitted to
Nephrology,
a section of the journal
Frontiers in Medicine
Received: 24 February 2021
Accepted: 22 April 2021
Published: 17 May 2021
Citation:
Cai H, Zhu X, Lu J, Zhu M, Liu S,
Zhan Y, Ni Z, Gu L, Zhang W and
Mou S (2021) A Decreased Level of
Soluble Klotho Can Predict
Cardiovascular Death in No or Mild
Abdominal Aortic Calcification
Hemodialysis Patients.
Front. Med. 8:672000.
doi: 10.3389/fmed.2021.672000
A Decreased Level of Soluble Klotho
Can Predict Cardiovascular Death in
No or Mild Abdominal Aortic
Calcification Hemodialysis Patients
Hong Cai, Xuying Zhu, Jiayue Lu, Minxia Zhu, Shang Liu, Yaping Zhan, Zhaohui Ni,
Leyi Gu, Weiming Zhang*and Shan Mou*
Department of Nephrology, School of Medicine, Renji Hospital, Shanghai Jiao Tong University, Shanghai, China
Background: Soluble Klotho plays an important role in cardiovascular disease and death
in chronic kidney disease (CKD). We assessed the relationship between serum soluble
Klotho (sKL) level and outcome in MHD patients.
Methods: Soluble Klotho was detected by ELISA. Cox regression analysis and
Kaplan-Meier analysis showed the relationship between sKL and cardiovascular disease
(CVD) mortality in maintenance hemodialysis (MHD) patients.
Results: There were 45 cases (35.2%) of all-cause death and 36 cases (28.1%)
of CVD mortality. Multivariate linear regression analysis showed that Log[iPTH] (γ=
−0.224, P=0.015) was an independent predictor of sKL level. Cox regression showed
that lower sKL was associated with higher CVD mortality rate [OR =0.401, 95% CI
(0.183–0.867), P=0.022]. Kaplan-Meier analysis showed that the CVD mortality rate
increased significantly in patients with low sKL (P=0.006). Compared with high sKL
patients, low sKL patients with no or mild vascular calcification [aortic calcification score
(AACs) ≤4] had no significant difference in all-cause mortality rate. The CVD mortality
rate was significantly lower in high sKL patients (P=0.004) than in those with low sKL. In
the severe calcification group (AACs ≥5), all-cause and CVD mortality rates were similar
between different sKL groups (P=0.706 and 0.488, respectively). The area under the
receiver-operating characteristic curve (AUC) of soluble Klotho for predicting the CVD in
MHD patients with AACs ≤4 was 0.796 (0.647–0.946, P=0.017), sensitivity was 0.921,
and specificity was 0.50 for a cutoff value of 307.69 pg/ml.
Conclusions: Lower sKL was associated with higher CVD mortality rate. Lower sKL
concentration in MHD patients with no or mild calcification can predict CVD mortality.
Keywords: soluble Klotho, cardiovascular disease, death rate, abdominal aorta calcification, maintenance
hemodialysis
INTRODUCTION
Cardiovascular disease (CVD) is one of the main causes of mortality in maintenance hemodialysis
(MHD) patients. The CVD mortality rate is 10–20 times higher in MHD patients than in the
general population (1). Studies have shown that, in addition to traditional risk factors, some
non-traditional risk factors, such as micro-inflammatory state, oxidative stress, protein-energy
Cai et al. sKlotho Predict CVD in MHD
malnutrition, and imbalances of calcium and phosphorus
metabolism are contributing factors in vascular calcification (2).
In recent years, the Klotho gene has received much attention
as a novel biomarker that may predict the prognosis of MHD
patients. The Klotho gene, originally identified as an aging
suppressor gene in mice, encodes for 130 kDa of Klotho protein
and is widely expressed in the kidney, parathyroid, and brain.
Klotho exists in two forms: membrane-bound and secreted. A
secreted form of Klotho of 70 kDa is the product of alternative
splicing, which releases the extracellular domain of membrane
Klotho into blood, where it functions as a circulating substance
that exerts multiple systemic biological actions on distant
organs. This cleaved extracellular domain of membrane Klotho
is referred to as soluble Klotho (sKL) (3). In chronic kidney
disease (CKD), sKL play an important role in cardiovascular
disease and death (3–5). However, few studies have been done
in MHD patients. In this study, we prospectively observed the
relationship between sKL level and prognosis in patients with
MHD to investigate the role of sKL in predicting the prognosis
in MHD patients.
METHODS
Patients
This study protocol was approved by the Ethics Committee
on human of Renji Hospital, Jiao Tong University, and all
the patients provided written informed consent to participate
in this study. The methods were carried out in accordance
with the approved guidelines. Patient’s inclusion criteria include
signing informed consent, older than 18 years, without
residual kidney function on MHD between August 2010 and
December 2011 with the vintage of longer than 3-month. All
the patients were prospectively observed to November 2020.
