Abstract
Aim: Increased level of serum fibroblast growth factor 23 (FGF23) is a hallmark of abnormal phosphate metabolism in patients with chronic kidney disease (CKD) and is recently shown to be associated with the risk of cardiovascular disease even in those without CKD. This study investigated the association between serum FGF23 levels and vascular function in patients with type 2 diabetes.
Methods: This was a cross-sectional study involving 283 Japanese patients with type 2 diabetes. Flow-mediated dilatation (FMD) and nitroglycerin-mediated dilatation (NMD) of the brachial artery were measured via ultrasonography to evaluate vascular endothelial and smooth muscle functions, respectively. Serum intact FGF23 levels were determined via a sandwich enzyme-linked immunosorbent assay.
Results: The median values of FMD, NMD, and serum FGF23 were 6.0%, 14.0%, and 27.3 pg/mL, respectively. The serum FGF23 levels were inversely associated with NMD but not with FMD, and the association was independent of atherosclerotic risk factors, estimated glomerular filtration rate (eGFR), and serum phosphate levels. Furthermore, the relationship between serum FGF23 levels and NMD was modified by kidney function, which was pronounced in subjects with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2).
Conclusion: Serum FGF23 levels are independently and inversely associated with NMD in patients with type 2 diabetes, particularly in those with normal kidney function. Our results indicate that FGF23 is involved in vascular smooth muscle dysfunction and that increased serum levels of FGF23 may serve as a novel biomarker for vascular smooth muscle dysfunction in patients with type 2 diabetes.
Introduction
Bone-derived hormone fibroblast growth factor 23 (FGF23) is a key regulator of phosphate homeostasis that acts on the kidney to promote urinary phosphate excretion and inhibit vitamin D activation1). Serum FGF23 levels are significantly elevated with a tight correlation with serum creatinine and phosphate levels in patients with chronic kidney disease (CKD)2). Because serum FGF23 levels increase early in the course of CKD before serum phosphate levels rise, elevated serum FGF23 levels are considered an early marker of disordered mineral metabolism in patients with CKD3).
Several recent studies have consistently shown a link between elevated serum FGF23 levels and the risk of cardiovascular diseases (CVDs) and mortality not only in patients with CKD4, 5) but also in the general population6, 7). Furthermore, although the results are inconsistent, several studies have indicated an association between serum FGF23 levels and surrogate markers of CVD risk, such as carotid intima-media thickness8, 9), arterial stiffness10, 11), and vascular dysfunction11), in the community-based population. These findings suggest that FGF23 is involved in structural and/or functional vascular damage regardless of the CKD status, although the underlying mechanism remains unclear.
Type 2 diabetes is a leading cause of CKD and CVDs and is often accompanied by abnormal phosphate metabolism12, 13). Elevated serum FGF23 levels are observed in patients with type 2 diabetes, even with preserved kidney function, compared with those without diabetes12, 14). Of note, several recent studies have demonstrated that elevated FGF23 levels are associated with an increased risk of CVD events and mortality in patients with type 2 diabetes and preserved kidney function15-17). To date, no study has demonstrated the association between serum FGF23 levels and subclinical atherosclerosis in patients with type 2 diabetes.
Here, we hypothesized that FGF23 is involved in vascular dysfunction, an important feature of atherosclerosis18), in type 2 diabetes. Flow-mediated vasodilation (FMD) and nitroglycerin-mediated vasodilation (NMD) of the brachial artery are widely adopted to assess vascular endothelial and smooth muscle functions, respectively, and are suggested as surrogate markers for atherosclerosis and CVD outcomes18, 19).
Aim
In this study, we measured the serum FGF23 levels and examined their association with FMD and NMD in patients with type 2 diabetes.
Methods
Study Design and Participants
This was a single-center, cross-sectional study. We consecutively enrolled 283 Japanese patients with type 2 diabetes from our inpatients at the Diabetes Center of the Osaka Metropolitan University Hospital who required hospitalization for glycemic control, education, and/or evaluation of diabetic complications between January 2009 and July 2014. Type 2 diabetes was diagnosed based on the criteria of the American Diabetes Association20) and Japan Diabetes Society21). Patients with type 1 diabetes or other types were excluded in this study. Information of preexisting CVDs, including coronary artery disease, cerebrovascular disease, or peripheral artery disease, was corrected from medical records.
This study was conducted in accordance with the Declaration of Helsinki (1975, as revised in 2013) and the Ethical Guidelines for Medical and Health Research Involving Human Subjects (The Japanese Ministry of Health, Labour and Welfare, 2014). The study protocol was approved by the ethics committee of the institution (Approval No. 4417), and informed consent was obtained using the opt-out method on the department website (http://www.med.osaka-cu.ac.jp/interm2/research/optout.shtml).
Physical and Laboratory Measurements
Blood pressure was measured using an automatic sphygmomanometer (Terumo Co., Ltd., Tokyo, Japan) with a conventional cuff after the subjects had rested for at least 5 min. Blood was drawn after an overnight fast, and biochemical parameters were analyzed using a standard laboratory method at the Central Clinical Laboratory of the Osaka Metropolitan University Hospital22). Glycated hemoglobin A1c (HbA1c) was assessed as the National Glycohemoglobin Standardization Program equivalent value (NGSP, %) according to the guidelines of the Japan Diabetes Society21). Estimated glomerular filtration rate (eGFR) was calculated using the Japanese GFR-estimating equation23). Immunoreactive insulin levels were measured for participants not receiving insulin therapy (n=161) via electrochemiluminescence immunoassay [cobas 8000(502/602), Roche Diagnostics, K.K., Tokyo, Japan]. The homeostasis model assessment of insulin resistance (HOMA-R) was calculated as fasting insulin (µU/mL)×fasting glucose (mg/dL) / 405 22).
The serum levels of human intact FGF23 were measured using sandwich enzyme-linked immunosorbent assay kits (Kainos Laboratories, Tokyo, Japan)24). The intra-assay coefficient of variation for human intact FGF23 was ＜10%.
