Objectives: In patients with advanced-stage chronic kidney disease, renal insufficiency, arterial stiffness, and vascular calcification are strong predictors of cardiovascular risk. Signal peptide-CUB-EGF-like protein-1 (SCUBE1) levels increase during platelet activation and acute ischemic events. Here, we evaluated associations between SCUBE1 levels and electrocardiographic/echocardiographic findings, epicardial adipose tissue thickness, and carotid intima-media thickness in patients with chronic kidney disease.

Materials and Methods: Our study included 21 renal transplant recipients, 20 peritoneal dialysis patients, 20 hemodialysis patients, 20 predialysis patients with glomerular filtration rate < 30 mL/min, and 16 healthy volunteers.

Results: We found no differences in SCUBE1 levels between patient groups and healthy volunteers, regardless of history of diabetes mellitus, myocardial infarction, cerebrovascular events, and hypertension. SCUBE1 levels correlated with C-reactive protein in renal transplant recipients; magnesium in peritoneal dialysis patients; erythrocyte sedimentation rate in predialysis patients; and parathyroid hormone, platelet count, calcium-phosphate product, and calcium in hemo-dialysis patients. No associations were shown between SCUBE1 levels and electrocardiographic/echocardiographic findings. Elevated C-reactive protein in predialysis patients was associated with cardiac valvular pathologies. In hemodialysis patients, SCUBE1 levels increased after hemodialysis (P = .007). Levels were higher in healthy individuals with normal echocardiography and pre-dialysis patients with left ventricular diastolic dysfunction. Positive correlations were found between carotid intima-media thickness and SCUBE1 levels in dialysis patients (P < .05), but no study groups showed correlations regarding epicardial adipose tissue thickness.

Conclusions: Hemodialysis may contribute to cardio-vascular events because of increased SCUBE1 levels after hemodialysis; however, no association was shown between SCUBE1 and electrocardiography/ echocardiography findings. We found no correlations between epicardial adipose tissue thickness and SCUBE1 levels, and levels were significantly higher in healthy patients and in predialysis patients without left ventricular diastolic dysfunction. However, cor-relations were shown between SCUBE1 levels and carotid intima-media thickness and secondary hyperparathyroidism markers, indicating associations with atherosclerosis and bone mineral disease in dialysis patients.

Key words : Cardiovascular disease, Chronic kidney disease, SCUBE1

Introduction

Chronic kidney disease (CKD) is a global health problem with a large socioeconomic impact.¹ It is known that most patients with renal failure die from cardiovascular disease before reaching hemodialysis or kidney transplant, regardless of their stage.² Inflammation is associated with chronic renal failure.³ High-sensitive C-reactive protein (CRP) is increased in patients with CKD and is associated with poor prognosis in this population.^4,5 Arterial stiffness and vascular calcification are strong predictors of cardiovascular risk in patients with chronic renal failure and renal replacement therapy.

Patients receiving renal replacement therapy are also prone to thrombotic complications. Signal peptide-CUB-EGF-like protein-1 (SCUBE1) is a SCUBE gene family-bound cell surface protein.⁶ SCUBE1 is found in platelet alpha granules and is presented to the cell surface by platelet stimulation and activation. SCUBE1 levels have been shown to be increased in platelet aggregation and acute ischemic events. In this study, our aim was to evaluate the relationship between SCUBE1 level, electrocardiography (ECG), echocardiography, epicardial adipose tissue thickness (EATT), and carotid intima-media thickness (CIMT) in patients with chronic renal failure (glomerular filtration rate < 30 mL/min) and receiving renal replacement therapy (hemodialysis, peritoneal dialysis, and renal transplant).

Materials and Methods

Our study included a total of 97 people: 21 renal transplant recipients, 20 peritoneal dialysis patients, 20 hemodialysis patients, 20 patients with stage 4 chronic renal failure, and 16 healthy volunteers. Acute myocardial infarction in the last 6 months, cerebrovascular event, angina pectoris, history of coronary artery bypass grafting and other surgeries, active infection, acute trauma, active heart failure, hepatitis, cirrhosis, malignant disease, rheumatologic disease, chronic obstructive pulmonary disease, and pregnancy and postpartum period were defined as exclusion criteria. Written informed consent was obtained from all patients regarding voluntary participation in the study.

