Association of suPAR with progression of cardiovascular calcification and cardiovascular outcomes in peritoneal dialysis patients

Abstract

Objective

Cardiovascular calcification (CVC) is an important factor influencing cardiovascular outcomes in peritoneal dialysis (PD). We investigated the association of serum soluble urokinase plasminogen activator receptor (suPAR) with CVC progression and cardiovascular outcomes in PD patients.

Methods

A total of 226 PD patients were recruited in our study. CVC assessments include abdominal aortic calcification (AAC), coronary artery calcification (CAC), and cardiac valvular calcification (ValvC). An increase in calcification score or the appearance of a new ValvC after approximately 24 months were defined as CVC progression. The endpoints were CVC progression, cardiovascular events (CVEs) and cardiovascular mortality.

Results

Of 226 PD patients, 111 had AAC, 155 had CAC and 26 had ValvC at baseline. At the end of follow-up, 41.1% of patients had AAC progression, 76.7% had CAC progression, and 6.4% had ValvC progression. Elevated serum suPAR was a significant risk factor for AAC progression and CAC progression but was no longer associated with ValvC progression by multivariate logistic regression analysis. The ROC curve showed that serum suPAR had a predictive value for CAC progression (AUC = 0.701). By multivariate Cox regression analysis, higher serum suPAR remained an independent risk factor for CVEs, but no longer an independent risk factor for cardiovascular mortality.

Conclusion

CVC is prevalent in PD patients. High levels of serum suPAR are associated with AAC progression and CAC progression in PD patients. Serum suPAR can be used as an independent predictor of CVEs in PD patients, but not yet for cardiovascular mortality.

Introduction

Cardiovascular disease (CVD) is the primary cause of death among peritoneal dialysis (PD) patients [1,2]. Cardiovascular calcification (CVC) plays a crucial role as a significant contributor to cardiovascular disease (CVD) among dialysis patients. The Kidney Disease Improving Global Outcomes (KDIGO) guideline indicates that patients with chronic kidney disease (CKD) stages 3-5D have the highest risk for cardiovascular events (CVEs) when CVC is present [3]. A large multicenter study conducted in China revealed that up to 65.1% of patients with PD exhibited varying degrees of CVC [4]. CVC progressed rapidly in patients undergoing dialysis, with different CVC types associated with different rates of prevalence and progression. Progression of coronary artery calcification (CAC) is associated with a higher risk of the composite of all-cause death and nonfatal CVEs [5]. Currently, there is a lack of effective treatment for CVC. Once it occurs, the condition is difficult to reverse, and even renal transplantation has been shown to be insufficient in halting the progression of CVC [6]. The early prediction of CVC progression remains challenging, and it is particularly important for risk factors associated with CVC to be prevented and controlled. Inflammation serves as a pivotal promotive factor in CVC, wherein inflammatory mediators stimulate the transformation of vascular smooth muscle cells and facilitate the remodeling of the extracellular matrix, providing a conducive microenvironment for calcification [7,8]. The soluble urokinase plasminogen activator receptor (suPAR), a recently identified inflammatory biomarker, is associated with CAC scores independently of the assessed systematic coronary risk. It offers prognostic information on CVD risk in the general population, extending beyond the predictive capabilities of the Framingham risk score [9,10]. Hemodialysis (HD) patients with high serum suPAR levels were found to have significantly higher CAC scores and a significantly higher incidence of CVEs and mortality [11,12]. One of our previous cross-sectional studies suggested that serum suPAR was associated with CVC at baseline in PD patients [13]. However, the effect of suPAR on CVC progression and cardiovascular outcomes in PD patients remains unclear. Thus, we conducted further follow-ups with patients to determine if serum suPAR was correlated with the progression of CVC and cardiovascular outcomes.