Patients with connective tissue disease, acute infection, trauma,
malignant tumor, severe malnutrition, mental illness, and those
who needed antibiotics, corticosteroids, immunosuppressive
FIGURE 1 | Flowchart describing sample selection. One hundred and forty-seven individual subjects were enrolled and excluded 19 subjects with different reasons. A
total of 128 cases were in analysis.
agents, or surgery within a month were excluded. Of 147
patients, 4 were excluded due to usage of corticosteroids or
immunosuppressive agents, 3 due to infection or antibiotic
use within a month, 2 due to surgery or trauma within
a month, 5 due to comorbid malignant tumors, 2 due
to severe malnutrition, and 2 due to mental illness or
mobility problems, 1 due to out of follow up. A total of
128 individuals were enrolled in the final analytic sample
(Figure 1).
Clinical data were recorded, including history of end-stage
renal disease cause, concomitant diseases (diabetes mellitus,
hypertension, CVD), smoking, medications, pre-dialysis blood
pressure, and dialysis duration. In addition, sKL, FGF23, height,
weight, and body mass index (BMI) were measured. All
patients were dialyzed for 500 ml/min bicarbonate dialysate
flow and F80 (Fresenius, Germany) or REXEED (Asahi Kasei
Corporation) polysulfide membrane dialyzers. Blood flows were
200–350 ml/min, with dialysis times of 12 h per week. Dialysate
calcium 1.5 mmol/l, low dialysate calcium 1.25 mmol/l, dialysate
magnesium 0.5 mmol/l. The ultrafiltration target was to achieve
the clinically estimated dry weight.
The primary end point was all-cause or CVD death.
CVD death was defined as death caused by acute myocardial
infarction, pericarditis, cardiac tamponade, cardiomyopathy,
coronary atherosclerotic heart disease, arrhythmia, valvular
heart disease, pulmonary embolism, cerebral infarction, or
cerebral hemorrhage. The cause of death was determined
by a physician who did not know the patient’s sKL level.
When patients died during hospitalization, the cause of death
was determined by the attending doctor and recorded in
the medical history. If the patients died outside the hospital,
the cause of death was based on the death certificate. CVD
history was defined as a history of angina pectoris, myocardial
infarction, angioplasty, coronary artery disease, peripheral
vascular disease, left ventricular hypertrophy, or congestive
heart failure (6).
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Cai et al. sKlotho Predict CVD in MHD
TABLE 1 | Baseline characteristics of community-living individuals and laboratory data by median of serum Klotho.
All (n=128) Soluble Klotho median,Klotho range (pg/mL) P-value
Low sKL groupsKL ≤567.8 (n=64) High sKL group sKL >567.8 (n=64)
Age (years, x ±s) 58.29 ±13.68 57.13 ±14.43 59.38 ±12.86 0.354
Male (n, %) 72 (56.3) 41 (64.1) 31 (48.4) 0.108
Smoking (n, %) 86 (67.2) 45 (70.3) 41 (64.1) 0.573
Diabetes (n, %) 30 (23.4) 17 (26.6) 13 (20.3) 0.532
CVD history, n(%) 62 (48.4) 32 (50.0) 30 (46.9) 0.860
Hypertension, n(%) 105 (82.0) 52 (81.3) 53 (82.8) 1.000
Primary disease (%)
CGN 44 (34.4) 25 (39.1) 19 (29.7) 0.352
DKD 10 (7.8) 3 (4.7) 7 (10.9) 0.324
HTN 11 (8.6 ) 5 (7.8) 6 (9.4) 1.000
Others 64 (50.0) 37 (57.8) 27 (42.2) 0.111
Dialysis duration [months, M(1/4,3/4)] 78.0 (28.0, 121.5) 78.0 (22.0, 122.0) 76.0 (30.0, 117.75) 0.977
