Introduction
Idiopathic nephrotic syndrome (INS) is a common renal disease during childhood characterized by proteinuria, edema and hypoalbuminemia (1). Prolonged duration of nephrotic status results in increased risk for cardiovascular disease due to the risk factors including hyperlipidemia, hypertension, using corticosteroids and immunosuppressive drugs, hypercoagulability, and oxidative stress (2). INS is considered an important risk factor for accelerated atherosclerosis in the adult population. However, the role of nephrotic syndrome in the development of atherosclerosis in children is not clear (3).
Endothelial dysfunction is an early pathophysiological feature and an independent predictor of poor prognosis in many forms of cardiovascular disease. The integrity of the endothelial function is highly dependent on normal nitric oxide (NO) production (4). Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase (NOS), inhibits vascular NO production. NO is responsible for various regulatory mechanisms in the cardiovascular system and defined as an anti-atherogenic molecule (3,4). Recent studies have shown that increased levels of serum ADMA was an independent risk factor for coronary heart disease (5). Serum ADMA levels increase in atherosclerotic disease and in conditions that are risk factors for coronary heart disease such as hypercholesterolemia, hypertriglyceridemia, hypertension, insulin resistance, diabetes, and hyperhomocysteinemia (6,7).
Carotid intima-media thickness (CIMT) measurement with B-mode ultrasound is a noninvasive, reliable, and relatively inexpensive method for detection of subclinical atherosclerosis (8). Epidemiologic studies have demonstrated the relationship of ADMA and CIMT with atherosclerosis (9-11).
The aim of this study was to investigate the relationship of ADMA with CIMT, an indicator of subclinical atherosclerosis, and atherosclerotic risk factors in children with INS.
Methods
This study was approved by the ethical committee of the İstanbul Medical Faculty (2013/706), in accordance with the Declaration of Helsinki. Informed consent was obtained from the parents of the patients and the controls. The study group comprised 41 children (25 boys, 16 girls) with INS who were under follow-up in our outpatient clinic.
INS was diagnosed in all patients in accordance with the criteria recommended by the International Study for Kidney Diseases in Children (12). Fifteen patients underwent kidney biopsy. The diagnosis was focal segmental glomerulosclerosis (FSGS) in five patients and minimal change disease in the remaining 10 patients.
The children were divided into three subgroups:
Group 1: Steroid-free remission (n=18);
Group 2: Steroid-induced remission, still on steroid therapy (n=11);
Group 3: Active proteinuria despite steroid and/or other immunosuppressive therapy (n=12).
In group 3, five patients were being treated with cyclosporine (n=4) or mycophenolate mofetil (n=1) for FSGS, the remaining seven patients were at the beginning of a new attack and still had active proteinuria although steroid had been initiated.
The control group consisted of 33 age- and sex-matched healthy subjects (17 boys and 16 girls) with no history of INS and/or proteinuria.
CIMT measurement with B-mode ultrasound was performed for the detection of subclinical atherosclerosis in the patient and control groups. Body mass index (BMI), systolic and diastolic blood pressure (BP), serum lipid profile, and serum homocysteine levels were assessed as risk factors for atherosclerosis.
Anthropometric Measurements
Weight (kg) and height (cm) of the subjects were measured by the same nurse. BMI was calculated as weight/height2 (kg/m2); the standard deviation scores (SDS) of BMI was also computed via the LMS method using BMI references for Turkish children. Patients with a BMI SDS >2 were defined as obese and those with a BMI SDS 1-2, overweight (13).
BP was measured using an average of 3 consecutive BP measurements with standard sphygmomanometer. Physical activity was avoided for at least 10 min before BP measurement. The tables of the task force report on high BP in children and adolescents were used to evaluate BP (14).
Ultrasound CIMT Measurements
Measurements were performed using a 12 Mhz probe and still image settings on the same device (Logiq 9 GE Healthcare, Milwaukee, WI, USA) by the same operator. The carotid arteries were scanned longitudinally with the patient lying in the supine position. The IMT of the right and left common carotid artery, bifurcation, and the first 2 cm of the internal carotid artery were measured from three different points, evaluating only the posterior wall. Three measurements were made in each patient, and the mean value was calculated (15).
Laboratory Measurements
Blood samples were drawn in the morning after an overnight fast (at least 12 h). Serum was immediately separated from blood cells using centrifugation at 3000 rpm for 5 min and stored at -20 °C until required for analysis. Serum ADMA level was measured using an enzyme-linked immunosorbent assay (MASSAY bio-medical assay, China).
