Low serum levels of vitamin D are associated with anxiety in children and adolescents with dialysis

Anxiety is a frequent and serious complication of children and adolescents receiving dialysis. Low serum vitamin D levels have been associated with anxiety in non-pediatric patients. This study sought to examine the possible association between serum vitamin D levels and the presence of anxiety in children and adolescents with dialysis in China. A total of 156 pediatric patients who were on hemodialysis or peritoneal dialysis and 100 healthy controls were included in the current study. Serum 25-hydroxyvitamin D [25(OH)D] levels were measured by using a competitive protein-binding assay. Anxiety was assessed by using the Chinese version of the Screen for Child Anxiety Related Emotional Disorders (SCARED, = 25 as cutoff). Among 156 patients, 110 had a current anxiety (70.5%) and 46 did not (29.5%). Serum levels of 25(OH)D were significantly lower in patients with anxiety than in normal controls (19.4 ± 10.3 vs. 38.6 ± 15.5 ng/ml, P < 0.001). Serum 25(OH)D levels (≤15.0 ng/ml) were independently associated with the existent of anxiety in children and adolescents receiving dialysis (OR 4.650, 95% CI 1.663–13.001, P = 0.003). Our research demonstrates that low serum levels of vitamin D are independently associated with anxiety among children and adolescents on dialysis, which needs to be confirmed in future experimental and clinical studies.


Characteristics of the sample per tertile of serum 25-hydroxyvitamin D. Patients in the lowest
tertile (≤15.0 ng/ml) were more likely to be girl (P = 0.003) and had higher intact parathyroid hormone (iPTH) (P < 0.001) and higher Screen for Child Anxiety Related Emotional Disorders (SCARED) scores (P = 0.005). No significant differences were observed in term of other variables between three tertiles of serum 25-hydroxyvitamin D [25(OH)D] (Table 1).
Differences in Vitamin D Level Tertiles of Subjects. Significant differences in 25(OH)D level tertiles of pediatric patients were observed between the patients with anxiety and the patients without anxiety (P = 0.002). Indeed, the proportion of patients in the lowest tertile (≤15.0 ng/ml) was significantly higher in the anxiety group (P = 0.001), whilst the proportion of patients in the highest tertile (≥25.1 ng/ml) was significantly lower in the anxiety group (P = 0.011) ( Table 2).

Independent Characteristics of Anxiety in Dialysis Patients.
With all patients taken as a whole, anxiety occurrence taken as a dependent variable and tertile 2 taken as the reference used for 25(OH)D levels in the logistic analysis, 25(OH)D levels (≤15.0 ng/ml) were independently associated with the existence of anxiety in pediatric patients with dialysis (OR 4.186, 95% CI 1.400-12.517, P = 0.010). Moreover, the number of hospitalizations was significantly associated with the presence of anxiety in children and adolescents treated with dialysis (OR 1.519, 95% CI 1.280-1.802, P < 0.001) ( Table 3).

Discussion
In the present study, we found that 70.5% of Chinese patients with dialysis presented with anxiety, which is similar to the findings of previous studies 3 . Compared with adult patients with dialysis, however, the data regarding the prevalence of anxiety among pediatric patients with dialysis remain scarce, thereby limiting the reliability of the reported findings. Our results also demonstrated a correlation between anxiety and the number of hospitalizations in children and adolescents with dialysis, which agrees with the results of earlier studies 3 .
To the best of our knowledge, this is the first study to explore the possible association between serum vitamin D levels and anxiety in children and adolescents on dialysis. We found that low serum levels of vitamin D were significantly associated with and anxiety in children and adolescents with dialysis. As mentioned earlier, a growing body of research has reported lower vitamin D levels in non-dialysis patients with anxiety [8][9][10][11][12] . Animal experiments have demonstrated a profound anxiolytic-like effect of vitamin D treatment in in female rats with long-term estrogen deficiency 14 . Moreover, study of patients with premenstrual syndrome-related mood disorders has shown positive effects of vitamin D supplementation on reduction in anxiety score 17 . The exact role of vitamin D in the pathophysiology of anxiety remains unknown. As a neurosteroid hormone across the blood-brain barrier 18 , vitamin D exerts its function via binding to VDR and vitamin D activating enzyme 1ahydroxylase, which are broadly present in neuronal and glial cells of the human brain 6,7 . Studies in animals have demonstrated that the VDR-deficient mice shown increased anxiety symptoms 13 , which suggested that the defects in the vitamin D-VDR system may directly result in the development of anxiety. Moreover, vitamin D plays a key role in modulating the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and C-reaction protein (CRP) 19 . A growing body of evidence suggests the involvement of cytokines in the process of anxiety by modulating the metabolism of neurotransmitters such as dopamine and serotonin [20][21][22][23] . Up-regulation of inflammatory cytokines and neutrophil activation has been found in non-infected pediatric patients with dialysis 24,25 . Therefore, these results suggest that vitamin D might play an important role in anxiety among children and adolescents receiving dialysis.
It is also possible that pediatric patients with anxiety take less vitamin D in the diet and are less exposed to sunlight; therefore, it is the anxiety which "causes" the low levels of vitamin D in pediatric patients receiving dialysis. The most important question in the present study is the nature and direction of the causal association between serum vitamin D levels and anxiety in children and adolescents with dialysis.   Several limitations of the present study should be noted. First, the seasonal variation of vitamin D status makes it preferable to perform all measurements on the same day. Second, the cross-sectional nature of the study did not allow us to explain the causal relationship between vitamin D and in anxiety among children and adolescents with dialysis. Third, subjects in our sample came from one only one clinic, which limited the generalization of the findings of the present study. Finally, the small sample size reduced the statistical power of the study.