Materials and methods

Population and study design

It was designed as a prospective observational study (Record NO. MR-33-22-013521). Patients with peritoneal dialysis from January 2022 to January 2023 at Shaoxing People’s Hospital were recruited. The recruitment criteria and patients were the same as in our previous study [13]. Inclusion criteria:1) age ≥ 18 years; 2) regular PD for ≥ 6 months; 3) agree to participate in the study and sign the informed consent form. Exclusion criteria were as follows: 1) treated with combined HD; 2) Patients with severe peripheral vascular disease, amputation; 3) patients with rheumatic heart disease, valve transplantation, congenital heart valve disease; 4) patients with malignant tumors, active inflammatory diseases, history of parathyroidectomy, life expectancy < 6 months; 5) pregnant women, lactating women, or women planning to become pregnant within 1 year. All patients were dialyzed using lactate-buffered glucose-based PD solutions (Baxter, China). Participants were followed from enrollment until death, switch to HD, renal transplantation, transfer to other hospitals or September 2024. This study received ethical approval from the Ethics Committee of Shaoxing People’s Hospital (Ethics Clearance No. 2021-K-Y-329-01) and strictly adhered to the principles outlined in the Helsinki Declaration.

Clinical and laboratory data

The study collected comprehensive data on participants’ demographic characteristics, medical history, and clinical parameters. Demographic and medical history data included age, sex, diabetes, PD regimen, comorbidity status, concomitant medications, and history of CVD. CVD was defined as congestive heart failure, ischemic heart disease, severe arrhythmia, and ischemic stroke. Laboratory parameters were systematically recorded, including hemoglobin, serum albumin, calcium, phosphorus, alkaline phosphatase, lipid profiles, high-sensitivity C-reactive protein (hsCRP), and intact parathyroid hormone (iPTH). The estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI equation [14]. Residual renal function (RRF) was estimated by calculating the average residual renal clearance of urea and creatinine as described by van Olden et al. [15]. Body composition analysis was performed using bioelectrical impedance analysis (BIA, Seca515, Hamburg) to determine appendicular skeletal muscle mass index (ASMI) and extracellular water/total body water ratio (ECW/TBW). Dialysis adequacy was evaluated through total weekly urea clearance (Kt/V) measurements and peritoneal membrane characteristics were assessed using the peritoneal equilibrium test to calculate the 4-h dialysate/plasma creatinine ratio (4 h D/Pcr). suPAR concentrations were quantified using the human uPAR Quantikine^® ELISA Kit (R&D Systems) in accordance with the manufacturer’s protocol.

Assessment of cardiovascular calcification

Patients underwent three distinct assessments to evaluate CVC: abdominal aortic calcification (AAC), coronary artery calcification (CAC), and cardiac valvular calcification (ValvC). For AAC evaluation, we employed a semiquantitative scoring system based on plain lateral lumbar radiographs, utilizing the validated 24-point aortic calcification scale initially developed by Kauppila et al. [16]. CAC assessment was conducted using multidetector computed tomography (MDCT, Aquilion Pure ViSION) following the methodology established by Agatston et al. [17]. The radiographs were reviewed by 2 radiologists blindly. For the inconsistent results, 2 radiologists re-scored and discussed together and then gave a unified result. ValvC was identified through echocardiography, characterized by the presence of bright echoes exceeding 1 mm on one or more cusps of the aortic valve, mitral valve, or mitral annulus. None of the echocardiographic technicians were aware of the patients’ detailed clinical conditions.

Definitions of cardiovascular calcification progression

CVC group was defined as calcification present in any 1 of the 3 measurements (AAC score >0, CAC score >0, or ValvC was detected). A second CVC assessment was performed at approximately 24 months after the baseline assessment. AAC progression and CAC progression were defined as an increase in AAC score or CAC score of >0 between assessments before and after, respectively. ValvC progression was defined as the appearance of a new point of calcification in the cardiac valves [18].

End points

The primary endpoint of the study was CVC progression. The secondary endpoints were cardiovascular events (CVEs) and cardiovascular mortality. CVEs were defined when patients exhibited congestive heart failure, ischemic heart disease, symptomatic arrhythmia, or ischemic stroke. Death caused by CVEs was defined as cardiovascular mortality.

Statistical analysis

Statistical analyses were performed using SPSS 26.0 and GraphPad Prism (version 10.1.2). Categorical variables were expressed as frequencies and analyzed using the Chi-square test. Continuous variables were presented as mean ± standard deviation (SD) for normally distributed data or median [Q1, Q3] for skewed distributions, with appropriate statistical tests (t-test or Mann-Whitney U test) applied accordingly. The relationship between suPAR and CVC was evaluated using Pearson or Spearman correlation analysis. To investigate the association between serum suPAR levels and CVC progression, we performed logistic regression analysis. Variables demonstrating p < 0.1 in univariate analysis and those deemed clinically significant were subsequently included in the multivariate logistic regression model. The ROC curve was plotted to calculate the area under the curve (AUC) for suPAR in identifying the progression of CAC. The incidence of CVEs and mortality were analyzed by the Kaplan-Meier method. The predictive value of suPAR in CVEs and mortality were analyzed by Cox proportional hazards regression model. All statistical tests were two-sided, and differences were considered statistically significant for a P value less than 0.05.

