Demographic, clinical, and biochemical data of subjects with and without T2DM are shown in Table 1. Four hundred and ninety-one patients were considered for enrolment in the study and 158 were excluded due to exclusion criteria. Therefore, 333 patients (220 male) were finally included (Table 1). One hundred and thirty-three subjects had T2DM. The mean age was 65.3 ± 10.9 years and the median BMI was 26.1 ± 3.2 kg/m2. There was no difference in demographics and comorbidities between the two groups except for the prevalence of current smokers, which was higher in the group of non-T2DM subjects (31.5% vs. 23.2%, P = 0.043). Significant CAD was present in 270 patients, with a similar prevalence in non-T2DM and T2DM subjects (83.5% vs. 77.4%, P = 0.17, respectively). Serum total cholesterol concentrations were also similar in both groups. Conversely, triglycerides (TG), triglyceride-glucose index (TyG), HDL- and LDL-cholesterol levels were lower in the subjects with T2DM; these differences might be explained by the higher percentage of subjects with T2DM treated with lipid lowering medications (52% vs. 63%, P = 0.04). The mean eGFR was 99.5 ± 3.3 mL/min/1.73m2, without differences between groups. ACR was higher in subjects with T2DM, although this difference did not reach statistical significance (5.82 [3.54–15.9] vs. 8.38 [4.08–16.96] mg/g; P = 0.08). None of the different inflammatory parameters analyzed, including inflammatory cytokines and the NLR or MLR, differed between groups. Of note, the levels of serum Klotho were significantly reduced (480.8 [332.2–651] vs. 421.9 [252-651.4] pg/mL; P = 0.04) in subjects with T2DM (Table 1 and Fig. 1A), whereas iFGF23 concentration did not differ between groups. When considering the differences in the stenosis of the epicardial arteries evaluated, the mean percentage of obstruction was similar in subjects with and without T2DM, except for the RCA, which was significantly higher in the group with T2DM (Table 2).
Table 1
Demographic and biochemical characteristics of patients included in the study.
|
Non-T2DM
|
T2DM
|
p
|
All subjects
|
Characteristics
|
|
|
|
|
N
|
200
|
133
|
|
333
|
Age (years)
|
64.6 ± 11.3
|
66.3 ± 10.2
|
0.27
|
65.3 ± 10.9
|
Sex (% male)
|
135 (67.5)
|
85 (63.9)
|
0.49
|
220 (66.1)
|
BMI (kg/m2)
|
25.3 ± 2.8
|
27.1 ± 2.1
|
0.09
|
26.1 ± 3.2
|
SBP (mm Hg)
|
123 ± 15.1
|
128 ± 11.5
|
0.12
|
127 ± 12.3
|
DBP (mm Hg)
|
74.5 ± 8.6
|
75.8 ± 9.7
|
0.13
|
74.9 ± 9.1
|
Comorbidities (%)
|
|
|
|
|
Significant CAD
|
167 (83.5)
|
103 (77.4)
|
0.17
|
270 (81.1)
|
Obesity
|
28 (14)
|
14 (10.5)
|
0.35
|
42 (12.6)
|
Hypertension
|
61 (30.5)
|
37 (27.8)
|
0.59
|
98 (29.4)
|
Former smokers
|
40 (20)
|
28 (21.1)
|
0.46
|
68 (20.4)
|
Current smokers
|
63 (31.5)
|
21 (23.3)
|
0.04
|
94 (28.2)
|
Dyslipidemia
|
88 (44)
|
54 (40.6)
|
0.54
|
142 (42.6)
|
Laboratory data
|
|
|
|
|
T-cholesterol (mg/dL)
|
172.6 (145-201.5)
|
168 (140–190)
|
0.14
|
171 (143–198)
|
HDL-C (mg/dL)
|
40.5 (33–48)
|
37 (31-43.5)
|
0.05
|
38 (32–46)
|
LDL-C (mg/dL)
|
103 (80–127)
|
94 (74.5–112)
|
< 0.01
|
100.4 (76-121.8)
|
TG (mg/dL)
|
125 (92.3-162.8)
|
142 (114.5-197.5)
|
< 0.001
|
135 (102–172)
|
TyG index
|