Assessment of Vascular Function
Vascular function was assessed by FMD and NMD of the brachial artery according to the guidelines of the International Brachial Artery Reactivity Task Force25) and the Japanese guidelines of the Vascular Failure Workshop Group26). FMD and NMD were measured using a vascular ultrasound system equipped with an edge-tracking system for two-dimensional imaging and automatic measurement (UNEXEF; Unex Co. Ltd., Nagoya, Japan.), as previously described22, 27, 28). In brief, the diameter of the brachial artery at rest was measured in the cubital region, and subsequently, the cuff was inflated to 50 mmHg above systolic blood pressure for 5 min and deflated. The diameter at the same point of the artery was continuously monitored, and the maximum dilatation from 45 s to 60 s after deflation was recorded.
The endothelium-independent NMD was measured following FMD measurement. After a 15-min interval for vessel recovery, 75 µg of glyceryl trinitrate was sublingually administered, and the maximum dilatation of the brachial artery at the same point was measured during at least 1 min after the initiation of maximum dilatation. The FMD and NMD values were calculated as follows: FMD or NMD (%)=(maximum diameter−diameter at rest)×100 / diameter at rest.
The measurements were performed in a quiet, air-conditioned room at 25℃ for inpatients who had been instructed to fast overnight and abstain from alcohol, caffeine, smoking, and exercise for at least 12 h prior to the measurements25, 26). Regarding the reproducibility of the FMD measurement using this system, the coefficient of correlation between examinations at different institutions was reported to be 0.84, and the intraobserver coefficient of variation was 10.1%–11.2%19).
Statistical Analysis
Data were expressed as numbers (%) or median with interquartile range. The association between vascular function and anthropometric or metabolic parameters was assessed using Spearman’s rank correlation tests and Wilcoxon’s rank-sum tests for continuous and categorical variables, respectively. Independent determinants of FMD and NMD after adjusting for potential confounders were determined via multiple regression analysis. To assess whether the effects of serum FGF23 levels on NMD are modified between participants with and without kidney dysfunction (eGFR ＜60 mL/min/1.73 m2), a cross-product term between FGF23 and with/without kidney dysfunction was included in the multiple regression analysis. P＜0.05 was considered to indicate statistical significance for all analyses. All data were statistically analyzed using JMP 14 (SAS Institute Inc., Cary, NC, USA).
Results
Clinical Characteristics, Serum FGF23 Levels, and Vascular Function
The clinical characteristics of the study subjects are presented in Table 1. The median age, duration of diabetes, body mass index, blood pressure, and HbA1c were 64 years, 11 years, 25.1 kg/m2, 130/74 mmHg, and 8.3%, respectively. The prevalence rates of current smokers, participants with CVDs, those taking renin-angiotensin system (RAS) inhibitors, those taking calcium channel blockers, and those taking statins were 25.1%, 19.4%, 40.6%, 39.9%, and 41.3%, respectively. The prevalence rate of participants receiving insulin therapy was 41.0%. Details of other antidiabetic drugs are presented in Table 1. The median eGFR, serum calcium level, and serum phosphate level were 66.0 mL/min/1.73 m2, 9.4 mg/dL, and 3.7 mg/dL, respectively.
Table 1. Clinical characteristics, serum intact FGF23 levels, and vascular function in all participants and in subgroups by kidney function
|
All participants |
eGFR ≥ 60 mL/min/1.73 m2
|
eGFR ＜60 mL/min/1.73 m2
|
p
|
| N (male/female) |
283 (170/113) |
172 (95/77) |
111 (75/36) |
0.039 |
| Age (year) |
64 [55–70] |
62 [51–68] |
68 [61–73] |
＜0.001 |
| Duration of diabetes (year) |
11 [5–20] |
10 [2–17] |
15 [9–20] |
＜0.001 |
| Body mass index (kg/m2)
|
25.1 [22.1–27.9] |
25.3 [21.8–28.5] |
24.9 [22.5–27.7] |
0.943 |
| Systolic blood pressure (mmHg) |
130 [118–144] |
125 [115–135] |
139 [124–156] |
＜0.001 |
| Diastolic blood pressure (mmHg) |
74 [69–81] |
74 [68–79] |
76 [69–84] |
0.047 |
| Current smoker (n, %)
|
71 (25.1) |
43 (25.0) |
28 (25.2) |
0.966 |
| Cardiovascular diseases (n, %)
|
55 (19.4) |
23 (13.4) |
32 (28.8) |
0.001 |
| Creatinine (mg/dL) |
0.82 [0.67–1.07] |
0.70 [0.62–0.81] |
1.20 [1.01–1.82] |
＜0.001 |
| eGFR (mL/min/1.73 m2)
|
66.0 [51.1–79.0] |
76.9 [68.9–87.3] |
43.2 [26.5–55.9] |
＜0.001 |
| Serum calcium (mg/dL) |
9.4 [9.1–9.7] |
9.5 [9.2–9.7] |
9.3 [9.0–9.7] |
0.004 |
| Serum phosphate (mg/dL) |
3.7 [3.4–4.1] |
3.7 [3.4–4.1] |
3.6 [3.4–4.2] |
0.752 |
| Fasting glucose (mg/dL) |
121 [104–147] |
127 [106–149] |
121 [100–145] |
0.307 |
| HbA1c (%) |
8.3 [7.3–9.7] |
8.6 [7.5–9.9] |
8.0 [7.1–9.2] |
0.012 |