Serum total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and homocysteine were assayed on a modular autoanalyzer (Roche). Serum low-density lipoprotein (LDL) cholesterol was calculated using Friedewald’s formula (16). Dyslipidemia was defined as a total cholesterol level of ≥200 mg/dL, triglycerides ≥150 mg/dL, and LDL cholesterol ≥130 mg/dL (17). Normal serum levels of homocysteine in childhood are considered 3.3-11.3 µmol/L (18). For all patients, urinalysis was evaluated, and in patients with active proteinuria, the spot urine protein/creatinine ratio was calculated. Proteinuria was defined as a urine protein/creatinine ratio of >0.2 mg/mgCr and nephrotic range proteinuria was defined as a urine protein/creatinine ratio of >2 mg/mgCr.
Statistical Analysis
Analyses were performed using SPSS v. 21.0 package program for Windows. The results are expressed as mean ± SD and median (lower and upper limit) for descriptive data. The normality of the parameters was tested using the Shapiro-Wilk test.
Parametric (Student’s t-test) and nonparametric tests (Mann-Whitney U test or Kruskal-Wallis test in cases of more than 2 groups) were used for between-group comparisons. Relationships between variables were analyzed using correlation (Pearson’s correlation coefficient and Spearman’s coefficient). A p value of less than 0.05 was considered statistically significant.
Results
Clinical Characteristics
The mean age of the patients and control group was 9.2±4.2 years (range, 3.5-17.8 years) and 10.3±3.2 years (range, 3-15.8 years), respectively. The two groups were similar for age and sex (p>0.05) (Table 1). Moreover, comparing the subgroups with each other, there was no significant difference for age and sex (p>0.05) (Table 2).
The mean age of INS onset was 5.0±3.1 years (1.5-14.5 years) and the mean follow-up duration was 49.0±42.5 months (1-192 months). The mean duration of remission was 5.6±12.0 months (0-50 months) in group 1 and 2. The mean number of relapses of patients, except FSGS, was 3.9±3.2 during follow-up. In five patients with FSGS, the number of relapses could not be calculated, because they were not in remission despite use of immunosuppressive drugs. In the patients with active proteinuria (group 3), the mean protein/creatinine ratio in spot urine was 4.0±4.4 mg/mgcr.
Risk Factors for Atherosclerosis
The patient and control groups were similar in terms of BMI, and systolic BP (p>0.05). The patients had significantly higher diastolic BP compared with healthy controls (p=0.018) (Table 1). The median diastolic BP was significantly higher in group 3 than in the controls (p=0.032), whereas group 3 and control group were not different for BMI and systolic BP (p>0.05). Groups 1 and 2 were compared with the control group in regards to BMI, and systolic and diastolic BP (Table 2).
Serum total cholesterol and triglycerides were significantly higher in the patients than in the controls (p=0.019 and p=0.004, respectively), although serum LDL and HDL cholesterol did not differ between the groups (Table 1 ). Also, in group 3, serum total cholesterol, LDL and HDL cholesterol, and triglycerides were significantly higher than in the controls (p<0.05). There was no difference in serum lipid profile between groups 1 and 2 and the controls (Table 2).
Serum homocysteine levels were not different between patient and control groups (Table 1), or between subgroups and control groups (Table 2).
Serum ADMA Levels and CIMT Measurements and Risk Factors for Atherosclerosis
When we compared the groups with each other, serum ADMA levels and CIMT were not significantly different between the patient and control groups (Table 1), or among the subgroups (Table 2). In addition, there was no correlation between serum levels of ADMA and CIMT measurements in Spearman’s test. CIMT measurement was positively correlated with diastolic BP in the patient group (r=0.332; p=0.034).
Serum levels of ADMA were not related with age, sex, BMI, BP, serum profile and homocysteine the patient group (p>0.05). Among the patients in group 3, serum ADMA levels were significantly higher in patients whose serum total cholesterol levels were >200 mg/dL (0.122 µmol/L vs 0.038 µmol/L; p=0.042) and a positive correlation was detected between serum levels of ADMA and total (Figure 1) and LDL cholesterol (Figure 2).