Conclusion
In spite of the limitations mentioned above, our research demonstrates an important association between serum vitamin D levels and anxiety in anxiety among children and adolescents with dialysis. Further prospective studies on larger populations should be encouraged to explore the exact relationship between them, which may provide a novel therapeutic target for anxiety in children and adolescents with dialysis.

Materials and Methods
Study design and population. Pediatric patients on chronic hemodialysis (HD) or peritoneal dialysis (PD) at the Dialysis Center of the First Affiliated Hospital of Jiaxing University between 15 May 2013 and 21 June 2016 were included in this cross-sectional study. Eligibility criteria were as follows: (1) Chinese ethnicity; (2) age between 8 and 18 years; (3) receipt of dialysis therapy for at least 3 months; (4) hospitalization within 14 days (exclusive of a hospital stay for HD or PD); (5) the willingness to give informed consent. Exclusion criteria included: (1) subjects with severe visual or auditory impairment or mental retardation; (2) renal transplant recipients; (3) subjects with a history of psychiatric disorders including anxiety and depression; (4) subjects with malignancy/leukaemia, chronic hepatic disease, autoimmune diseases or active infections; (5) subjects having a significant life event unrelated to their renal disease in the past 30 days, such as severe illness of a family member, family structure changes, losing a family member, changing the living place; (6) subjects with osteoporosis or receiving supplementation with ergocalciferol or cholecalciferol. All dialysis patients were maintained on a single dialysis modality. No patient switched. Meanwhile, 100 healthy volunteers without renal impairment, a history of psychiatric disorders, receiving supplementation with ergocalciferol or cholecalciferol, or osteoporosis, were recruited from a health survey. Written informed consents were obtained from all subjects and their parents before enrollment in the present study. The study was approved by the Ethics Committee of the First Affiliated Hospital of Jiaxing University and was conducted in compliance with the Declaration of Helsinki.
Clinical Variables. Demographic and clinical data including age, gender, body mass index (BMI), education, family structure, dialysis mode, dialysis duration, chemistry parameters and medications, were obtained from participant report and electronic medical records. BMI was calculated as weight (kg)/squared height (m 2 ). Blood samples were obtained for all subjects according to a standard protocol between 8 a.m. and 10 a.m. each morning and stored at −80 °C until measurement. The blood samples of the majority patients were collected at summer (44.2%) and fall (35.3%). All blood samples of control subjects were collected at the end of August 2015. Routine chemistry variables such as serum calcium, phosphorus, albumin, hemoglobin, and iPTH, were test by standard automated methods. Serum 25(OH)D was chosen to test vitamin D status for all participants because of its widespread clinical application, standardized ranges and testing protocol. Serum 25(OH)D levels were measured by using a competitive protein-binding assay and the intra-assay coefficient of variation was 7-10%. In the present study, serum 25(OH)D levels <10 ng/ml were considered deficiency and levels of 10-30 ng/ml were considered insufficiency. Serum 25(OH)D levels of all patients were divided into three tertiles (≤15.0, 15.1-25.0, and ≥25.1 ng/ml) owing to its skewed distribution. Because of ties, the number of patients in each tertile was not even.
Assessment of Anxiety. The Chinese version of SCARED 26,27 was used to assess the presence of anxiety in our study sample. SCARED is a 41-item self-report questionnaire of a broad range of anxiety symptoms that consists of five subscales which parallel the five subtypes of anxiety in the DSM-IV: panic disorder, generalized anxiety disorder, separation anxiety, school phobia, and social phobia. Each item is scored 0 to 2 (0 almost never, 1 sometimes, 2 often) with higher scores reflecting greater anxiety; a total score over 25 indicates the presence of clinically relevant anxiety. The Chinese version of SCARED has been shown to have good reliability and validity 27 . Statistical Analysis. Data are presented as mean ± standard deviation (SD) for normally distributed variables, medians (25th, 75th percentiles) for non-normally distributed variables, and number (percentage) for categorical variables. All patients with dialysis were stratified into two groups on the basis of the presence of anxiety (SCARED scores ≥25). Comparisons between the groups were performed using Student t test, one-way ANOVA, Pearson's χ 2 test, Fisher's exact test or Mann-Whitney U on the basis of the variable type and distribution. Associations were estimated using Spearman or Pearson linear coefficients. Binary logistic regression including age, gender, and the variables with P < 0.05 in the univariate analysis, was estimated to determine the correlates of anxiety in children and adolescents with dialysis. The abnormally distributed parameters were log-transformed for satisfying the log-linearity assumption. The results were described as adjusted odds ratios (OR) with the corresponding 95% confidence intervals (CI). All analyses were performed by using SPSS 17.0 (Chicago, IL). Significance level was defined as P < 0.05. Data availability. The datasets analyzed during the current study are available from the corresponding author on reasonable request.