| Immunoreactive insulin (μU/mL)＊
|
7.1 [4.9–10.2] |
7.0 [4.9–10.4] |
7.4 [4.6–10.1] |
0.953 |
| HOMA-R＊
|
2.2 [1.4–3.1] |
2.2 [1.4–3.1] |
2.0 [1.4–3.1] |
0.576 |
| Triglycerides (mg/dL) |
117 [91–154] |
114 [86–143] |
134 [97–183] |
0.002 |
| Total cholesterol (mg/dL) |
178 [151–202] |
180 [153–199] |
174 [148–203] |
0.688 |
| HDL-cholesterol (mg/dL) |
40 [35–55] |
42 [36–52] |
39 [34–47] |
0.015 |
| LDL-cholesterol (mg/dL) |
107 [86–131] |
107 [88–134] |
105 [86–129] |
0.426 |
| RAS inhibitors (n, %)
|
115 (40.6) |
57 (33.1) |
58 (52.7) |
0.001 |
| Calcium channel blockers (n, %)
|
113 (39.9) |
48 (27.9) |
65 (58.6) |
＜0.001 |
| Statins (n, %)
|
117 (41.3) |
69 (40.1) |
48 (43.2) |
0.602 |
| Sulfonylureas (n, %)
|
105 (37.1) |
67 (39.0) |
38 (34.2) |
0.422 |
| Metformin (n, %)
|
97 (34.3) |
74 (43.0) |
23 (20.7) |
＜0.001 |
| DPP-4 inhibitors (n, %)
|
77 (27.2) |
47 (27.3) |
30 (27.0) |
0.956 |
| Pioglitazone (n, %)
|
32 (11.3) |
22 (12.8) |
10 (9.0) |
0.327 |
| α-Glucosidase inhibitors (n, %)
|
44 (15.5) |
29 (16.9) |
15 (13.5) |
0.448 |
| Insulin (n, %)
|
116 (41.0) |
54 (31.4) |
62 (55.9) |
＜0.001 |
| Oral vitamin D (n, %)
|
10 (3.5) |
5 (2.9) |
5 (4.5) |
0.477 |
| FGF23 (pg/mL) |
27.3 [12.4–48.3] |
20.6 [9.6–35.4] |
41.9 [21.0–72.6] |
＜0.001 |
| FMD (%) |
6.0 [3.8–8.4] |
6.6 [3.9–9.3] |
5.7 [3.4–7.7] |
0.028 |
| NMD (%) |
14.0 [9.4–19.3] |
15.6 [11.2–20.1] |
10.6 [7.5–16.3] |
＜0.001 |
Data are median [interquartile range] or n (%). P-values were determined by χ2-test or Wilcoxon rank-sum test for comparison between the group with normal kidney function and the group with impaired kidney function.
Abbreviations: eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin A1c; HOMA-R, homeostasis model assessment of insulin resistance; HDL, high-density lipoprotein; LDL, low-density lipoprotein; RAS, renin-angiotensin system; DPP, dipeptidyl peptidase; FGF, fibroblast growth factor; FMD, flow-mediated vasodilatation; NMD, nitroglycerin-mediated vasodilatation. ＊, N = 161 for participants not receiving insulin therapy
The median values of serum FGF23 level, FMD, and NMD were 27.3 pg/mL (range, 0.006–751.0 pg/mL), 6.0% (range, 0.4%–24.6%), and 14.0% (range, 0.5%–33.7%), respectively.
Association between Serum FGF23 Levels and Vascular Function
We first examined the unadjusted correlations between FMD or NMD and cardiometabolic risk factors, serum calcium, phosphate, and FGF23 levels (Table 2A, Fig.1). The serum FGF23 levels were not (ρ=−0.105, P=0.079), but systolic blood pressure and eGFR were, significantly correlated with FMD. On the other hand, the serum FGF23 levels were significantly correlated with NMD (ρ=−0.282, P＜0.001), along with systolic blood pressure, eGFR, and HbA1c. Of note, neither the serum calcium levels nor the serum phosphate levels showed a significant correlation with FMD or NMD. In participants not receiving insulin therapy (N=161), neither FMD nor NMD was correlated with HOMA-R. For categorical variables, male sex, RAS inhibitor use, and calcium channel blocker use were associated with lower FMD, whereas calcium channel blocker use and pioglitazone use were associated with lower NMD (Table 2B).
Table 2. Associations of FMD and NMD with cardiometabolic risk factors and medications
|
FMD |
NMD |
| ρ |
p
|
ρ |
p
|
| A. Continuous variables
|
| Age |
–0.074 |
0.217 |
–0.241 |
＜0.001 |
| Body mass index |
–0.019 |
0.752 |
–0.027 |
0.648 |
| Systolic blood pressure |
–0.117 |
0.049 |
–0.304 |
＜0.001 |
| eGFR |
0.132 |
0.026 |
0.296 |
＜0.001 |
| Serum calcium |
0.078 |
0.193 |
0.066 |
0.272 |
| Serum phosphate |
0.086 |
0.138 |
–0.024 |
0.685 |
| HOMA-R＊
|
0.011 |
0.885 |
0.065 |
0.371 |
| LDL-cholesterol |
–0.057 |
0.342 |
0.033 |
0.585 |
| Triglycerides |
–0.011 |
0.858 |
–0.045 |
0.448 |
| HDL-cholesterol |
–0.005 |
0.936 |
0.009 |
0.880 |
| HbA1c |
–0.102 |
0.086 |
0.129 |
0.030 |
| FGF23 |
–0.105 |
0.079 |
–0.282 |
＜0.001 |
| B. Categorical variables
|
|
FMD (medians, %) |
NMD (medians, %) |
|
Yes |
No |
p
|
Yes |
No |
p
|
| Male |
5.6 |
6.9 |
0.007 |
13.9 |
14.6 |
0.565 |
| Current smoker |
5.8 |
6.1 |
0.412 |
14.2 |
14.0 |
0.468 |
| RAS inhibitors |
5.3 |
6.6 |
0.021 |
13.4 |
14.2 |
0.186 |
| Calcium channel blockers |
5.6 |
6.4 |
0.022 |
11.0 |
15.4 |
＜0.001 |
| Insulin therapy |
6.1 |
5.9 |
0.648 |
13.0 |
14.5 |
0.218 |
| Sulfonylureas |
5.9 |
6.2 |
0.458 |
13.2 |
14.2 |
0.935 |
| Metformin |
6.3 |
5.9 |
0.137 |
14.8 |
13.7 |
0.089 |
| DPP-4 inhibitors |
5.4 |
6.1 |
0.133 |
13.2 |
14.2 |
0.969 |
| Pioglitazone |
5.7 |
6.1 |
0.296 |
10.6 |
14.3 |
0.049 |
| α-Glucosidase inhibitors |
7.2 |
5.8 |
0.110 |
12.6 |
14.4 |
0.315 |
ρ, Spearman’s rank correlation coefficient. P values were determined by Spearman’s rank correlation test and Wilcoxon rank sum test for continuous variables and categorical variables, respectively.