Follow up (months) 120.0 (69.0–120.0) 120.0 (64.5–123.0) 120.0 (71.75–120.0) 0.679
BMI [kg/m2, x ±s] 21.04 ±3.03 20.95 ±3.22 21.14 ±2.83 0.718
hsCRP [mg/L, M(1/4,3/4)] 1.71 (0.83, 4.25) 1.97 (0.80, 5.89) 1.46 (0.87, 3.47) 0.365
Kt/v (x ±s) 1.73 ±0.36 1.73 ±0.34 1.72 ±0.37 0.794
TC (mmol/L, x ±s) 4.36 ±1.22 4.28 ±1.05 4.46 ±1.31 0.388
HDL (mmol/L, x ±s) 1.06 ±0.44 1.04 ±0.45 1.07 ±0.44 0.735
TG [mmol/L, M(1/4,3/4)] 1.44 (1.02, 2.36) 1.32 (0.98, 1.94) 1.68 (1.13, 2.70) 0.045
LDL [mmol/L, M(1/4,3/4)] 2.26 (1.67, 2.97) 2.16 (1.67, 2.77) 2.39 (1.67, 3.12) 0.160
Hb (g/L, x ±s) 110.35 ±16.88 108.59 ±17.10 112.42 ±16.68 0.203
Hct (%, x ±s) 0.34 ±0.05 0.33 ±0.05 0.34 ±0.05 0.418
Scr (mmol/L, x ±s) 1070.16 ±252.29 1070.51 ±257.42 1069.76 ±248.41 0.987
Adjust Ca (mmol/L, x ±s) 2.38 ±0.28 2.38 ±0.28 2.39 ±0.29 0.823
P(mmol/L, x ±s) 2.03 ±0.58 1.97 ±0.58 2.08 ±0.58 0.307
IPTH [ng/L, M(1/4,3/4)] 394.0 (169.0, 667.0) 347.0 (154.5, 703.5) 437.5 (187.5, 664.75) 0.462
Alb (g/L, x ±s) 39.43 ±4.87 39.50 ±5.03 38.92 ±4.46 0.494
FGF23 [ng/l, M(1/4,3/4)] 6777.97 (2061.98, 9895.29) 5977.77 (1506.80, 9999.71) 7537.41 (2914.11, 9512.51) 0.388
sKlotho [pg/ml, M(1/4,3/4)] 567.82 (364.76, 804.19) 387.19 (233.51, 503.06) 819.12 (704.67, 1135.81) <0.001
AACs [M(1/4,3/4)] 4 (0, 11) 8 (0, 13.5) 3 (0, 9) 0.045
SBP (mmHg, x ±s) 139.45 ±20.68 139.42 ±20.16 139.52 ±21.53 0.979
DBP (mmHg, x ±s) 74.67 ±13.02 75.68 ±13.39 73.97 ±12.78 0.460
MBP (mmHg, x ±s) 96.28 ±13.44 96.93 ±13.58 95.53 ±13.35 0.559
Calcium carbonate (n, %) 113 (88.3) 60 (93.8) 53 (82.8) 0.097
ACEI/ARB (n, %) 79 (61.7) 42 (65.6) 37 (57.8) 0.467
Calcidiol (n, %) 56 (43.8) 31 (48.4) 25 (39.1) 0.725
All-cause death (n, %) 45 (35.2) 28 (43.8) 17 (26.6) 0.064
CVD death (n, %) 36 (28.1) 27 (42.2) 9 (14.1) 0.001
The low sKL group had higher AACs and higher CVD mortality compared with high sKL group. The results showed no significant differences in basic demographic data, all-cause
mortality, and laboratory data between the groups. CGN, Chronic glomerulonephritis; DKD, Diabetes kidney disease; HTN, Hypertensive nephrosclerosis; hsCRP, Hypersensitive C-
reactive protein; TG, Triglyceride; TC, Cholesterol; HDL, High-density lipoprotein; LDL, Low density lipoprotein; Hb, Hemoglobin; Hct, Hematocrit; Scr, Serum creatinine; Ca, Calcium; P,
Phosphorus; iPTH, Immunoreactive parathyroid hormone; Alb, Albumin; FGF23, Fibroblast growth factor 23; AACs, Abdominal aortic calcification score; SBP, Systolic blood pressure;
DBP, Diastolic blood pressure; MBP, Mean blood pressure.
Plain Radiography of the Abdominal Aorta
A plain lateral radiograph of the abdomen was obtained that
included the last two thoracic vertebrae and the first two sacral
vertebrae. The aorta was identified as the tubular structure
coursing in front of the anterior surface of the spine. A semi-
quantitative scoring system was utilized as suggested in the
original manuscript by Kauppila et al. (7). Only the segments
of abdominal aorta in front of the first to the fourth lumbar
vertebrae were considered. Calcific deposits in the posterior
and anterior walls of the abdominal aorta adjacent to each
lumbar vertebra were assessed separately, using the midpoint
of the intervertebral space above and below as the boundaries.