Results

Demographics and baseline characteristics

A total of 226 patients were recruited for our study, comprising 175 cases in the CVC group and 51 cases in the non-CVC group.111 (49.1%) patients had AAC, 155 (68.6%) patients had CAC and 26 (11.5%) patients had ValvC at baseline. About 175 patients had at least one type of calcification, 73 patients had both types of calcification, and 22 patients had all three types of calcification. There were significant differences in age, BMI, diabetes, phosphorus, hsCRP, suPAR, ECW/TBW, dialysis duration, Kt/V, ultrafiltration, volume of urine, RRF, total volume of dialysate, and glucose exposure between the two groups (Table 1).

Table 1.

Comparison of baseline characteristics of patients.

Variables	CVC (n = 175)	Non-CVC (n = 51)	P
Age (year)	63 ± 11	52 ± 10	<0.001
Male (n, %)	83(47.4%)	20 (39.2%)	0.300
BMI (kg/m2)	24.0 ± 3.6	22.1 ± 3.2	0.001
History of CVD (n, %)	33(18.9%)	4 (7.8%)	0.061
Diabetes (n, %)	59 (33.7%)	5(9.8%)	0.001
MAP (mmHg)	96 ± 10	95 ± 11	0.899
Hemoglobin (g/L)	99.3 ± 16.7	97.2 ± 18.4	0.444
Albumin (g/L)	31.9 ± 4.2	32.6 ± 3.9	0.308
Calcium (mmol/L)	2.25 ± 0.19	2.21 ± 0.18	0.262
Phosphorus (mmol/L)	1.62 ± 0.46	1.46 ± 0.40	0.022
iPTH (pg/ml)	176[60, 300]	158 [96, 241]	0.808
Alkaline phosphatase (U/L)	87.3 ± 32.8	89.7 ± 44.4	0.680
Cholesterol (mmol/L)	4.57 ± 1.36	4.64 ± 1.11	0.331
Triglycerides (mmol/L)	1.66[1.15, 2.69]	1.58 [1.20, 2.52]	0.858
HDL/LDL	0.39 ± 0.17	0.41 ± 0.16	0.701
hs-CRP (mg/L)	2.01[0.77, 5.53]	0.83 [0.41, 2.37]	0.001
eGFR (ml/min)	4.79 ± 2.40	5.52 ± 3.36	0.085
suPAR (pg/ml)	7930.1 ± 2541.5	6579.3 ± 2199.7	0.001
ASMI (kg/m2)	7.21 ± 2.09	7.05 ± 1.32	0.619
ECW/TBW	48.16 ± 4.39	46.14 ± 3.12	0.004
Dialysis duration (month)	35.7 ± 27.6	25.3 ± 19.1	0.012
CAPD (n, %)	92(52.6%)	22 (43.1%)	0.263
Kt/V (per week)	2.03 ± 0.43	2.21 ± 0.56	0.013
4 h D/Pcr	0.65 ± 0.11	0.65 ± 0.12	0.991
Ultrafiltration (ml/d)	520 [270,865]	200[10,635]	<0.001
Volume of urine (ml/d)	300 [0,878]	712 [372,1010]	0.001
RRF (ml/min)	1.29 [0,3.58]	2.2 [1.09,4.95]	0.002
Total volume of dialysate (L/d)	7.5 ± 1.7	6.6 ± 1.5	0.001
Low-calcium dialysate (n, %)	67 (38.3%)	13 (25.5%)	0.093
Glucose exposure (g/d)	131.6 ± 43.9	108.2 ± 35.8	<0.001
P-binder (Ca) (n,%)	33 (18.9%)	9 (17.6%)	0.845
Vitamin D intake (n, %)	78 (44.6%)	21 (41.2%)	0.667
Statins (n,%)	29 (16.6%)	8 (15.7%)	0.881

CVC: cardiovascular calcification; BMI: body mass index; MAP: mean arterial pressure; CAPD: continuous ambulatory peritoneal dialysis; iPTH: intact-parathyroid hormone; HDL/LDL: high-density lipoprotein and low-density lipoprotein ratio; hs-CRP: high-sensitivity C reaction protein; eGFR: estimated glomerular filtration rate; ASMI: appendicular skeletal muscle mass index; ECW/TBW: extra-cellular water/total body water; Kt/V: total weekly urea clearance; 4 h D/Pcr: 4 h dialysate/plasma creatinine ratio; RRF: residual renal function; P-binder (Ca): calcium-based phosphate binder.