4.68 (4.53–4.79)
|
5.04 (4.9–5.25)
|
< 0.001
|
4.8 (4.61–5.02)
|
FG (mg/dL)
|
90 (86–95)
|
158 (138–200)
|
< 0.001
|
97 (89-147.5)
|
Hb1ac (%)
|
5.37 ± 0.37
|
7.57 ± 0.42
|
< 0.001
|
6.28 ± 1.46
|
eGFR (ml/min/1.73 m2)
|
101.1 ± 9.2
|
97.5 ± 8.3
|
0.11
|
99.5 ± 3.3
|
Creatinine (mg/dL)
|
0.89 (0.75–1.05)
|
0.82 (0.7–1.02)
|
0.04
|
0.86 (0.74–1.04)
|
ACR (mg/g)
|
5.82 (3.54–15.9)
|
8.38 (4.08–16.96)
|
0.08
|
6.83 (3.68–16.04)
|
Uric acid (mg/dL)
|
5.54 (4.58–6.69)
|
5.36 (4.46–6.21)
|
0.29
|
5.43 (5.5–6.6)
|
Calcium (mg/dL)
|
9.32 ± 0.31
|
9.33 ± 0.38
|
0.56
|
9.32 ± 0.34
|
Phosphate (mg/dL)
|
3.54 ± 0.53
|
3.55 ± 0.67
|
0.71
|
3.54 ± 0.71
|
NLR (/mL)
|
2.3 (1.69–3.24)
|
1.78 (2.32–3.61)
|
0.28
|
2.31 (1.74–3.33)
|
MLR (/mL)
|
0.33 (0.26–0.49)
|
0.32 (0.24–0.44)
|
0.06
|
0.33 (0.24–0.46)
|
hs-CRP (mg/L)
|
3.2 (1.92–6.23)
|
3.3 (2-6.7)
|
0.74
|
3.2 (2-6.6)
|
TNFα (pg/mL)
|
1.99 (1.35–2.55)
|
2.09 (1.52–2.99)
|
0.12
|
2.06 (1.43–2.77)
|
IL6 (pg/mL)
|
6.12 (3.49–10.35)
|
6.36 (3.3-12.14)
|
0.52
|
6.3 (3.3-10.76)
|
Klotho (pg/mL)
|
480.8 (332.2–651)
|
421.9 (252-651.4)
|
0.04
|
466.3 (290.5-651.4)
|
iFGF23 (pg/mL)
|
583.3 (471.7-790.1)
|
570.7 (427.3-706.1)
|
0.11
|
577 (443.1-766.1)
|
Medication
|
|
|
|
|
Statin (%)
|
104 (52)
|
84 (63.2)
|
0.04
|
188 (56.5)
|
ACEI/ARB (%)
|
80 (40)
|
61 (45.9)
|
0.29
|
141 (42.3)
|
T2DM, type 2 diabetes mellitus; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; CAD, coronary artery disease; HDL-C high density lipoprotein cholesterol; LDL-C low-density lipoprotein cholesterol; TG, triglycerides; TyG, triglyceride glucose index; FG, fasting glucose; Hb1ac, glycated hemoglobin; eGFR, estimated glomerular filtrate rate; ACR, urine albumin-to-creatinine ratio; hs-CRP high sensitivity C-reactive protein, TNFα, tumor necrosis factor alpha; IL, interleukin; NLR, neutrophil lymphocyte ratio; MLR, monocyte lymphocyte ratio; iFGF23, intact fibroblast growth factor 23; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor antagonist. |
Table 2
Coronary angiography study results.
|
Non-T2DM
|
T2DM
|
p
|
All subjects
|
SSI
|
30.13 (16.1-44.94)
|
33.5 (16.13–54.13)
|
0.39
|
32.25 (16.1-51.13)
|
Obstruction (%)
|
|
|
|
|
LCA
|
55.12 ± 29.1
|
54.2 ± 29.21
|
0.61
|
54.75 ± 29.1
|
RCA
|
25.86 ± 34.66
|
33.16 ± 37.4
|
0.02
|
28.78 ± 35.91
|
LAD
|
33.43 ± 38.92
|
34.59 ± 40.05
|
0.33
|
33.89 ± 39.32
|
CA
|
14.54 ± 30.82
|
14.77 ± 31.1
|
0.94
|
14.63 ± 30.89
|
T2DM, type 2 diabetes mellitus; SSI, severity stenosis index; LCA, left main coronary artery; RCA, right coronary artery; LAD, left anterior descending artery; CA, circumflex artery. |
The sub-analysis of the group of subjects with T2DM attending to the occurrence of CAD revealed that Klotho was significantly higher in subjects with significant CAD (Klotho percent increase: 45.6%; P = 0.02) (Table 3 and Fig. 1B), without differences in non-T2DM subjects (Fig. 1C). The subjects with T2DM and CAD also presented higher SBP values (P = 0.04), higher concentrations of serum inflammatory parameters (hs-CRP, TNFα, and IL6), and a trend to increased levels of ACR. No differences were observed in the TyG index between T2DM subjects according to the presence of significant CAD.