Abbreviations are as in Table 1. ＊, N = 161 for participants not receiving insulin therapy
We next conducted a multiple regression analysis to explore the association between serum FGF23 levels and FMD or NMD with adjustment for age, sex, serum phosphate levels, and traditional CVD risk factors (Table 3). The serum FGF23 levels (β=−0.131, P=0.046), along with age, systolic blood pressure, and use of calcium channel blockers, were independently and negatively associated with NMD. On the other hand, male sex, RAS inhibitor use, and HbA1c, but not serum FGF23 levels (β=−0.018, P=0.793), were independently associated with FMD. None of the antidiabetic drugs, inulin (β=−0.028, P=0.650), sulfonylureas (β=0.014, P=0.811), metformin (β=0.014, P=0.813), DPP-4 inhibitors (β=0.004, P=0.947), pioglitazone (β=−0.110, P=0.060), or α-glucosidase inhibitors (β=0.057, P=0.307), showed a significant effect on NMD, nor did they virtually affect the relationship between serum FGF23 levels and NMD.
Table 3. Multivariate analyses for the determinants of FMD and NMD in all participants
|
FMD |
NMD |
| β |
p
|
β |
p
|
| Age |
–0.137 |
0.076 |
–0.187 |
0.012 |
| Sex (male= 1, female= 0) |
–0.132 |
0.043 |
–0.068 |
0.281 |
| Body mass index |
–0.106 |
0.120 |
–0.082 |
0.216 |
| Systolic blood pressure |
–0.107 |
0.102 |
–0.167 |
0.008 |
| Current smoker (yes= 1, no= 0) |
–0.066 |
0.279 |
–0.004 |
0.942 |
| RAS inhibitors (yes= 1, no= 0) |
–0.135 |
0.028 |
0.020 |
0.731 |
| Calcium channel blockers (yes= 1, no= 0) |
–0.043 |
0.516 |
–0.130 |
0.045 |
| Insulin therapy (yes= 1, no= 0) |
0.069 |
0.290 |
–0.028 |
0.650 |
| eGFR |
0.114 |
0.149 |
0.061 |
0.421 |
| Serum calcium |
0.039 |
0.518 |
0.024 |
0.680 |
| Serum phosphate |
0.049 |
0.472 |
0.078 |
0.236 |
| HbA1c |
–0.212 |
0.001 |
0.035 |
0.576 |
| LDL-cholesterol |
–0.028 |
0.675 |
0.035 |
0.583 |
| Triglycerides |
–0.008 |
0.909 |
–0.081 |
0.236 |
| HDL-cholesterol |
–0.050 |
0.463 |
–0.054 |
0.410 |
| FGF23 |
–0.018 |
0.793 |
–0.131 |
0.046 |
| R2 (p)
|
0.126 (0.002) |
0.191 (＜0.001) |
β, standardized regression coefficient by multiple regression analysis; R2, multiple coefficients of determination. Abbreviations are the same as Table 1.
Because serum FGF23 levels are strongly affected by kidney function29, 30), an interaction analysis was conducted to determine whether the presence or absence of kidney dysfunction modifies the association between serum FGF23 levels and NMD. The interaction analysis indicated a potential effect modification by the presence or absence of kidney dysfunction on the association between serum FGF23 levels and NMD (P for interaction =0.015). Therefore, the association between FGF23 levels and NMD was then separately analyzed for the group with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2) and the group with impaired kidney function (eGFR ＜60 mL/min/1.73 m2). As expected, the impaired kidney function group exhibited significantly higher serum FGF23 levels and lower NMD than the normal group (Table 1). The unadjusted correlation between serum FGF23 levels and NMD was found to be statistically significant in both groups (Fig.2). Finally, multivariate analysis by subgroup revealed that, although both were statistically significant, the association between serum FGF23 levels and NMD was more pronounced in the group with normal kidney function (β=−0.237, P=0.001) than in the group with impaired kidney function (β=−0.247, P=0.040) (Table 4).
Table 4. Multivariate analysis for the determinants of NMD stratified by kidney function
|
eGFR ≥ 60 mL/min/1.73 m2 (N = 172)
|
eGFR ＜60 mL/min/1.73 m2 (N = 111)
|
| β |
p
|
β |
p
|
| Age |
–0.426 |
＜0.001 |
0.056 |
0.635 |
| Sex (male= 1, female= 0) |
–0.129 |
0.122 |
0.061 |
0.578 |
| Body mass index |
–0.259 |
0.003 |
0.174 |
0.125 |
| Systolic blood pressure |
–0.132 |
0.064 |
–0.153 |
0.153 |
| Current smoker (yes= 1, no= 0) |
–0.005 |
0.944 |
–0.038 |
0.711 |
| RAS inhibitors (yes= 1, no= 0) |
–0.005 |
0.944 |
0.102 |
0.331 |
| Calcium channel blockers (yes= 1, no= 0) |
–0.141 |
0.076 |
–0.105 |
0.336 |
| Insulin therapy (yes= 1, no= 0) |
–0.081 |
0.290 |
0.021 |
0.843 |
| Serum calcium |
0.061 |
0.377 |
0.030 |
0.779 |
| Serum phosphate |
0.053 |
0.501 |
0.040 |
0.727 |
| HbA1c |
0.082 |
0.307 |
0.007 |
0.947 |
| LDL-cholesterol |
–0.060 |
0.450 |
0.176 |
0.140 |
| Triglycerides |
–0.175 |
0.036 |
0.040 |
0.741 |
| HDL-cholesterol |
–0.051 |
0.515 |
–0.053 |
0.656 |
| FGF23 |
–0.237 |
0.001 |
–0.247 |
0.040 |
| R2 (p)
|
0.306 (＜0.001) |
0.149 (0.356) |
β, standardized regression coefficient by multiple regression analysis; R2, multiple coefficients of determination. Abbreviations are the same as Table 1.