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Cai et al. sKlotho Predict CVD in MHD
TABLE 2 | The relationship between sKL and clinical indicators.
sKlotho FGF23 Age Dialysis duration MBP Alb Hb LDL Adjust Ca PhsCRP LogiPTH AAC
sKlotho 1 −0.032 −0.174 −0.152 −0.032 −0.052 0.061 0.001 0.014 0.001 0.079 −0.205 −0.213
– 0.724 0.048 0.025 0.716 0.561 0.491 0.993 0.876 0.995 0.372 0.021 0.015
FGF23 1 −0.204 0.382 −0.080 0.109 0.075 −0.051 0.508 0.470 −0.055 0.316 0.237
– 0.025 0.001 0.381 0.235 0.412 0.583 0.001 0.001 0.550 0.001 0.009
Age 1 −0.161 −0.188 −0.355 −0.091 0.149 −0.129 −0.275 0.164 −0.241 0.227
– 0.068 0.033 0.001 0.303 0.094 0.146 0.002 0.064 0.006 0.010
Dialysis duration 1 −0.162 −0.082 0.181 −0.164 0.248 0.138 −0.009 0.316 0.251
– 0.066 0.357 0.040 0.064 0.005 0.118 0.923 0.001 0.004
MAP 1 0.135 −0.004 0.043 0.019 −0.014 0.083 0.013 −0.109
– 0.126 0.969 0.633 0.829 0.879 0.348 0.886 0.219
Alb 1 0.06 −0.149 0.239 0.123 −0.277 0.105 −0.094
– 0.499 0.094 0.006 0.166 0.001 0.244 0.287
Hb 1 −0.095 0.026 0.070 −0.093 0.037 0.768
– 0.286 0.767 0.432 0.295 0.678 0.129
LDL 1 −0.134 0.153 0.030 −0.124 0.033
– 0.131 0.084 0.733 0.169 0.708
Adjust Ca 1 0.036 0.016 −0.032 −0.018
– 0.684 0.854 0.719 0.839
P 1 −0.208 0.298 0.104
– 0.018 0.001 0.239
hsCRP 1 −0.016 0.039
– 0.855 0.658
LogiPTH 1 0.200
– 0.025
AACs 1
–
sKL levels were inversely correlated with log[IPTH], AACs, dialysis duration and age. Multiple liner regression analysis showed that Log[IPTH] was an independent risk factor for sKL level.
MBP, Mean blood pressure; Alb, Albumin; Hb, Hemoglobin; LDL, Low density lipoprotein; Ca, Calcium; P, Phosphorus; hsCRP, Hypersensitive C-reactive protein; iPTH, Immunoreactive
parathyroid hormone.
Lesions were graded as follows: 0, no aortic calcific deposits;
1, small scattered calcific deposits occupying less than one-
third of the length of the corresponding vertebra; 2, moderate
quantity of calcific deposits of one-third to less than two-thirds
of the corresponding vertebral length; 3, marked calcification
of more than two-thirds of the corresponding vertebral length.
With this numerical grading, the abdominal aortic calcification
score (AACs) could vary from a minimum of 0 to a maximum
of 24 points. All radiographs were read and graded by two
investigators. The average of the two scores was considered to be
the final score. On the basis of the CORD study (8), patients were
divided into a none-or-mild calcification group (AACs ≤4), or
moderate-to-severe calcification group (AACs ≥5).
Laboratory Tests
Biochemical data were obtained using routine laboratory
methods. Serum markers relating to mineral metabolism,
including total calcium, phosphate, and intact parathyroid
hormone (IPTH), were measured, as were hemoglobin, albumin,
fasting glucose, C-reactive protein, and lipid levels. Total serum
calcium was adjusted for albumin levels using the conversion
factor: corrected calcium=calcium+0.02 mmol/L ×(40 –
albumin).
Blood samples were collected after taking the plain lateral
abdominal film and at the time of pre-dialysis. Serum
and plasma were separated and frozen at −80◦C. Soluble
Klotho and FGF23 were measured in plasma using a solid-
phase sandwich enzyme- linked immunosorbent assay (ELISA)
(Klotho: Immuno-Biological Laboratories, Takasaki, Japan,
FGF23: Kainos, Japan).
According to the median distribution of sKL levels, patients
were divided into two groups: group I, with sKL levels below the
median; group II, sKL levels above the median.
Statistical Analysis
The Kolmogorov–Smirnov test was used to estimate the Gaussian
distribution of the data, and P>0.05 was considered to indicate
normal distribution. For normally distributed data, continuous
variables were expressed as means and standard deviations,
while for skewed data, variables were expressed as medians
and interquartile ranges. Categorical variables were expressed
as numbers (or percentages). We categorized patients based on
the median sKL level within our study population. Differences
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Cai et al. sKlotho Predict CVD in MHD
FIGURE 2 | Relationship between soluble Klotho level and all-cause mortality
in MHD patients. For all-cause death, patients in high soluble Klotho level (sKl
>567.8 ng/L) had a higher survival time than in the low soluble Klotho level
(sKl ≤567.8 ng/L). But Kaplan-Meier analysis with log-rank test revealed no
significant difference between groups (P=0.174).