Complete blood count, fasting blood glucose, blood urea nitrogen, creatinine, sodium, potassium, calcium, phosphorus, magnesium, aspartate amino-transferase, alanine aminotransferase, total protein, albumin, parathyroid hormone (PTH), vitamin D, C reactive protein (CRP), and erythrocyte sedimen-tation rate (ESR) were measured. Serum SCUBE1 levels were measured using the enzyme-linked immunosorbent assay method with a commercial kit according to the manufacturer’s instructions. Absorbents were measured with a microplate reader at 450 nm. Serum SCUBE1 concentrations of patient serum samples were calculated with standard graphics (results are given in ng/mL).

Twelve-lead ECG was performed with ECG device standardized by the biomedical laboratory. Trans-thoracic echocardiography was performed according to the American Society of Echocardiography and European Association of Echocardiography guidelines⁷ by a cardiologist blinded to the clinical details of patients. A 3-MHz sector probe was used with the Generic Electric VIVID-S5 (model 050684VS5N) echocardiography device (GE Health-care, California, USA) for all patients. M mode, two-dimensional, pulse-wave Doppler, and tissue Doppler parameters were used in the echo-cardiographic evaluations. Epicardial adipose tissue thickness was measured in the parasternal long-axis diastolic phase. The same device was used to measure CIMT from the right carotid artery using a 12-MHz linear probe.

Electrocardiographic and echocardiographic find-ings were evaluated by the same cardiologist. Patho-logic findings in the ECG included ST segment and T-wave changes, right or left bundle branch block, and left ventricular hypertrophy (LVH) findings. According to the current guidelines, patho-logic findings in echocardiography include LVH and left ventricular diastolic dysfunction (LVDD), left ventricular systolic dysfunction (ejection fraction < 45%), right ventricular systolic dysfunction (right ventricular S wave < 10 m/s, tricuspid annular plane systolic excursion < 15 mm), pericardial effusion, significant valve pathology (at least moderate), and pulmonary hypertension (pulmonary artery pressure > 40 mm Hg).

This study was approved by the Çukurova University Faculty of Medicine Ethics Committee (2/12/2016 meeting no. 4).

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, IBM Corporation, Armonk, NY, USA). Data are presented as means and standard deviation or as medians. P < .05 was considered statistically significant.

Results

The mean age of the patient groups (81 patients) was 47.90 ± 14.35 years, and the mean age of the control group (16 healthy people) was 44.87 ± 5.48 years; 49 patients (60.50%) and 9 healthy volunteers (56.30%) were males. Age distribution and SCUBE1 levels of the patient groups and the healthy control group are summarized in Table 1. No statistically significant difference was found between patient and control groups in terms of age and sex.

Associations between biochemical parameters and SCUBE1 levels in study groups are summarized in Table 2. SCUBE1 levels were similar in all patients regardless of history of diabetes mellitus, myocardial infarction, cerebrovascular events, and hypertension. SCUBE1 levels in hemodialysis patients were significantly increased after dialysis versus before dialysis (P = .007). Associations between SCUBE1 level and EATT and CIMT in study groups are shown in Table 3.

Electrocardiography and echocardiography findings
Although we did not observe any pathologic ECG findings in the healthy control group, we detected ST-segment depression (4 patients), T-wave changes (6 patients), LVH (2 patients), right branch block (5 patients), left branch block (4 patients), and normal sinus rhythm (60 patients) in the patient groups. There were no significant differences between CRP and SCUBE1 values in patients with and without pathologic ECG findings in the patient groups. However, median SCUBE1 values were higher in patients with ECG pathology in the peritoneal dialysis, predialysis, and hemodialysis patient groups, although these were not statistically significant. The echocardiographic evaluation revealed 5 healthy individuals with LVDD; we also observed 36 patients with LVH, 58 patientswith LVDD, 1 patient with pericardial effusion, 24 patients with right ventri-cular systolic dysfunction, 7 patients with significant valve pathology, and 21 patients with pulmonary hypertension. No statistically significant differences were observed between patient groups and the healthy group in terms of pathologic ECG and echocardiographic findings and EATT versus SCUBE1 levels. Table 4 shows the CRP levels of patients and control groups with and without ECG and echocardiograph pathology. Table 5 shows SCUBE1 levels and comparisons of patient and healthy control groups with and without ECG and echocardiograph pathology.