Correlation between serum suPAR levels and cardiovascular calcification

Pearson or Spearman correlation was used to analyze the correlation between serum suPAR and CVC. Serum suPAR was positively correlated with ValvC (r = 0.362, p < 0.001), AAC scores (r = 0.254, p < 0.001), and CAC scores (r = 0.301, p < 0.001).

Relationship between serum suPAR and progression of cardiovascular calcification

After a 2-year follow-up period, 190 patients completed the assessment for AAC progression, of whom 41.1% exhibited AAC progression. AAC scores increased by an average of 9.4 in the AAC progression group. A total of 176 patients completed the assessment for CAC progression, with 76.7% demonstrating CAC progression. CAC scores increased by an average of 246.4 in the CAC progression group. Additionally, 202 patients completed the assessment for ValvC progression, and 6.4% of these patients had ValvC progression. Logistic regression analysis revealed that serum suPAR levels were associated with the progression of CVC across all types prior to adjustment. Following adjustment using model 1 and model 2, elevated serum suPAR remained a significant risk factor for AAC and CAC progression but was no longer associated with ValvC progression (Table 2).

Table 2.

Logistic regression analysis of serum suPAR and cardiovascular calcification progression.

suPAR (100 pg/ml)	Unadjusted		Model 1		Model 2
	OR (95% CI)	P	OR (95% CI)	P	OR (95% CI)	P
AAC Progression	1.015 (1.003–1.028)	0.016	1.019 (1.005–1.033)	0.006	1.017 (1.003–1.031)	0.020
CAC Progression	1.041 (1.020–1.063)	<0.001	1.039 (1.017–1.062)	<0.001	1.035 (1.008–1.062)	0.010
ValvC Progression	1.022 (1.002–1.041)	0.029	1.022 (0.999–1.046)	0.063	1.010 (0.978–1.043)	0.544

Model 1: adjusted by age, body mass index, diabetes, history of CVD; Model 2: Model 1+ high-sensitivity C reaction protein, calcium, phosphorus, intact-parathyroid hormone, estimated glomerular filtration rate, dialysis duration, total volume of dialysate, volume of urine, total weekly urea clearance, baseline calcification score.

ROC assessment of coronary artery calcification progression by suPAR

The ROC was plotted to calculate the area under the curve for suPAR to identify the progression of CAC. The optimal cutoff value of serum suPAR for the prediction of CAC progression in PD patients was 6289.4 pg/ml, with a sensitivity of 71.9% and a specificity of 65.9%, corresponding to an area under the curve of 0.701 (Figure 1).

Figure 1.

ROC curve of serum suPAR for coronary artery calcification progression.

Comparison of cardiovascular events and cardiovascular mortality

As of September 30, 2024, the median follow-up duration was 25.4 months. Eight patients underwent kidney transplantation, 18 switched to hemodialysis, and 22 were transferred to other hospitals. A total of 50 cardiovascular events were recorded, comprising 39 cases of congestive heart failure, 3 cases of acute myocardial infarction, 1 case of rapid atrial fibrillation, and 7 cases of ischemic stroke. A total of 28 patients died, with 19 patients (67.9%) died due to CVEs suggesting that cardiovascular death was the main cause of death in PD patients. According to the optimal cutoff value of serum suPAR for the prediction of CAC progression, patients were divided into high suPAR group (>6289.4 pg/ml) and low suPAR group (≤6289.4 pg/ml). The incidence of CVEs in the high suPAR group was significantly higher than that in the low suPAR group (Figure 2(A)), but the difference of cardiovascular mortality between the two groups was not statistically significant (Figure 2(B)).

Figure 2.

Comparison of incidence of cardiovascular events (A) and cardiovascular mortality (B).