Discussion
In this cross-sectional study, we investigated the association between serum FGF23 levels and vascular function in patients with type 2 diabetes. Our results indicated that serum FGF23 levels were inversely associated with NMD, independently of age, sex, serum phosphate levels, and traditional CVD risk factors. Furthermore, the association between FGF23 and NMD was more pronounced in those with normal kidney function than in those with impaired kidney function. On the other hand, no significant association was observed between serum FGF23 levels and FMD. To the best of our knowledge, this is the first study to associate increased levels of FGF23 with vascular smooth muscle dysfunction in patients with type 2 diabetes.
This study first demonstrated an independent association between higher levels of serum FGF23 and higher NMD, a marker of vascular smooth muscle function, in patients with type 2 diabetes. A few studies previously investigated the association between FGF23 and vasodilatory function in a community11) or a hospital31, 32) setting. Our results are consistent with a previous report by Mirza, et al.11), which associated higher serum FGF23 levels with impaired endothelium-independent vasodilatation in a community-based elderly cohort. Unlike our study, the study by Yilmaz, et al.32) failed to find an association between serum FGF23 levels and NMD, probably because the study included only patients with severe CKD (stages 3–4). One previous study in patients with type 2 diabetes, although the method of assessing smooth muscle function was different from ours, associated higher FGF23 levels with impaired myocardial blood flow following vasodilator-induced stress by adenosine31), which may support our results.
Contrary to the prior studies11, 32), our study failed to identify the association between FGF23 and FMD, an endothelium-dependent vasodilatation. Evidence from experimental studies suggests that FGF23 directly impairs endothelium-dependent vasodilatation by reducing nitric oxide bioavailability33-35). The association between increased serum FGF23 levels and endothelial dysfunction was demonstrated in the general population11). Considering that our study only included patients with type 2 diabetes with inadequate glycemic control and that hyperglycemia is a major factor that attenuates nitric oxide bioavailability in the endothelium18), we might have failed to associate serum FGF23 levels with FMD as endothelial function was significantly impaired due to chronic hyperglycemia. The association between FGF23 and FMD was also shown in patients with advanced CKD32). Although CKD is a risk factor for endothelial dysfunction36, 37), the patients in the study32) were younger (mean age, 45 vs. 64 years) and had greater FMD (mean, 6.8% vs. 6.0%) than those in our study. Thus, the association between FGF23 and endothelial function may differ depending on the age and/or medical conditions related to the CVD risk of the study subjects.
This study further demonstrated that kidney function affected the relationship between FGF23 levels and NMD, which was more prominent in those with normal kidney function than in those with impaired kidney function. Previous studies have also shown results similar to ours11, 32). In a study by Mirza, et al.11), serum FGF23 levels were associated with endothelium-independent vasodilatation in a subgroup of 759 patients with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2) but not in 208 patients with impaired kidney function. Another study by Yilmaz, et al.32) failed to find an association between serum FGF23 levels and NMD in 183 patients with stage 3–4 CKD (mean eGFR, 33 mL/min/1.73 m2). Of importance, we previously reported that the advanced CKD stage preferentially and adversely affected NMD, rather than FMD, in patients with type 2 diabetes27). Collectively, the attenuated association between FGF23 and NMD in the group with kidney dysfunction could be explained by a loss of power due to subgrouping or by significantly impaired vascular smooth muscle function due to CKD (Table 1).
There are several possible mechanisms for the close relationship between increased FGF23 levels and vascular smooth muscle dysfunction suggested in this study. Although the direct effect of FGF23 on cardiovascular tissue is still controversial, there are reports indicating the expression of fibroblast growth factor (FGF) receptors and its coreceptor, Klotho, in human aortic tissue and vascular smooth muscle cells34, 38). The FGF receptor signaling in vascular smooth muscle cells is shown to induce conversion from the normal contractile phenotype to the proliferative phenotype, which plays a central role in the pathogenesis of atherosclerosis39, 40). Furthermore, in a study evaluating the vasoreactivity of the mouse aortic ring, FGF23 stimulated aortic contractions and increased reactive oxygen species in vascular smooth muscle cells41). These reports suggest that FGF23 plays a direct role in the impairment of dilatory function in vascular smooth muscle cells.
Considering that FGF23 is a hallmark of abnormal phosphate metabolism12, 30), factors related to phosphate metabolism may also be involved in the impairment of vascular smooth muscle function. First, postprandial increase in serum phosphate levels due to high phosphate intake, which increases circulating FGF23 levels42), may also contribute to vascular dilatory dysfunction43). The experimental evidence has indicated a direct and adverse effect of excess phosphate in calcification44, 45) and de novo cholesterol synthesis46) in vascular smooth muscle cells. Second, an excess of phosphate and calcium in circulation promotes the formation of calciprotein particles (CPPs) that can induce inflammation and calcification in vascular smooth muscle cells in individuals with CKD47, 48). Considering a recent report indicating that serum calcification propensity, or the maturation time of CPPs in serum, is associated with HbA1c in patients with type 2 diabetes49), CPPs may play a role in vascular smooth muscle dysfunction in type 2 diabetes even without CKD. Third, vitamin D and parathyroid hormone, which are major regulators of calcium–phosphate metabolism in CKD, might have contributed to vascular dysfunction50). Fourth, increased inflammatory cytokines42) and dysregulated adipocytokines51) are reportedly associated with increased FGF23 levels in the context of obesity and may also contribute to the impairment of smooth muscle function.
