| Abstract|| |
The two most common causes of death in patients with chronic kidney disease (CKD) are cardiovascular diseases and infections, and both have been linked to impaired vitamin D levels and dysregulated immune response. The aim of this work is to study the relation between vitamin D levels in children with end-stage renal disease (ESRD) on regular hemodialysis (HD) and their immune status. This case-control study was conducted at the Nephrology Unit, Department of Pediatrics, the Zagazig University Hospital, from April 2010 to August 2010. We studied 27 children with ESRD on regular HD (group-A) whose mean age was 8 ± 1.3 years; there were 15 males and 12 females. The study patients were divided into two groups depending on the degree of vitamin D deficiency; group-A1 had 12 patients, all of whom had vitamin D deficiency defined as serum concentration of 25-hydroxy vitamin D3 [25(OH) D3] of 15-30 ng/mL. Group-A2 had 15 patients with more severe vitamin D deficiency (<15 ng/mL). Twenty healthy age- and sex-matched children served as the control group (group-B); their mean age was 7.8 ± 1.6 years and they included 12 males and eight females. All subjects underwent thorough history taking, clinical examination and the following investigations: complete blood count, lymphocyte count, blood urea, serum creatinine, total serum calcium, ionized calcium, serum phosphorus, plasma 25(OH)D3, intact para-thormone (iPTH), serum interleukin-10 (IL-10) and soluble IL-2 receptor (SIL-2R). We found that the vitamin D level was significantly lower in the patient group (group-A) than in the control-group (group-B). The IL-10 level was significantly lower in group-A than in group-B, and the SIL-2R level was significantly higher in group-A than in group-B. We found a significant positive correlation between serum 25(OH)D3 levels and serum IL-10, while there was a negative correlation between 25(OH)D3 levels and SIL-2R; this correlation was not significant. Our findings suggest that 25(OH)D3 levels affect the immune state in patients through their effect on both limbs of immunity, the anti-inflammatory and the pro-inflammatory, but the effect was higher on the anti-inflammatory IL-10. We conclude that the serum levels of vitamin D are lower in children with ESRD than in age-matched controls, and that it is significantly positively related to the anti-inflammatory IL-10 and negatively related to the pro-inflammatory SIL-2R. Further studies are required to throw more light on the role of vitamin D supplementation in children with ESRD in maintaining immune balance.
|How to cite this article:|
Youssef DM, Elshal AS, Abo Elazem AA. Assessment of immune status in relation to vitamin D levels in children on regular hemodialysis. Saudi J Kidney Dis Transpl 2012;23:267-73
|How to cite this URL:|
Youssef DM, Elshal AS, Abo Elazem AA. Assessment of immune status in relation to vitamin D levels in children on regular hemodialysis. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2013 Dec 8];23:267-73. Available from: http://www.sjkdt.org/text.asp?2012/23/2/267/93148
| Introduction|| |
Chronic kidney disease (CKD) is characterized by a loss of kidney function and dysregulation of vitamin D metabolism.  Patients with CKD have low levels of 1,25-dihydroxy vitamin D3 [1,25(OH)2D3] as well as low circulating 25-hydroxyvitamin D3 [25(OH)D3] levels. The latter further contributes to low 1,25(OH)2D3 levels in association with disease progression. 
Vitamin D insufficiency is a term used to describe the biochemical evidence of deficiency without obvious clinical signs or symptoms, such as rickets or osteomalacia. Although there is continuing discussion about the precise levels of 25(OH)D3, which define the different categories of vitamin D status, there is general agreement that vitamin D insufficiency or hypovitaminosis D is prevalent in many populations across the globe. Vitamin D insufficiency is diagnosed by a serum concentration of 25(OH)D3 between 15 and 30 ng/mL; levels <15 ng/mL are defined as vitamin D deficiency, with concentrations <5 ng/mL being severely deficient and associated with osteomalacia and rickets. 
The potential role of vitamin D and its active metabolite 1,25(OH)2D3 in modulating the immune response has been recognized for a long time based on several discoveries: the presence of vitamin D receptors (VDRs) in macrophages, dendritic cells (DCs) and activated T and B lymphocytes; the ability of macrophages, DCs and activated T and B cells to express CYP27B1, the enzyme that produces 1,25(OH)2D3; and the ability of 1,25(OH)2D3 to regulate the proliferation and function of these cells. 