in demographic data and clinical variables between the groups
were analyzed with the independent samples t-test or Mann-
Whitney U-test for continuous variables, and with the chi-square
test or Fisher’s exact test for categorical variables. Cox regression
analysis was performed to assess whether sKL level was a risk
factor for CVD death in MHD patients. A Kaplan-Meier survival
curve was used to analyze the relationship between the sKL level
and MHD, AACs, and CVD death. ROC curves under the curve
(AUC) of sensitivity and specificity were used to predicting the
risk of CVD and all-cause mortality. P<0.05 was considered
statistically significant. SPSS 15.0 (SPSS Inc., Chicago, IL, USA)
was used for all statistical analyses and figures.
RESULTS
Patient Characteristics
Among the 128 MHD patients, 72 were men, the mean age was
58.29 ±13.68 years, and the mean dialysis duration was 78.0
(28.0, 121.5) months. There were 45 (35.2%) all-cause deaths,
and 36 (28.1%) CVD deaths. Based on the median sKL level, the
patients were divided into two groups, with group I having sKL
≤567.8 ng/L, and group II having sKL >567.8 ng/L.
Table 1 shows the basic demographic characteristics. The low
sKL group had higher AACs and higher CVD mortality (P=
0.045 and 0.001, respectively). The results showed no significant
differences in basic demographic data, all-cause mortality, and
laboratory data between the groups.
FIGURE 3 | Relationship between soluble Klotho level and CVD mortality in
MHD patients. For CVD death, patients in high soluble Klotho level (sKl >
567.8 ng/L) had a significant long survival time than in the low soluble Klotho
level (sKl ≤567.8 ng/L). Kaplan-Meier analysis with log-rank test revealed a
significant difference between groups (P=0.006).
Correlation of sKL and Clinical Laboratory
Data
Pearson correlation analysis showed that patients’ sKL levels were
inversely correlated with Log[iPTH] (γ= −0.205, P=0.021),
AACs (γ= −0.213, P=0.015), dialysis duration (γ= −0.152, P
=0.025), and age (γ= −0.174, P=0.048) (Table 2). Multivariate
linear regression analysis showed that Log[iPTH] (γ= −0.224, P
=0.015) was an independent predictor of sKL level.
Relationship Between sKL Level and
Prognosis in Patients With MHD
For all-cause deaths, patients with high sKL levels had higher
survival times than those in the low sKL group, but the difference
was not statistically significant [(97.51 ±4.54) months vs. (104.06
±4.24) months, P=0.174]. For CVD deaths, the group with high
sKL levels had significant longer survival times than the low sKL
level group [(98.79 ±4.42) months vs. (113.24 ±3.17) months, P
=0.006] (Figures 2, 3).
Analysis of Risk Factors for CVD Death in
MHD Patients
Univariate analysis showed increased sKL levels to correlate with
a reduction in CVD death in MHD patients. Cox regression
analysis showed that sKL level (OR =0.401, 95% CI 0.183–0.867,
P=0.022), age (OR =2.176, 95% CI 1.074–4.406, P=0.031),
male (OR =5.445, 95% CI 1.484–19.972, P=0.011), and levels
of hemoglobin (OR =0.396, 95% CI 0.187–0.840, P=0.016) and
AAC score (OR =3.100, 95% CI 1.421–6.764, P=0.004) to be
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Cai et al. sKlotho Predict CVD in MHD
TABLE 3 | Analysis of risk factors for CVD death in MHD patients (COX regression analysis).