This study has several limitations. First, we cannot exclude the possibility that unmeasured factors related to abnormal phosphate metabolism, such as dietary phosphate intake, CPPs, vitamin D, parathyroid hormone, inflammatory cytokines, and adipocytokines, affected NMD. Second, our results should be carefully interpreted because the sample size was rather small to draw reliable conclusions, particularly in the multivariate analysis stratified by kidney function. Third, because our study consecutively included older adults with type 2 diabetes, poor glycemic control, and a relevant prevalence of macrovascular complications, our results are not applicable to the entire population of patients with type 2 diabetes. Lastly, due to the cross-sectional nature of this study, a causal relationship cannot be drawn.
Conclusion
This study demonstrated that serum FGF23 levels are independently and negatively associated with NMD in patients with type 2 diabetes and that the association is prominent in those with normal kidney function. Our data suggest that FGF23 is involved in the impairment of vascular smooth muscle function and that increased serum levels of FGF23 may serve as a novel biomarker for vascular smooth muscle dysfunction in patients with type 2 diabetes. Further interventional studies are warranted to determine whether dietary phosphate restriction or phosphate-lowering agents improve serum FGF23 levels and NMD in patients with type 2 diabetes.
Acknowledgements
The authors acknowledge Ms. Chiaki Murakami for technical assistance at the laboratory of the Department of Metabolism, Endocrinology, and Molecular Medicine, Osaka Metropolitan University Graduate School of Medicine.
Results of this study were presented at the 53rd Annual Scientific Meeting of the Japan Atherosclerosis Society in Kyoto, Japan (Oct. 23-24, 2021), the 19th International Symposium on Atherosclerosis in Kyoto, Japan (Oct. 24-27, 2021), the 65th Annual Meeting of the Japan Diabetes Society in Kobe, Japan (May 12-14, 2022), and the 95th Annual Congress of the Japan Endocrine Society in Oita, Japan (June 2-4, 2022).
This study was supported in part by research grants from Takeda Pharmaceutical Co., Ltd. (to TM) (Grant No. TKDS20180423012), Novo Nordisk Pharma Ltd. (to TM) (Grant No. RA2020A000124472) and Eli Lilly Japan K.K. (to TM) (Grant No. LGO-2100543). None of the funders were involved in the study design, collection, analysis, and interpretation of data, drafting the report, or the decision to submit the article for publication.
Conflict of Interest
The authors declare no conflict of interest related to this study.
References
- 1) Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, Takeuchi Y, Fujita T, Fukumoto S and Yamashita T: Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A, 2001; 98: 6500-6505
- 2) Imanishi Y, Inaba M, Nakatsuka K, Nagasue K, Okuno S, Yoshihara A, Miura M, Miyauchi A, Kobayashi K, Miki T, Shoji T, Ishimura E and Nishizawa Y: FGF-23 in patients with end-stage renal disease on hemodialysis. Kidney Int, 2004; 65: 1943-1946
- 3) Isakova T, Wahl P, Vargas GS, Gutiérrez OM, Scialla J, Xie H, Appleby D, Nessel L, Bellovich K, Chen J, Hamm L, Gadegbeku C, Horwitz E, Townsend RR, Anderson CA, Lash JP, Hsu CY, Leonard MB and Wolf M: Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease. Kidney Int, 2011; 79: 1370-1378
- 4) Gao S, Xu J, Zhang S and Jin J: Meta-Analysis of the Association between Fibroblast Growth Factor 23 and Mortality and Cardiovascular Events in Hemodialysis Patients. Blood Purif, 2019; 47 Suppl 1: 24-30
- 5) Kendrick J, Cheung AK, Kaufman JS, Greene T, Roberts WL, Smits G, Chonchol M and Investigators H: FGF-23 associates with death, cardiovascular events, and initiation of chronic dialysis. J Am Soc Nephrol, 2011; 22: 1913-1922
- 6) Marthi A, Donovan K, Haynes R, Wheeler DC, Baigent C, Rooney CM, Landray MJ, Moe SM, Yang J, Holland L, di Giuseppe R, Bouma-de Krijger A, Mihaylova B and Herrington WG: Fibroblast Growth Factor-23 and Risks of Cardiovascular and Noncardiovascular Diseases: A Meta-Analysis. J Am Soc Nephrol, 2018; 29: 2015-2027
- 7) Xiao Y, Luo X, Huang W, Zhang J and Peng C: Fibroblast growth factor 23 and risk of all-cause mortality and cardiovascular events: a meta-analysis of prospective cohort studies. Int J Cardiol, 2014; 174: 824-828
- 8) Hu X, Ma X, Luo Y, Xu Y, Xiong Q, Pan X, Bao Y and Jia W: Contribution of fibroblast growth factor 23 to Framingham risk score for identifying subclinical atherosclerosis in Chinese men. Nutr Metab Cardiovasc Dis, 2017; 27: 147-153
- 9) Kestenbaum B, Sachs MC, Hoofnagle AN, Siscovick DS, Ix JH, Robinson-Cohen C, Lima JA, Polak JF, Blondon M, Ruzinski J, Rock D and de Boer IH: Fibroblast growth factor-23 and cardiovascular disease in the general population: the Multi-Ethnic Study of Atherosclerosis. Circ Heart Fail, 2014; 7: 409-417