The inhibitory effects of 1,25(OH)2D3 on interferon-γ (IFN-γ), interleukin (IL)-6, IL-2 and tumor necrosis factor (TNF)-α production, together with the stimulatory effects on IL-10 release, are all factors proposed as mechanisms contributing to protective effects of vitamin D on the cardiovascular system. Additionally, the persistent systemic inflammatory response observed in CKD contributes to a further loss of renal function, and vitamin D is believed to play a significant role in this process as well. Vitamin D deficiency was reported to be associated with increased renal inflammation in a variety of renal diseases. 
The aim of this work is to study the correlation between vitamin D levels in children with end-stage renal disease (ESRD) on regular hemodialysis (HD) and their immune state.
| Patients and Methods|| |
This case-control study was conducted at the Nephrology Unit, Department of Pediatrics, the Zagazig University Hospital, from April 2010 to August 2010. We studied 27 children with ESRD on regular HD (group-A); their mean age was 8 ± 1.3 years and there were 15 males and 12 females. Dialysis was performed with Fresenius 2008K machines using hollow fiber polysulfone dialyzers (Fresenius, Bad Homburg, Germany) and standard citrate dialysate solution. The dialysis prescription was as follows: three sessions per week, 3-5 h per session, blood flow 300 mL/min, with target urea reduction ratio (URR) of >65%. URR was calculated as follows:
URR = (pre-dialysis urea - post-dialysis urea) / pre-dialysis urea
We subdivided group-A into A1 (12 patients) in whom vitamin D insufficiency, with serum concentration of 25(OH)D3 between 15 and 30 ng/mL, was found and A2 (15 patients) who had vitamin D deficiency with serum 25(OH) D3 <15 ng/mL, as classified by the National Kidney Foundation.  Twenty healthy age- and sex-matched children were used as the control group (group-B); their mean age was 7.8 ± 1.6 years and included 12 males and eight females.
The exclusion criteria for participation in this study included acute infections and severe cardiovascular events less than six months before the study. The study was approved by the ethics committee of the Zagazig University and informed consent was obtained from all subjects. All subjects underwent detailed history taking and thorough clinical examination.
Blood sample collection
Blood samples for laboratory tests were collected before the mid-week dialysis sessions, and the following investigations were performed: complete blood count, lymphocyte count, blood urea, serum creatinine, total serum calcium that was corrected for serum albumin, ionized calcium, serum phosphorus and intact parathormone (iPTH, pg/mL); this was determined by second-generation immunoradiometric assay (Bio-Rad Laboratories Inc., Hercules, Ca, USA).
Measurement of plasma 25(OH)D3
This was measured by high-performance liquid chromatography (HPLC) according to Brunetto et al, 2004.  Reagents and standards All solvents and chemicals used were HPLC or analytical reagent grade. Acetonitrile, methanol, ethanol, isopropanol, potassium dihydrogen phosphate and potassium hydrogen phosphate were purchased from JT Baker (Phillipsburg, NJ, USA). Tetrahydrofuran was obtained from OmniSolv (Gibbstown, NJ, USA). High-purity water was obtained through a Millipore Milli-Q system (Bedford, MA, USA). 25-OHD3 was supplied by Sigma (St. Louis, MO, USA). Individual stock standard solution of 25OHD3 (500 μg/mL) was prepared in methanol and stored at -20°C. Working solutions were prepared every week by an appropriate dilution of concentrated stock standard solutions in methanol.
Sample pre-treatment and methodology
A HPLC method with automated column switching and UV-diode array detection has been described for the simultaneous determination of 25OHD3 in a sample of human plasma. The system uses a BioTrap pre-column for the online sample clean up. A sample of 1 mL of human plasma was treated with 2 mL of a mixture of ethanol-acetonitrile [2:1 (v/v)]. Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250 μL of a solution of methanol 5 mmol/L phosphate buffer, pH 6.5 [4:1 (v/v)], and a 200 μL aliquot of this solution was injected onto the BioTrap pre-column. After washing for 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol/L phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the back-flush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B [Solvent A: acetonitrile-phosphate buffer 5 mmol/L, pH 6.5; 20:80 (v/v); solvent B: methanol-acetonitrile-tetrahydrofuran, 65:20:15 (v/v)]. The compounds of interest were detected at 265 nm. The method was linear in the range 3.0-32.0 ng/mL, with a quantification limit of 3.0 ng/mL. Quantitative recoveries from spiked plasma samples were between 91 and 98%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day assay precision was ≤2.80%. The proposed method permitted the simultaneous determination of 25OHD3 in 16 min, with adequate precision and sensitivity.