Unadjusted Adjusted1 Adjusted2
OR 95% CI POR 95% CI POR 95% CI P
sKlotho 0.362 0.170–0.769 0.008 0.333 0.156–0.712 0.005 0.401 0.183–0.867 0.022
Age 2.925 1.466–5.836 0.014 2.176 1.074–4.406 0.031
Male 2.852 1.239–6.565 0.014 5.445 1.484–19.972 0.011
Dialysis duration 0.970 0.669–1.406 0.872 1.003 0.596–1.689 0.990
Smoking 1.327 0.627–2.808 0.460 0.809 0.279–2.341 0.695
DM 1.449 0.678–3.099 0.339 1.570 0.553–4.452 0.397
FGF23 1.052 0.356–3.110 0.927
Kt/V 1.796 0.683–4.724 0.235
iPTH 1.164 0.501–2.705 0.725
P 0.996 0.414–2.397 0.993
Adjusted Ca 1.338 0.478–3.748 0.580
Alb 0.855 0.246–2.968 0.805
Hb 0.396 0.187–0.840 0.016
hsCRP 1.497 0.647–3.464 0.346
TG 0.981 0.398–2.421 0.967
TC 0.929 0.263–3.279 0.909
HDL 2.027 0.990–4.1154 0.053
LDL 1.551 0.433–5.550 0.500
AACs 3.100 1.421–6.764 0.004
Statistical model used COX regression analysis for the risk of CVD death in MHD patients adjusting for demographic data and clinical data. Soluble Klotho level was associated with
CVD death. This finding remained consistent in models that adjusted for age and sex, demographic data and clinical data (set sKlotho>567.8 pg/mL =1, sKlotho ≤567.8 pg/ml =
0; Age ≤58.29(y) =0, Age >58.29(y) =1; Male =1, Female =0; dialysis duration >78.0(m) =1, dialysis duration ≤78.0(m) =0; Smoking =1, no smoking =0; Diabetes =1,
non-diabetes =0; FGF23 >6777.97 ng/l =1, FGF23 ≤6777.97 ng/l =0; Kt/v >1.73 =1, Kt/v ≤1.73 =0; iPTH >394.0 ng/l =1, iPTH ≤394.0 ng/l =0; P >2.03 mmol/l =1, P
≤2.03 mmol/l =0; Adjusted Ca >2.38 mmol/l =1, Adjusted Ca ≤2.38 mmol/l =0; Alb >39.43 g/l =1, Alb ≤39.43 g/l =0; Hb ≤110.35 (g/l) =0, Hb >110.35(g/l) =1; hsCRP
>1.71 mg/l =1, hsCRP ≤1.71 mg/l =0; TG ≤1.44 mmol/l =0, TG >1.44 mmol/l =1; TC ≤4.36 mmol/l =0, TC >4.36 mmol/l =1; HDL ≤1.06 mmol/l =0, HDL >1.06 mmol/l
=1; LDL ≤2.26 mmol/l =0, LDL >2.26 mmol/l =1; AACs >4=1, AACs ≤4=0). hsCRP, Hypersensitive C-reactive protein; TG, triglyceride; TC, Cholesterol; HDL, High-density
lipoprotein; LDL, Low density lipoprotein; Hb, Hemoglobin; Ca, Calcium; P, Phosphorus; iPTH, Immunoreactive parathyroid hormone; Alb, Albumin; FGF23, Fibroblast growth factor 23;
AACs, Abdominal aortic calcification score.
independent risk factors for CVD death in MHD patients after
adjusting for demographics and biochemical indexes (Table 3).
The Relationship Between AACs, sKL
Level, All-Cause Death and CVD Death
For patients with no or mild calcification (AACs ≤4), the risk
of all-cause death in the high sKL group was lower than in the
low sKL group, but the difference had no statistical significance
(P=0.077). The risk of CVD death in patients with high sKL was
significantly lower than in those with low sKL (P=0.004). With
mild or severe calcification (AACs ≥5), the risks of all-cause
death and CVD death were not significantly different between the
high and low sKL level groups (P=0.706 and 0.488, respectively)
(Figures 4, 5).
Cox regression analysis showed that for patients with no or
mild calcification (AACs ≤4), lower levels of sKL [OR =0.061
95% CI (0.008–0.483), P=0.008] and age >60 years [OR =9.863,
95% CI (2.062–47.168), P=0.004] were independent risk factors
for CVD death.
The areas under the curve (AUC) for sKl to predict CVD death
were 0.634 (95% CI 0.528–0.740, P=0.019) and 0.796 (95% CI
0.647–0.946, P=0.017) in MHD patients and in MHD patients
with AACs ≤4 respectively, which showed that sKl concentration
had a high accuracy for predicting CVD death especially in MHD
patients with AACs ≤4. A cut off value of 566.52 and 307.69
pg/ml yielded to good sensitivity and specificity of predicting
CVD death in MHD and MHD patients with AACs ≤4 by
sKl level. The sensitivity and specificity were 57, 92.1, 69.4, and
50%, respectively. In MHD patients and in MHD patients with
AACs ≤4, the AUC for sKl to predicting all-cause death were no
significantly different between the high and low sKl level group
(P=0.484 and 0.397, respectively) (Figures 6, 7).
DISCUSSION
Mineral bone disease, which includes arterial calcification, is
a common complication in CKD, especially in MHD patients.
