This cross-sectional study screened 389 patients and ultimately enrolled 206 maintenance HD patients at Beijing Luhe Hospital Affiliated with Capital Medical University between June and July 2023. Patients were eligible for inclusion if they (1) were >18 years old, (2) had been on dialysis for at least 3 months, and (3) agreed to participate in this study. The exclusion criteria were as follows: (1) active malignant tumors or systemic diseases, (2) acute or chronic infectious diseases, (3) a survival period of less than 6 months, (4) inability to cooperate with lateral abdominal X-ray due to limb mobility impairment, (5) incomplete clinical data, or unwilling to participate in this study. Figure 1 shows a flowchart of patient recruitment for the study. We enrolled 41 matched control subjects who underwent routine health checkups at the health medical centre in Beijing Luhe Hospital, Affiliated with Capital Medical University. All participants signed an informed consent form before participating in the study. This clinical study was approved by the Ethics Committee of Beijing Luhe Hospital (2023-LHKY-012-02).

HD patients were treated 3 times per week (3.5–4 h per session) with standard bicarbonate dialysis using a Revaclear 400 dialyzer (with a membrane area of 1.8 m²). The vascular access of HD patients is arteriovenous fistula, graft or central venous catheter, with a blood flow rate ranged from 200 to 300 ml/min; the flow rate of the dialysate was 500 ml/min.

For all participants, demographics such as age, sex, and the presence of diabetes mellitus were recorded. The following laboratory data were collected at baseline: haemoglobin, leukocytes, platelets, C-reactive protein (CRP), serum albumin, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum potassium, serum sodium, total carbon dioxide (tCO2), serum creatinine, serum urea, serum uric acid, serum corrected calcium and serum phosphorus.

For HD patients, the following additional information was collected: height, body weight, dialysis vintage, primary kidney disease, history of cardio-cerebrovascular disease (CVD), smoking history, and supplemental biochemical parameters [serum ferritin, intact parathyroid hormone (iPTH), and fractional urea clearance (Kt/V)]. The body mass index (BMI) was calculated as weight/height² (kg/m²). Blood pressure was obtained from a single pre-dialysis reading, and pulse pressure was calculated as the difference between systolic and diastolic blood pressure. CVD was recorded if one of the following conditions was present: coronary artery disease, coronary atherosclerotic heart disease, congestive heart failure, transient ischaemic attack, stroke, or peripheral arterial disease (10). The height and body weight were measured.

Blood samples were collected from all participants for measurement of follistatin. For HD patients, blood samples were obtained before haemodialysis treatment. Following centrifugation, the serum was collected and stored at -80 °C until analysis. Serum follistatin concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. The sample was placed in a well of a microplate pre - coated with human follistatin monoclonal antibody and incubated. Subsequently, follistatin antibodies labeled with biotin were added. These antibodies combined with Streptavidin - HRP to form an immune complex. After incubation, the well was washed to remove the unbound enzyme. Then, Chromogen Solution A and B were added.

VC was imaged within 2 weeks in enrolled HD patients: abdominal aortic calcification (AAC) by lateral lumbar radiography with Kauppila scoring. Patients were allocated to the high-AAC-score group if they had an AAC score of ≥ 4 (moderate or severe calcification) and to the low-AAC-score group if they had an AAC score of < 4 (no or minor calcification) (11).

All analyses were performed using IBM SPSS 26.0 (IBM, USA). Normally distributed quantitative data are presented as the mean ± standard deviation (SD), and nonnormally distributed quantitative data are presented as the median (interquartile range). We performed normality tests on all parameters. For normally distributed data, comparisons between quantitative variables were performed using Student’s t-test or one-way analysis of variance (ANOVA). The Mann–Whitney U test was used to compare two sets of skewed distribution data. The χ² test was used to compare categorical variables. Binary logistic regression analysis was performed to determine the independent factors that affect VC (using forward LR). Variables with p values < 0.1 in the univariate logistic regression analysis (serum follistatin, age, with hypertension, pulse pressure, serum TC, serum sodium, serum corrected calcium) were selected. Additionally, variables suspected to be correlated with vascular calcification in clinical practice (even if they had p values > 0.1 in the univariate logistic regression analysis, such as dialysis vintage, BMI, serum phosphorus and iPTH) were included in our multivariate logistic regression analysis to identify independent associated factors for a high AAC score in HD patients. A two - tailed p value of < 0.05 was considered to indicate a significant difference.