Discussion
Endothelial dysfunction and increased IMT are the most important changes in early-stage subclinical atherosclerotic disease. ADMA, an endogenous inhibitor of NOS, was found to be correlated with endothelial dysfunction and proteinuria in patients with chronic renal disease (19,20). Vallance et al. (21) showed that endothelial function was impaired by exogenous ADMA administration. Moreover, it has been shown that endothelial dysfunction in chronic renal failure was improved by removing ADMA with hemodialysis (22). Few studies have associated ADMA with INS. Although serum ADMA levels in Schimke-immuno-osseous dysplasia were not different from those of the controls (23), Lücke et al. (24) found significantly higher ADMA levels in patients with sporadic FSGS, and suggested that ADMA might have a role in the pathophysiology of a subgroup of INS. Hyla-Klekot et al. (25) showed that serum ADMA levels were not different in remission from the active phase of INS.
Our study was designed to investigate a possible relationship of ADMA with CIMT, an indicator of atherosclerosis, and the risk factors for atherosclerosis such as BMI, dyslipidemia, BP, and homocysteine in children with INS.
According to our results, there was no difference in serum levels of ADMA between the patients and controls, as well as in the INS subgroups. The reason why serum ADMA levels were not elevated in INS may be that CIMT was also normal, which suggests that there was no remarkable atherosclerosis in our study group.
In some of the studies evaluating CIMT in children with INS, CIMT was found to be higher compared with controls, whereas other studies reported that there was no difference. CIMT has been reported to be increased in majority of children who were currently being treated for steroid-resistant NS (SRNS) and steroid-dependent NS (SDNS) (26-28). The majority of our patients had steroid-sensitive NS (SSNS) and they were in remission, as with another study that found normal IMT (29-31). Moreover, the mean follow-up duration of our patients was shorter than in studies that reported increased CIMT in INS (49 months vs. 93.7-94.5 months), which suggests that longer duration of nephrotic condition may be needed for emergence of atherosclerosis. In addition, the number of relapses was small as once or twice during follow-up in nearly half of our children. It may be expected that CIMT, and therefore ADMA, were not high in our patients because of the transient nature of metabolic changes and steroid exposure in SSNS.
When we evaluated the relationship between ADMA and risk factors for atherosclerosis, there was no difference between patients with INS and the control group in terms of BMI, systolic BP, and homocysteine, and there was no relationship between serum ADMA levels and these factors. Diastolic BP, total cholesterol, LDL cholesterol, and triglyceride levels were higher in patients with INS compared with the controls. This difference appears to be caused by the patients with active proteinuria (group 3). The presence of dyslipidemia is expected in the acute phase of INS. In addition, serum ADMA levels were significantly higher in patients whose serum total cholesterol levels were >200 mg/dL, and serum ADMA levels were positively correlated with total cholesterol, and LDL cholesterol in this group. It has been demonstrated that plasma concentration of ADMA was elevated due to hypercholesterolemia (32). Moreover Kitova et al. (33) reported that plasma level of ADMA was found to be increased in patients with non-treated asymptomatic hypercholesterolemia. However, diastolic BP was not correlated with serum ADMA levels, although it was found to be correlated with CIMT.
It has been reported that there was no increased risk for cardiovascular disease in young adults with relapsing childhood SSNS (3). Supporting this study, our findings suggest that serum ADMA levels are not a useful marker to indicate endothelial dysfunction in children with INS. These results should be interpreted while considering that the majority of our patients was steroid-sensitive and in remission at the time of study.
Regression of proteinuria, recovery of the lipid metabolism, and cessation of steroid therapy may result in the elimination of risk factors and remission of early atherosclerotic changes in SSNS. Dyslipidemia and BP should be closely monitored in patients with SDNS and SRNS.
Conclusion
In conclusion, children with INS did not show signs of endothelial damage assessed by ADMA and CIMT. This may be due to short duration of the nephrotic state. A long-term follow-up of children with INS is needed to determine atherosclerosis.
Authorship Contributions
Surgical and Medical Practices: B.A. Concept: B.A. Design: B.A., S.E. Data Collection or Processing: B.A., Z.N.Y., C.P. Analysis or Interpretation: B.A., Ü.D.M.D., O.B.E. Literature Search: B.A. Writing: B.A., A. Y.
Conflict of Interest: The authors declared that they have no conflict of interest.
Financial Disclosure: This study was supported by Istanbul University Scientific Research Project Foundation grant number of 35351 dated 2014.