Vascular calcification is closely correlated with poor outcomes,
including CVD death, in MHD patients. Klotho is an aging
suppressor and encodes 130 kDa of Klotho protein. Klotho
influences vascular calcification in CKD and directly inhibits
vascular calcification. Klotho gene knockout animals exhibit
a wide range of vascular calcification patterns and short life
expectancy (3). sKL plays an important role in the prevention
and treatment of acute kidney injury, reduces renal fibrosis
induced by unilateral ureteral obstruction and slow the transition
Frontiers in Medicine | www.frontiersin.org 6May 2021 | Volume 8 | Article 672000
Cai et al. sKlotho Predict CVD in MHD
FIGURE 4 | AAC ≤4, relationship between soluble Klotho level and CVD
mortality in MHD patients. For patients with no or mild calcification (AAC ≤4),
the high level of soluble Klotho level (sKl >567.8 ng/L) patients had a lower
risk of CVD death than in those with low level of soluble Klotho level (sKl ≤
567.8 ng/L). Kaplan-Meier analysis with log-rank test revealed a significant
difference between groups (P=0.004).
FIGURE 5 | AAC ≤4, Relationship between soluble Klotho level and All-cause
mortality in MHD patients. For patients with no or mild calcification (AAC ≤4),
the high level of soluble Klotho level (sKl >567.8 ng/L) patients had a lower
risk of all-cause death than in those with low level of soluble Klotho level (sKl ≤
567.8 ng/L). But Kaplan-Meier analysis with log-rank test revealed no
significant difference between groups (P=0.077).
FIGURE 6 | The role of soluble Klotho in predicting the CVD mortality in MHD
patients. The receiver operating characteristic curve illustrates soluble Klotho.
Areas under the curves are 0.634 (95% CI 0.528–0.740, P=0.019) for the
soluble Klotho. A cut off valuable of 566.52 pg/ml yielded to good sensitivity
and specificity. The sensitivity and specificity are 57 and 69.4%, respectively.
FIGURE 7 | The role of soluble Klotho in predicting the CVD mortality in MHD
patients with AAC ≤4. The receiver operating characteristic curve illustrates
soluble Klotho. Areas under the curves are 0.796 (95% CI 0.647–0.946, P=
0.017) for the soluble Klotho. A cut off valuable of 307.69 pg/ml yielded to
good sensitivity and specificity. The sensitivity and specificity are 92.1 and
50%, respectively.
Frontiers in Medicine | www.frontiersin.org 7May 2021 | Volume 8 | Article 672000
Cai et al. sKlotho Predict CVD in MHD
of AKI to CKD (9). There have been few studies on the
relationship between sKL and prognosis in MHD patients.
This study shows that sKL concentration can predict CVD
death especially in MHD patients with no or mild abdominal
aortic calcification.
Patients with low levels of sKL have a higher risk of
CVD death, which may be related to vascular calcification
in MHD patients. Animals lacking Klotho show upregulated
expression of the phosphate transporters Pit1/2 and the
key osteogenic transcription factor Runx2. Cells in high-
phosphorus and uremic environments will upregulate their
Pit1/2 activity. Both transporters can promote the influx of
extracellular phosphorus into the cell, and promote cellular
calcification (10). At the same time, Klotho can also release
NO to reduce the vasoconstriction caused by FGF23 and
phosphorus (11). Recently, studies have found that rapamycin
can inhibit the mammalian target of rapamycin (mTOR)
receptor to reduce vascular calcification and that it can also
inhibit the mTOR-like receptor to increase the membrane
and secretory Klotho concentrations. Importantly, rapamycin
failed to reduce vascular calcification in the absence of
Klotho by using either siRNA knockdown of Klotho or
Klotho knockout mice, suggesting that Klotho may be in
mediating the observed decrease in calcification by rapamycin
in vitro and in vivo (12). In CKD patients, klotho knockdown
potentiated the development of accelerated calcification through
a Runx2 and myocardin-serum response factor-dependent
pathway (13). The decreased level of soluble sKL in patients
with CKD is an independent risk factor for vascular dysfunction,
manifesting as arterial stiffness. Further studies are needed
on whether CKD patients with arterial stiffness can be
improved by Klotho supplementation (14). sKL level correlates
with vascular calcification in MHD patients. Our previous
studies found that sKL was closely related to abdominal
aortic calcification in patients with MHD, and had diagnostic
value in patients with severe calcification. Low levels of sKL
were 4.5 times more prevalent than high levels in patients
with moderate or severe abdominal aortic calcification (15).
Therefore, Klotho is closely associated with vascular calcification
and may be one of the reasons for vascular calcification in
MHD patients.
Recently, studies have demonstrated that vascular calcification
is closely related to CVD death. Among patients with coronary
artery calcification, CVD death is 2.66 times more common
than non-CVD death (16). Vascular calcification can be used
as a predictor of CVD mortality in patients with MHD.