- 10) Hsu JJ, Katz R, Ix JH, de Boer IH, Kestenbaum B and Shlipak MG: Association of fibroblast growth factor-23 with arterial stiffness in the Multi-Ethnic Study of Atherosclerosis. Nephrol Dial Transplant, 2014; 29: 2099-2105
- 11) Mirza MA, Larsson A, Lind L and Larsson TE: Circulating fibroblast growth factor-23 is associated with vascular dysfunction in the community. Atherosclerosis, 2009; 205: 385-390
- 12) van der Vaart A, Yeung SMH, van Dijk PR, Bakker SJL and de Borst MH: Phosphate and fibroblast growth factor 23 in diabetes. Clin Sci (Lond), 2021; 135: 1669-1687
- 13) Yoda K, Imanishi Y, Yoda M, Mishima T, Ichii M, Yamada S, Mori K, Emoto M and Inaba M: Impaired response of FGF-23 to oral phosphate in patients with type 2 diabetes: a possible mechanism of atherosclerosis. J Clin Endocrinol Metab, 2012; 97: E2036-2043
- 14) Wahl P, Xie H, Scialla J, Anderson CAM, Bellovich K, Brecklin C, Chen J, Feldman H, Gutierrez OM, Lash J, Leonard MB, Negrea L, Rosas SE, Anderson AH, Townsend RR, Wolf M, Isakova T and Group obotCRICS: Earlier Onset and Greater Severity of Disordered Mineral Metabolism in Diabetic Patients With Chronic Kidney Disease. Diabetes Care, 2012; 35: 994-1001
- 15) Silva AP, Mendes F, Carias E, Gonçalves RB, Fragoso A, Dias C, Tavares N, Café HM, Santos N, Rato F, Leão Neves P and Almeida E: Plasmatic Klotho and FGF23 Levels as Biomarkers of CKD-Associated Cardiac Disease in Type 2 Diabetic Patients. Int J Mol Sci, 2019; 20:
- 16) Tuñón J, Fernández-Fernández B, Carda R, Pello AM, Cristóbal C, Tarín N, Aceña Á, González-Casaus ML, Huelmos A, Alonso J, Lorenzo Ó, González-Parra E, Hernández-González I, Mahíllo-Fernández I, López-Bescós L and Egido J: Circulating fibroblast growth factor-23 plasma levels predict adverse cardiovascular outcomes in patients with diabetes mellitus with coronary artery disease. Diabetes Metab Res Rev, 2016; 32: 685-693
- 17) Yeung SMH, Binnenmars SH, Gant CM, Navis G, Gansevoort RT, Bakker SJL, de Borst MH and Laverman GD: Fibroblast Growth Factor 23 and Mortality in Patients With Type 2 Diabetes and Normal or Mildly Impaired Kidney Function. Diabetes Care, 2019; 42: 2151-2153
- 18) Tomiyama H and Yamashina A: Non-invasive vascular function tests: their pathophysiological background and clinical application. Circ J, 2010; 74: 24-33
- 19) Maruhashi T, Kajikawa M, Kishimoto S, Hashimoto H, Takaeko Y, Yamaji T, Harada T, Han Y, Aibara Y, Mohamad Yusoff F, Hidaka T, Kihara Y, Chayama K, Nakashima A, Goto C, Tomiyama H, Takase B, Kohro T, Suzuki T, Ishizu T, Ueda S, Yamazaki T, Furumoto T, Kario K, Inoue T, Koba S, Watanabe K, Takemoto Y, Hano T, Sata M, Ishibashi Y, Node K, Maemura K, Ohya Y, Furukawa T, Ito H, Ikeda H, Yamashina A and Higashi Y: Diagnostic Criteria of Flow-Mediated Vasodilation for Normal Endothelial Function and Nitroglycerin-Induced Vasodilation for Normal Vascular Smooth Muscle Function of the Brachial Artery. J Am Heart Assoc, 2020; 9: e013915
- 20) American Diabetes Association: Standards of medical care in diabetes--2014. Diabetes Care, 2014; 37 Suppl 1: S14-80
- 21) Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus, Seino Y, Nanjo K, Tajima N, Kadowaki T, Kashiwagi A, Araki E, Ito C, Inagaki N, Iwamoto Y, Kasuga M, Hanafusa T, Haneda M and Ueki K: Report of the committee on the classification and diagnostic criteria of diabetes mellitus. J Diabetes Investig, 2010; 1: 212-228
- 22) Hayashi M, Morioka T, Hatamori M, Kakutani Y, Yamazaki Y, Kurajoh M, Motoyama K, Mori K, Fukumoto S, Shioi A, Shoji T, Emoto M and Inaba M: Plasma omentin levels are associated with vascular endothelial function in patients with type 2 diabetes at elevated cardiovascular risk. Diabetes Res Clin Pract, 2019; 148: 160-168
- 23) Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta K, Yamagata K, Tomino Y, Yokoyama H, Hishida A and Collaborators developing the Japanese equation for estimated GFR: Revised equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis, 2009; 53: 982-992
- 24) Yamazaki Y, Okazaki R, Shibata M, Hasegawa Y, Satoh K, Tajima T, Takeuchi Y, Fujita T, Nakahara K, Yamashita T and Fukumoto S: Increased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia. J Clin Endocrinol Metab, 2002; 87: 4957-4960
- 25) Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J and Vogel R: Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol, 2002; 39: 257-265
- 26) Inoue T, Matsuoka H, Higashi Y, Ueda S, Sata M, Shimada KE, Ishibashi Y and Node K: Flow-mediated vasodilation as a diagnostic modality for vascular failure. Hypertens Res, 2008; 31: 2105-2113
- 27) Kawano N, Emoto M, Mori K, Yamazaki Y, Urata H, Tsuchikura S, Motoyama K, Morioka T, Fukumoto S, Shoji T, Koyama H, Okuno Y, Nishizawa Y and Inaba M: Association of endothelial and vascular smooth muscle dysfunction with cardiovascular risk factors, vascular complications, and subclinical carotid atherosclerosis in type 2 diabetic patients. J Atheroscler Thromb, 2012; 19: 276-284