Relationship between SCUBE1 and epicardial adipose tissue and carotid intima-media thickness
The mean value of EATT was 5.54 ± 1.69 mm in the renal transplant group, 4.79 ± 1.59 mm in the peritoneal dialysis group, 5.37 ± 1.69 mm in the predialysis group, 5.09 ± 2.35 mm in the hemo-dialysis group, and 4.43 ± 1.44 mm in the healthy control group. The right CIMT was 0.55 ± 0.13 mm in the renal transplant group, 0.58 ± 0.12 mm in the peritoneal dialysis group, 0.63 ± 0.10 mm in the predialysis group, 0.53 ± 0.13 mm in hemodialysis group, and 0.55 ± 0.08 mm in the healthy control group. When correlations between SCUBE1 levels and EATT and CIMT were examined, we observed a significant relationship between CIMT and SCUBE1 level in the hemodialysis and the peritoneal dialysis groups. The relationships between EATT and right CIMT and SCUBE1 level are summarized in Table 3.

Discussion

Chronic kidney disease increases the risk of cardio-vascular disease by 1.5 to 3.5 times. Both diseases are characterized by increased inflammation. Increased levels of CRP in patients with CKD can predict cardiovascular risk.⁸ SCUBE1 was found to be stored in alpha granules in inactive platelets and secreted into small soluble fragments and incorporated into thrombus.⁹ One study showed that SCUBE1 levels were increased in acute ischemic stroke and acute coronary syndrome with platelet aggregation and activation, and it was stated that this protein may be a good marker in acute thrombotic events.¹⁰

In our study, we aimed to investigate whether a relationship existed between SCUBE1 levels and ECG and echocardiography findings, EATT, and CIMT in patients on renal replacement therapy. There was no significant difference in mean SCUBE1 values between the groups. Male sex is an invariable risk factor for cardiovascular disease.¹¹ In a study evaluating the correlations between SCUBE1 levels and soluble CD-40 ligand inducing platelet-activating factor synthesis in endothelial cells and smooth muscle cells, predialysis SCUBE1 levels were significantly higher in male than in female patients; therefore, SCUBE1 was thought to be one of the factors contributing to the predisposition to cardiovascular events in males.¹² In our study, no statistically significant difference was found in SCUBE1 levels between patient and healthy control groups. However, SCUBE1 was found to be higher in healthy controls and hemodialysis patients, whereas, in the other patient groups, both mean and median values were higher in women. This may be related to increased inflammation and/or cardio-vascular risk in CKD women before dialysis, those on peritoneal dialysis, and those who have received renal transplant.

C-reactive protein, a significant inflammation marker, and ESR values were significantly higher in the peritoneal dialysis and hemodialysis groups. As expected, hematocrit, calcium, and vitamin D levels were lower and PTH and phosphorus levels were higher in the peritoneal dialysis and hemodialysis patient groups. Secondary hyperparathyroidism has also been implicated in the frequency and severity of cardiovascular disease in patients with CKD. The prevalence of vascular calcification was high in dialysis patients. In a cross-sectional study of patients with high risk of cardiovascular disease, an inverse relationship was found between vitamin D levels and vascular calcification degree.¹³ In our evaluation of laboratory values versus SCUBE1 levels, we observed a correlation between SCUBE1 levels and CRP in the renal transplant group; magnesium in the peritoneal dialysis group; PTH, ESR, platelet count, and calcium-phosphorus product in the predialysis group; and calcium levels in the hemodialysis group. We suggest that secondary hyperparathyroidism is associated with SCUBE1 levels in patients on dialysis and in patients predialysis.