Influence of serum suPAR on cardiovascular outcomes

Serum suPAR was included as a continuous variable in multi-model COX regression analysis. Higher serum suPAR was a risk factor for CVEs and cardiovascular mortality before adjusting for multivariate. After multivariate adjusted by model 1 and model 2, higher serum suPAR remained an independent risk factor for CVEs, but no longer an independent risk factor for cardiovascular mortality (Table 3). Additionally, Cox regression analysis did not identify CVC progression as a risk factor for CVEs and cardiovascular mortality.

Table 3.

Cox proportional hazard ratio for cardiovascular outcomes by serum suPAR.

suPAR (100 pg/ml)	Cardiovascular events		Cardiovascular mortality
	HR (95% CI)	P	HR (95% CI)	P
Unadjusted	1.018 (1.00–1.027)	<0.001	1.023 (1.009–1.038)	0.001
Model 1	1.012 (1.001–1.023)	0.031	1.009 (0.994–1.024)	0.236
Model 2	1.012 (1.001–1.024)	0.040	1.002 (0.983–1.022)	0.824

Model 1: adjusted by age, diabetes, history of CVD; Model2: Model 1 + high-sensitivity C reaction protein, hemoglobin, serum albumin, low-density lipoprotein, phosphorus, intact-parathyroid hormone, estimated glomerular filtration rate, dialysis duration, total weekly urea clearance.

Discussion

In the present study, up to 77.4% of PD patients exhibited various forms of CVC at baseline. Among these patients, 49.1% presented with AAC, 68.6% demonstrated CAC, and 11.5% showed ValvC. During 2 years of follow-up, we found that many patients had progression of CVC, with a particularly high rate of 76.7% of patients with CAC progression. We further found that elevated serum suPAR was closely associated with the progression of CVC in PD patients and was an independent risk factor for AAC progression and CAC progression. Higher serum suPAR was also an independent risk factor for CVEs in PD patients.

The incidence of CVC remains significantly elevated in dialysis patients. While traditional risk factors including age, diabetes, and hypertension have been well-established, recent research has shifted focus toward nontraditional contributors. Emerging evidence highlights the critical role of calcium and phosphorus metabolism disorders, oxidative stress, malnutrition, and chronic inflammation in the pathogenesis of CVC among this patient population [19,20]. CVC represents a form of ectopic tissue mineralization that frequently coexists with various pathological conditions, including aortic stenosis, atherosclerosis, renal failure, and chronic inflammation. Both in vitro and clinical studies have shown that a series of active osteogenic processes are key to CVC formation, and that these osteogenic processes are often triggered by inflammation. suPAR is a novel inflammatory biomarker that reflects levels of immune activation and inflammation. It is the soluble form of the urokinase plasminogen activator receptor (uPAR). Stimulation by inflammatory mediators and immune activation induce the gene expression of uPAR and release of suPAR, via major inflammatory transcriptional pathways regulated by NF-kB and activator protein 1 (AP1) and increase the blood concentration of suPAR [21]. Immune dysregulation and inflammation are known to play a significant role in the development of CVC and atherosclerosis. It is believed that suPAR can modulate the function of monocytes to promote atherosclerosis [22]. uPAR is highly expressed in atherosclerotic plaques, at levels that correlate to inflammatory activity within the lesion [23]. A study by Wu W et al. demonstrated a significant correlation between serum suPAR levels and CVC in hemodialysis patients. The research revealed that patients with higher CAC scores exhibited substantially elevated serum suPAR levels compared to those with lower CAC scores [12]. Our previous cross-sectional study of PD patients was also observed that patients with high suPAR levels not only had higher CAC scores, but also higher AAC scores [13]. Furthermore, higher serum suPAR was found to be a risk factor for CVC progression in this prospective study, especially for CAC progression. In an Italian study involving 369 PD patients, 77% of the patients had CVC at baseline. Seventy-three percent of the patients exhibited CVC progression after 3 years follow-up [24]. The China Dialysis Calcification Study (CDCS) [5] included 1489 dialysis patients, including 321 PD patients, and found that 86.5% of the patients had CVC progression through 4-year follow-up, of which 69.6% had CAC progression, 72.4% had AAC progression, and 33.4% had ValvC progression. Our study observed that the corresponding types of calcification progression rates were 76.7%, 41.1%, and 6.4%, respectively. The difference may be related to the length of follow-up, the definition of calcification progression, and the type of population included. The cohort of our study was all patients with PD, while the CDCS included patients on hemodialysis. Current evidence suggests that persistent low-grade systemic inflammation is closely interrelated with and promotes CVC. Inflammatory cytokines can induce EMT by downregulating bone morphogenetic protein receptor type 2 and exacerbate CVC by upregulating tissue-nonspecific alkaline phosphatase activity [25]. The interaction between uPAR and αMβ2 integrin (Mac1) regulates cellular motility. uPAR binds to β1 integrins, which in turn activates the MAPK/ERK pathways and phosphorylates FAK, inhibiting collagen fibrillogenesis and thereby allowing cellular invasion and migration [26]. uPAR plays an important role in mesenchymal stem cells-osteoblast differentiation controlling the regulation of the C5aR expression with subsequent activation of the NFkB transcriptional program. uPAR-C5aR axis via the NFkB transcriptional program controls osteogenic differentiation with functional impact on vascular calcification [27].