Table 3
Sub-analysis of demographic and biochemical characteristics of subjects with T2DM according to the presence of significant CAD.
|
No CAD
|
CAD
|
p
|
Characteristics
|
|
|
|
N
|
30
|
103
|
|
Age (years)
|
63 (54.8–73)
|
68 (61–75)
|
0.16
|
Sex (% male)
|
20 (66.7)
|
65 (63.1)
|
0.83
|
BMI (kg/m2)
|
26.8 ± 2.2
|
27.3 ± 2.9
|
0.13
|
SBP (mm Hg)
|
129 ± 10.1
|
132 ± 11.1
|
0.04
|
DBP (mm Hg)
|
75.3 ± 7.7
|
75.9 ± 9.1
|
0.47
|
Comorbidities
|
|
|
|
Obesity
|
4 (13.3)
|
10 (9.7)
|
0.52
|
Hypertension (%)
|
6 (20)
|
31 (30.1)
|
0.36
|
Former smoker (%)
|
7 (23.3)
|
21 (20.4)
|
0.12
|
Current smokers (%)
|
7 (23.3)
|
19 (18.4)
|
0.21
|
Dyslipidemia (%)
|
10 (33)
|
44 (42.7)
|
0.41
|
Laboratory data
|
|
|
|
T-cholesterol (mg/dL)
|
160.5 (134.5-188.5)
|
168 (142–193)
|
0.47
|
HDL-C (mg/dL)
|
34 (27.3–42.8)
|
38 (31–44)
|
0.26
|
LDL-C (mg/dL)
|
90.5 (70.3-118.5)
|
95 (75–110)
|
0.84
|
TG (mg/dL)
|
142.5 (114.5–200)
|
142 (114–197)
|
0.97
|
TyG index
|
5.08 (4.9–5.26)
|
5.03 (4.9–5.25)
|
0.76
|
FG (mg/dL)
|
133.8 (67.2–222)
|
162 (142–196)
|
0.28
|
Hb1ac (%)
|
7.42 ± 0.2
|
7.58 ± 0.21
|
0.11
|
eGFR (ml/min/1.73 m2)
|
99.8 ± 10.3
|
96.9 ± 9.3
|
0.16
|
Creatinine (mg/dL)
|
0.84 (0.71–0.99)
|
0.82 (0.7–1.1)
|
0.64
|
ACR (mg/g)
|
5.76 (3.22–13.84)
|
9 (4.3–19)
|
0.08
|
Uric acid (mg/dL)
|
5.36 (4.35–6.89)
|
5.36 (4.45–6.11)
|
0.99
|
Calcium (mg/dL)
|
9.3 ± 0.34
|
9.36 ± 0.28
|
0.85
|
Phosphate (mg/dL)
|
3.55 ± 0.71
|
3.55 ± 0.42
|
0.98
|
NLR (/mL)
|
2.58 (1.62–3.89)
|
2.2 (1.8–3.52)
|
0.69
|
MLR (/mL)
|
0.33 (0.23–0.46)
|
0.32 (0.24–0.43)
|
0.86
|
hs-CRP (mg/L)
|
2.75 (1.25–5.9)
|
3.9 (2.1–6.8)
|
0.03
|
TNFα (pg/mL)
|
1.87 (1.34–2.72)
|
2.22 (1.57–3.1)
|
0.04
|
IL6 (pg/mL)
|
5.03 (2.48–8.97)
|
7.5 (3.51-12-25)
|
0.03
|
Klotho (pg/mL)
|
314.5 (6.15-562.81)
|
458.97 (275.2-667.2)
|
0.02
|
iFGF23 (pg/mL)
|
571.2 (444.5-675.4)
|
570.21 (421.3-707.3)
|
0.91
|
Medication
|
|
|
|
Statin (%)
|
21 (70)
|
63 (61.2)
|
0.38
|
ACEI/ARB (%)
|
11 (36.7)
|
50 (48.5)
|
0.25
|
CAD, coronary artery disease; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL-C high density lipoprotein cholesterol; LDL-C low-density lipoprotein cholesterol; TG, triglycerides; TyG, triglyceride glucose index; FG, fasting glucose; Hb1ac, glycated hemoglobin; eGFR, estimated glomerular filtrate rate; ACR, urine albumin-to-creatinine ratio; hs-CRP high sensitivity C-reactive protein, TNFα, tumor necrosis factor alpha; IL, interleukin; NLR, neutrophil lymphocyte ratio; MLR, monocyte lymphocyte ratio; iFGF23, intact fibroblast growth factor 23; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor antagonist. |
Bivariate correlation analysis showed that Klotho was significantly and positively related with SSI values in the sub-group of T2DM patients (r = 0.223, P < 0.01), particularly with the percentage of stenosis observed in the two major epicardial arteries: LCA (r = 0.212, P = 0.01) and RCA (r = 0.182, P = 0.04) (Table 4). Klotho was also inversely related with MLR values (r = -0.184, P = 0.03). Importantly, these correlations were absent in the group of non-T2DM subjects.