In total, 206 HD patients were recruited according to the inclusion and exclusion criteria. The mean age of those patients was 58.02 ± 11.90 years. The median HD duration was 46.50 (17.00, 83.00) months. The primary renal diseases were diabetes (75 patients, 36.41%), chronic glomerulonephritis (64 patients, 31.07%), hypertensive glomerulosclerosis (36 patients, 17.47%), autosomal dominant polycystic kidney disease (11 patients, 5.34%), and others (20 patients, 9.71%). The vascular access were arteriovenous fistula (195 patients, 94.66%), arteriovenous graft(7 patients, 3.40%) and central venous catheter (4 patients, 1.94%). Table 1 shows the demographic and clinical characteristics of those patients and 41 healthy controls. HD patients had higher serum follistatin levels than the controls (1.25 ± 0.53 ng/ml vs. 0.88 ± 0.30 ng/ml, p < 0.001). Besides, HD patients had lower haemoglobin, platelet, albumin, TC, HDL-C, LDL-C, sodium, tCO2, and corrected calcium levels but higher serum CRP, TG, serum uric acid, serum creatinine, serum urea, serum potassium, and serum phosphorus levels.

We divided all dialysis patients into a high-serum-follistatin group (higher than or equal to the mean level) and a low-serum-follistatin group (lower than the mean level) according to the mean follistatin level. As shown in Table 2, compared with those in the low-follistatin group, patients in the high-follistatin group had higher AAC scores [9.00 (1.00, 16.00) vs. 2.00 (0.00, 9.00), p = 0.019]. A significant difference was observed in the prevalence of moderate or severe vascular calcification (AAC score ≥ 4) between the high and low-follistatin groups (67.82% vs. 42.86%, p < 0.001). Besides, patients in the high-follistatin group had longer dialysis vintage, lower BMI, platelets, and serum LDL-C levels. There was no difference in gender, age, diabetes mellitus status, hypertension, CVD history, haemoglobin, leukocyte count, serum CRP, TG, TC, HDL-C, serum albumin, serum sodium, serum potassium, serum tCO2, serum creatinine, serum urea, serum acid, serum corrected calcium, serum phosphate, serum iPTH, spKt/V or serum ferritin between the two groups. Despite non-significantly higher serum albumin levels in the high follistatin group, the prevalence of hypoproteinemia (serum albumin < 40 g/L) was notably higher in this group at 60.92%, compared to 45.38% in the low follistatin group, p = 0.027. There was no significant difference in the use of noncalcium phosphate binders, renin-angiotensin-aldosterone system inhibitors, calcitriol, or cinacalcet.

The median calcification score of 206 HD patients was 4.00 (interquartile range: 0.00 - 12.00). Of these, 110 patients exhibited moderate to severe vascular calcification, as defined by an AAC score ≥ 4 points. Compared with the low-AAC-score group, the high-AAC-score group had a higher serum follistatin concentration (1.37 ± 0.57 ng/ml vs. 1.12 ± 0.46 ng/ml, p < 0.001). HD patients with high AAC scores had lower serum sodium levels than patients with low AAC scores (p < 0.05). However, age, CVD history, pulse pressure, and serum corrected calcium were significantly greater in HD patients with a high AAC score than in those with a low AAC score (p < 0.05). In addition, there were no differences in dialysis duration, gender, BMI, haemoglobin, leukocyte count, platelets, serum CRP, TG, TC, HDL-C, LDL-C, serum albumin, serum potassium, serum creatinine, serum urea, serum phosphorus, serum iPTH, or serum ferritin between the high-AAC-score group and the low-AAC-score group. In terms of medication history, 74.55% of patients in the high-AAC-score group were taking noncalcium phosphate binders, which was significantly greater than the percentage in the low-AAC-score group (39.58%). There was no difference in the use of renin-angiotensin-aldosterone system inhibitors, calcitriol, or cinacalcet (Table 3).

To eliminate the impact of confounding factors on vascular calcification, a multivariate logistic regression model was used to identify the independent associated factors for vascular calcification in HD patients. Variables with p values < 0.1 in the univariate logistic regression analysis were selected. Additionally, variables suspected to be correlated with vascular calcification in clinical practice (even if they had p values > 0.1 in the univariate logistic regression analysis, such as dialysis vintage, BMI, serum phosphorus and iPTH) were included in our multivariate logistic regression analysis to identify independent associated factors for a higher AAC score in HD patients. In the analysis, higher serum follistatin, higher pulse pressure, lower serum TC, lower serum sodium, and higher serum corrected calcium were independently associated with vascular calcification in HD patients (Table 4).

The ROC curve analyzing serum follistatin concentration for predicting vascular calcification in HD patients yielded an AUC of 0.655 (95% CI: 0.580-0.729; p < 0.001). At the optimal cut-off value of 1.26 ng/mL, the sensitivity was 53.6% and the specificity was 71.9% (Figure 2).