High levels of serum suPAR increase the incidence and mortality of CVD in patients with CKD [28]. In a study of 4994 patients with CKD, suPAR levels were independently associated with mortality and cardiovascular outcomes, and this association was not affected by renal function indicators. This finding is consistent with the independent role of suPAR in the pathogenesis of atherosclerosis [29]. An Italian multicenter study, which covers 1,038 hemodialysis patients at 35 dialysis centers, revealed an important association between serum suPAR levels and risk of death. The findings indicate patients with elevated serum suPAR levels have increased their risk of cardiovascular mortality by 1.47 times and their risk of all-cause mortality by 1.91 times. [30]. The relationship between suPAR and CVD in PD patients has rarely been reported in the previous literature. In a retrospective study of 64 PD patients, Pawlak K et al. [31] reported that serum suPAR levels were lower in PD patients with CVD than in the non-CVD group. This is contrary to what has been observed in the vast majority of studies of patients with CKD or hemodialysis. Due to the weaknesses of this study, such as a small sample size and its retrospective nature, the bias in the results cannot be ignored. In our prospective study, we found that high serum suPAR is an independent risk factor for CVEs in PD patients, although it is not yet an independent risk factor for cardiovascular mortality, which may be related to the short follow-up time and low number of cardiovascular deaths in this cohort. CVC is an important risk factor for poor cardiovascular outcomes in dialysis patients. One of our previous studies showed that ValvC is an independent risk factor for CVEs and cardiovascular mortality in PD patients [32]. The CDCS [5] has also shown that CAC progression is a risk factor for non-fatal CVEs and all-cause mortality in dialysis patients. We found that patients with high serum suPAR levels had more severe CVC and progressed more rapidly in calcifications, which may partly explain the higher incidence of CVEs in patients with high serum suPAR levels. The suPAR has demonstrated its capacity to indicate vascular inflammation, subclinical organ damage, and endothelial dysfunction, implying a close association with the atherosclerotic process [33]. Hindy et al. [22] found that elevated suPAR levels can regulate monocyte function, thereby promoting the development of atherosclerosis, which is strongly associated with the development of CVD.

There are also some limitations in our study. Firstly, although our study found that suPAR is associated with calcification progression in PD patients, the exact mechanism is still unclear and needs to be further explained through basic research. Secondly, because it was a real-world observational study, not all recruited patients completed assessments for all types of CVC progression. Serum suPAR levels were only measured at baseline, without dynamic monitoring of changes over time. In addition, due to the time limit of the study, our follow-up time is relatively short, and the number of cases of cardiovascular death is relatively small, which may affect the assessment of the predictive value of suPAR on cardiovascular mortality. However, as far as we know, our study is the first to establish an association between suPAR and CVC progression in PD patients, which could provide a new pathway to investigate the causes of high prevalence of CVEs in PD patients.

In summary, CVC is prevalent in PD patients. High levels of serum suPAR are associated with AAC progression and CAC progression in PD patients. Serum suPAR can be used as an independent predictor of CVEs in PD patients, but not yet for cardiovascular mortality.

Funding Statement

This study was supported by the Health Science and Technology Program of Zhejiang Province of China (Grant No. 2022KY396; Grant No. 2022KY1300).

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethical statement

This research protocol strictly follows the ethical guidelines of the Declaration of Helsinki and has been reviewed and approved by the Ethics Committee of Shaoxing People’s Hospital (2021-K-Y-329-01). All subjects participating in the study have signed an informed consent form.