- 28) Morioka T, Emoto M, Yamazaki Y, Kawano N, Imamura S, Numaguchi R, Urata H, Motoyama K, Mori K, Fukumoto S, Koyama H, Shoji T and Inaba M: Leptin is associated with vascular endothelial function in overweight patients with type 2 diabetes. Cardiovasc Diabetol, 2014; 13: 10
- 29) Imanishi Y, Kobayashi K, Kawata T, Inaba M and Nishizawa Y: Regulatory mechanisms of circulating fibroblast growth factor 23 in parathyroid diseases. Ther Apher Dial, 2007; 11 Suppl 1: S32-37
- 30) Larsson T, Nisbeth U, Ljunggren O, Jüppner H and Jonsson KB: Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers. Kidney Int, 2003; 64: 2272-2279
- 31) Sørensen MH, Bojer AS, Jørgensen NR, Broadbent DA, Plein S, Madsen PL and Gæde P: Fibroblast growth factor-23 is associated with imaging markers of diabetic cardiomyopathy and anti-diabetic therapeutics. Cardiovasc Diabetol, 2020; 19: 158
- 32) Yilmaz MI, Sonmez A, Saglam M, Yaman H, Kilic S, Demirkaya E, Eyileten T, Caglar K, Oguz Y, Vural A, Yenicesu M and Zoccali C: FGF-23 and vascular dysfunction in patients with stage 3 and 4 chronic kidney disease. Kidney Int, 2010; 78: 679-685
- 33) Guo LW, Wang YK, Li SJ, Yin GT and Li D: Elevated Fibroblast Growth Factor 23 Impairs Endothelial Function through the NF-κB Signaling Pathway. J Atheroscler Thromb, 2023; 30: 138-149
- 34) Richter B, Haller J, Haffner D and Leifheit-Nestler M: Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells. Pflugers Arch, 2016; 468: 1621-1635
- 35) Silswal N, Touchberry CD, Daniel DR, McCarthy DL, Zhang S, Andresen J, Stubbs JR and Wacker MJ: FGF23 directly impairs endothelium-dependent vasorelaxation by increasing superoxide levels and reducing nitric oxide bioavailability. Am J Physiol Endocrinol Metab, 2014; 307: E426-436
- 36) Li Y, Cui R, Liu K, Eshak ES, Cui M, Dong J, Imano H, Muraki I, Kiyama M, Kitamura A, Okada T, Yamagishi K, Umesawa M, Ohira T and Iso H: Relationship between Endothelial Dysfunction and Prevalence of Chronic Kidney Disease: The Circulatory Risk in Communities Study (CIRCS). J Atheroscler Thromb, 2021; 28: 622-629
- 37) Zoccali C: Endothelial dysfunction and the kidney: emerging risk factors for renal insufficiency and cardiovascular outcomes in essential hypertension. J Am Soc Nephrol, 2006; 17: S61-63
- 38) Donate-Correa J, Mora-Fernandez C, Martinez-Sanz R, Muros-de-Fuentes M, Perez H, Meneses-Perez B, Cazana-Perez V and Navarro-Gonzalez JF: Expression of FGF23/KLOTHO system in human vascular tissue. Int J Cardiol, 2013; 165: 179-183
- 39) Chen PY, Qin L, Li G, Tellides G and Simons M: Fibroblast growth factor (FGF) signaling regulates transforming growth factor beta (TGFβ)-dependent smooth muscle cell phenotype modulation. Sci Rep, 2016; 6: 33407
- 40) Qi M and Xin S: FGF signaling contributes to atherosclerosis by enhancing the inflammatory response in vascular smooth muscle cells. Mol Med Rep, 2019; 20: 162-170
- 41) Six I, Okazaki H, Gross P, Cagnard J, Boudot C, Maizel J, Drueke TB and Massy ZA: Direct, acute effects of Klotho and FGF23 on vascular smooth muscle and endothelium. PLoS One, 2014; 9: e93423
- 42) Gutierrez OM, Wolf M and Taylor EN: Fibroblast growth factor 23, cardiovascular disease risk factors, and phosphorus intake in the health professionals follow-up study. Clin J Am Soc Nephrol, 2011; 6: 2871-2878
- 43) Shuto E, Taketani Y, Tanaka R, Harada N, Isshiki M, Sato M, Nashiki K, Amo K, Yamamoto H, Higashi Y, Nakaya Y and Takeda E: Dietary phosphorus acutely impairs endothelial function. J Am Soc Nephrol, 2009; 20: 1504-1512
- 44) Giachelli CM: The emerging role of phosphate in vascular calcification. Kidney Int, 2009; 75: 890-897
- 45) Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H and Giachelli CM: Phosphate regulation of vascular smooth muscle cell calcification. Circ Res, 2000; 87: E10-17
- 46) Zhou C, He Q, Gan H, Zeng T, Liu Q, Moorhead JF, Varghese Z, Ouyang N and Ruan XZ: Hyperphosphatemia in chronic kidney disease exacerbates atherosclerosis via a mannosidases-mediated complex-type conversion of SCAP N-glycans. Kidney Int, 2021; 99: 1342-1353
- 47) Disthabanchong S: Phosphate and Cardiovascular Disease beyond Chronic Kidney Disease and Vascular Calcification. Int J Nephrol, 2018; 2018: 3162806
- 48) Kuro-o M: Phosphate as a Pathogen of Arteriosclerosis and Aging. J Atheroscler Thromb, 2021; 28: 203-213
- 49) Mencke R, van der Vaart A, Pasch A, Harms G, Waanders F, Bilo HJG, van Goor H, Hillebrands JL and van Dijk PR: Serum calcification propensity is associated with HbA1c in type 2 diabetes mellitus. BMJ Open Diabetes Res Care, 2021; 9:
- 50) Rostand SG and Drüeke TB: Parathyroid hormone, vitamin D, and cardiovascular disease in chronic renal failure. Kidney Int, 1999; 56: 383-392
- 51) Natsuki Y, Morioka T, Fukumoto S, Kakutani Y, Yamazaki Y, Ochi A, Kurajoh M, Mori K, Shoji T, Imanishi Y, Inaba M and Emoto M: Role of adiponectin in the relationship between visceral adiposity and fibroblast growth factor 23 in non-diabetic men with normal kidney function. Endocr J, 2022; 69: 121-129