Table 4
Bivariate correlation study of serum Klotho protein levels with coronary artery stenosis determinations in patients with and without T2DM.
|
Non-T2DM
|
T2DM
|
|
r
|
p
|
r
|
p
|
SSI
|
-0.3
|
0.675
|
0.233
|
0.007
|
Obs LCA (%)
|
-0.074
|
0.297
|
0.212
|
0.014
|
Obs RCA (%)
|
0.028
|
0.696
|
0.182
|
0.036
|
Obs LAD (%)
|
-0.063
|
0.379
|
0.152
|
0.082
|
Obs CA (%)
|
0.114
|
0.108
|
0.029
|
0.739
|
T2DM, type 2 diabetes mellitus; SSI, severity stenosis index; Obs, obstruction; LCA, left main coronary artery; RCA, right coronary artery; LAD, left anterior descending artery; CA, circumflex artery. |
The correlation between Klotho and SSI was also observed even after additional adjustment for potential confounders: age, sex, HT, current smokers, dyslipidemia, ACR, body mass index (BMI), phosphorus, iFGF23, TNFα, IL6, and hs-CRP. Thus, the results of a forward stepwise multiple regression analysis performed with the SSI as the dependent variable showed the independent and positive association between the severity of stenosis with soluble Klotho, dyslipidemia and ACR (adjusted R2 = 0.153, P < 0.01) (Table 5). Again, the association between SSI and Klotho was not present in the group of non-T2DM subjects, where TNFα and ACR were the variables significantly and independently associated with the SSI (adjusted R2 = 0.045, P < 0.05). Notably, the ACR remained significantly associated with SSI in both groups.
Table 5
Multiple stepwise regression analysis for stenosis severity index as the dependent variable in T2DM and non-T2DM subjects.
Stenosis severity index
|
Adjusted R2
|
ß
|
SE
|
t
|
p
|
Non-T2DM subjects
|
0.045
|
|
|
|
< 0.05
|
TNFα (pg/mL)
|
|
0.130
|
0.228
|
1.837
|
0.048
|
ACR (mg/g)
|
|
0.129
|
0.178
|
1.849
|
0.046
|
T2DM subjects
|
0.153
|
|
|
|
< 0.01
|
Dyslipidemia
|
|
0.206
|
3.852
|
2.469
|
0.015
|
Klotho (pg/mL)
|
|
0.184
|
0.007
|
2.201
|
0.030
|
ACR (mg/g)
|
|
0.228
|
0.225
|
2.735
|
0.007
|
T2DM, type 2 diabetes mellitus; TNFα, tumor necrosis factor alpha; ACR, urine albumin-to-creatinine ratio |
Finally, we performed a multivariate logistic regression analysis for the presence of significant CAD as the dependent variable and serum Klotho levels as an independent variable (Table 6). Traditional risk factors for CAD (age, HT, current smokers, and dyslipidemia) were entered as covariates (model 1). The model was additionally adjusted for markers of kidney function (eGFR and ACR) and iFGF23 (model 2), and inflammatory variables (MLR, hs-CRP, IL6 and TNFα) (model 3). The results showed that Klotho was positively associated with CAD, even after additional adjustment for all the potential confounders (OR [95% CI]: 1.001 [1.0–1.003], P < 0.05) (Table 6).
Table 6
Multivariate logistic regression analysis for the presence of significant CAD in the group of subjects with T2DM with serum Klotho levels as independent variable.
|
Unadjusted
|
Model 1
|
Model 2
|
Model 3
|
|
OR (95% CI)
|
p
|
OR (95% CI)
|
p
|
OR (95% CI)
|
p
|
OR (95% CI)
|
p
|
Klotho (pg/mL)
|
1.003
(1.002–1.003)
|
< 0.001
|
1.001
(1.0-1.003)
|
0.037
|
1.001
(1.0-1.003)
|
0.039
|
1.001
(1.0-1.003)
|
0.041
|