Studies have shown that scores higher than 3 for vascular
calcification in the pelvis indicate a 3.6-fold increase in CVD
death risk, a 2.8-fold increase in cardiovascular hospitalizations
and a 2.3-fold increase in non-fatal CVD events compared
with scores below 3 (17). Calcification in other areas, such
as the abdominal aorta and aortic arch, is an independent
risk factor for CVD death in dialysis patients (18–20). The
results of this study show that the risk of CVD death
was significantly higher in the low sKL group than that in
the high sKL group, especially the AACs was significantly
low. Recently some studies have shown that high levels of
soluble Klotho can reduce the risk of cardiovascular events
and cardiovascular death by 61% in MHD patients compared
with low levels of soluble Klotho patients. Even adjusted for
age, gender, diabetes, cardiac function, dialysis vintage, serum
hemoglobin, albumin, FGF23 and other factors, the risk still
reduced by 14% (21,22). However, these studies did not take
into account the effect of vascular calcification on survival in
MHD patients. Patients with low soluble Klotho may have
severe vascular calcification (15,23). Vascular calcification is
a strong predictor of death in MHD patients (24). Therefore,
these studies did not clarify the role of vascular calcification
and soluble Klotho in predicting death in patients with MHD.
Our study demonstrates that low soluble Klotho in Patients
without or mild calcification can also predict cardiovascular
death in MHD patients, which is a full complement and proof
of previous studies.
Klotho may directly affect cardiac function. Hui et al. (25)
showed that aging-related augmentation of inflammatory
responses and cardiac dysfunction were associated with
relative Klotho deficiency. Treatment with recombinant
Klotho suppresses the inflammatory response and improves
cardiac function in aging endotoxemia mice. In CKD, sKL
can protect the myocardium from pathological stimuli, such
as uremic toxins or FGF23 (26), even though myocardial
hypertrophy still occurs in low sKL CKD mice in which
phosphorus and FGF23 levels are controlled. Exogenous
sKL can significantly improve cardiac hypertrophy in CKD
mice. sKL may be a risk factor for cardiomyopathy in
uremic patients, independent of FGF23 and phosphate
(5). Klotho may inhibit apoptosis of cardiomyocytes by
inhibiting the P38 and JNK signaling pathways (27). For
patients with no or mild calcification in this study, the high
risk of CVD death was significantly higher in the low sKL
group compared with the high sKL group. It is suggested
that sKL can affect the risk of CVD death independent of
vascular calcification, and may directly affect and improve
myocardial function.
This study has some limitations. First, vitamin D, which
is regulated by FGF23 and Klotho, was not measured. The
relationship between vitamin D, FGF23, and Klotho in the
prognosis of MHD patients requires further study. Second, in
the uremic environment, renal secretion of Klotho is reduced,
and compensatory secretion is induced in other organs, such
as the parathyroid glands, and cerebral choroid epithelial cells.
The effect on patients’ Klotho levels is unclear. However, animal
studies have shown that parathyroid Klotho secretion decreased
in uremia, suggesting that secretion may be reduced in all organs
in uremia, but further studies are needed (28). Third, the sample
size of this study is small, limiting the conclusions that can
be drawn.
sKL and CVD death are closely related in MHD patients.
A high sKL level is associated with low risk of CVD death, and
may be an independent risk factor for CVD death in MHD
patients. In MHD patients with no or mild calcification, low
sKL levels have value in predicting CVD mortality. Soluble sKL
concentration may be a biomarker that can predict CVD death in
MHD patients especially with lower AACs.
Frontiers in Medicine | www.frontiersin.org 8May 2021 | Volume 8 | Article 672000
Cai et al. sKlotho Predict CVD in MHD
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding authors.
ETHICS STATEMENT
The studies involving human participants were reviewed and
approved by Ethics Committee on Human of Renji Hospital,
Jiao Tong University. The patients/participants provided their
written informed consent to participate in this study. Written
informed consent was obtained from the individual(s) for the
publication of any potentially identifiable images or data included
in this article.
AUTHOR CONTRIBUTIONS
HC wrote the manuscript, conceived the study, and participated
in its design. WZ and SM planned and supervised the study.
JL, MZ, SL, and YZ collected and enrolled patients. ZN and
LG reviewed and edited the manuscript. All authors read and
approved the final manuscript.
FUNDING
This study was supported by National Natural Science
Foundation of China (81770668), Shanghai Municipal
Health Commission (ZXYXZ-201904), Shanghai Jiaotong
University School of Medicine (DGDZXYJHZD190111),
Clinical Innovation and Multi Discipline Integrated Medical
Construction project of South Campus, Renji Hospital, School
of Medicine, Shanghai Jiao Tong University (2014MDT02).
ACKNOWLEDGMENTS
This work was supported by the Renal Division of Renji Hospital
affiliated of Shanghai JiaoTong University. The authors thank
physicians and nurses of the HD center for technical assistance.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Cai, Zhu, Lu, Zhu, Liu, Zhan, Ni, Gu, Zhang and Mou. This is an
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