As an indication of the role of SCUBE1 in the inflammatory process, an in vitro study indicated that SCUBE1 concentrations change with interleukin 1β and tumor necrosis factor α treatment.¹⁴ Because hemodialysis triggers inflammation, SCUBE1 levels increased after hemodialysis in our study. Hemo-concentration may also contribute to this condition. Similarly, in a study conducted with hemodialysis patients, SCUBE1 levels after dialysis were found to be higher than those before dialysis, and the authors suggested that hemodialysis treatment led to platelet activation.¹⁴ In our study, SCUBE1 levels showed a parallel increase with inflammation markers (CRP and ESR) and the number of platelets in renal transplant recipients and predialysis patients, respectively.

In addition to traditional risk factors, there are some other factors that increase cardiovascular risk in patients with CKD. These include left ventricular mass, carotid-femoral pulse-wave velocity, left ventricular systolic and diastolic dysfunction, and vascular calcification. Diastolic dysfunction is frequently found in patients with CKD.^15-17 When we examined echocardiography findings, significant pathologies, including LVH and LVDD, pericardial effusion, right ventricular systolic dysfunction, valve pathologies, and pulmonary hypertension, were detected. Some healthy patients showed LVDD. However, these echocardiography findings were not significantly related to SCUBE1 and CRP levels. This may be because of inclusion of clinically stable patients with no acute problems and the small number of patients in our study. However, we suggest that SCUBE1 does not increase without acute conditions in these patients with endothelial dysfunction and platelet dysfunction.

Although our healthy control patients showed no pathologic ECG findings, there were significant ECG abnormalities in the patients with CKD, including ST segment and T-wave changes, LVH, and right or left bundle branch block. However, findings in patients with and without ischemic ECG results were not significantly related to CRP and SCUBE1 levels. We did observe median SCUBE1 levels to be higher (but not significantly) in patients with ECG pathology in the peritoneal dialysis, predialysis, and hemodialysis groups.

In our study, we observed no significant dif-ferences between patients and healthy volunteers in terms of EATT and CIMT and no correlations between SCUBE1 levels and EATT in both CKD patients and healthy individuals. A positive correlation between SCUBE1 levels and CIMT was only shown in the dialysis groups.

Both CRP and SCUBE1 levels did not differ between healthy controls and patients with dialysis, predialysis, and renal transplant without acute clinical findings. In renal transplant recipients, SCUBE1 and CRP levels were correlated. In patients on peritoneal dialysis, SCUBE1 levels were correlated with ESR and number of platelets. The effect of secondary hyperparathyroidism markers on SCUBE1 levels in dialysis patients may support that bone mineral disease may be a pathogenetic factor in cardiovascular events. The increase in SCUBE1 levels after hemodialysis suggests that hemodialysis may activate platelets and may be a factor that increases cardiovascular events. However, in nonacute conditions, SCUBE1 levels were not associated with ECG or echocardiographic findings. Interestingly, CRP levels were higher in patients with valvular pathology. Inflammatory causes of valvular diseases may also have contributed to this condition. Because no correlation was shown between SCUBE1 levels and EATT, which is an important finding in cardiovascular diseases, and because SCUBE1 was significantly higher in predialysis patients without LVDD and in healthy individuals without pathologic echocardiographic findings, we are not able to comment on cardiovascular events with single SCUBE1 measurement. In contrast to healthy indi-viduals, the increase of SCUBE1 levels may be an important finding (although not statistically significant) in women with CKD, except for those on hemodialysis. Carotid intima-media thickness, which is a good marker of atherosclerosis, and secondary hyperparathyroidism markers were also correlated with SCUBE1 levels in dialysis patients. Therefore, SCUBE1 levels may be associated with atheros-clerosis and bone mineral disease in dialysis patients. Future studies should examine theoretical and practical applications.

Limitations

Limitations to our study include the small number of patients. Our results may be different in larger-scale studies. Another limitation is the cross-sectional design of our study.

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