The serum SlL-2R was measured using enzyme-linked immunosorbent assay (ELISA) (Cellfree, T Cell Sciences, Cambridge, MA, USA). The serum was stored at -70°C until the assay was performed; normal value was 246- 742 U/mL.
The serum lL-10 was measured using ELISA (RayBio Human IFN-γ kit, Norcross, GA, USA). The serum was stored at -70°C until the assay was performed; normal value was 2-20 pg/mL.
| Statistical Analysis|| |
Statistical analysis was performed using a computer-based program SPSS version 19. The quantitative data are presented as mean ± standard deviation. Unpaired independent t test is statistical test of significance for two groups, and the ANOVA test was applied for comparing more than two groups. P-value less than 0.05 indicated statistical significance.
| Results|| |
Our results showed that there was no significant difference between group-A1 (vitamin D insufficiency) and group-A2 (vitamin D deficiency) in age, duration of disease, duration on dialysis, blood urea, serum creatinine, hemoglobin, calcium, serum phosphorus, iPTH or SIL-2R. However, there were significant lower levels of serum IL-10 in group-A2 than in group-A1.
By comparing the three study groups with the ANOVA test, we found a highly significant difference in the urea, creatinine, hemoglobin, serum calcium, serum phosphorus, iPTH, SIL-2R and IL-10 levels [Table 1].
|Table 1: Comparison of various parameters between the two patient sub-groups A1 and A2 and the control group B.|
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The correlation between vitamin D levels and the various parameters studied is given in [Table 2]. [Figure 1] and [Figure 2] show the correlation between vitamin D levels and SIL-2R and IL-10, respectively, in the study groups.
|Figure 1: A and B: Scatter diagram showing a negative correlation between SIL-2R and vitamin D levels in all the studied subjects (A, R2 = -0.244) and a stronger negative correlation between the parameters in the patient group (B, R2 = -0.064).|
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|Figure 2: A and B: Scatter diagram showing a positive correlation between IL-10 and vitamin D levels in all studied subjects (A, R2 = 0.662) and a stronger positive correlation between the parameters in the patient group (B, R2 = 0.577).|
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|Table 2: Correlation between vitamin D levels and different parameters studied.|
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| Discussion|| |
CKD is characterized by a state of chronic low-grade inflammation, which translates into an increased risk for cardiovascular disease in patients with ESRD. Remarkably, the two most common causes of death in patients with CKD are cardiovascular diseases and infections, and both have been linked to impaired vitamin D levels and dysregulated immune responses.  Our study included 27 patients with ESRD on HD (group-A) and 20 healthy age- and sex-matched children who served as controls (group-B). We found significantly lower levels of vitamin D in group-A than in group-B, which is in accordance with most previous studies. 
There was significantly higher serum levels of SIL-2R in group-A patients (814 ± 327 IU/mL) than in group-B subjects (398 ± 98.1 IU/mL); this finding matches the results of Shu et al in 1998,  Takemasa et al in 2001,  Abou-Shousha et al in 2006  and Costa et al in 2008.  Their studies revealed more SIL-2R positive cells with higher SIL-2R density than in controls. It has been suggested that a deficient IL-2 activity in uremic patients may, in part, be due to an increased absorption by IL-2 receptors and that CKD per se is the major cause of the pre-activation of T lymphocytes. The modes of treatment and various clinical variables in these patients have no significant influence on the serum SIL-2R levels.  In the current study, we found significantly lower levels of serum IL-10 in group-A subjects (4 ± 1.9 pg/mL) when compared with group-B subjects (8.9 ± 3.3 pg/mL). These results are in agreement with a previously reported impaired IL-10 synthesis by monocytes exposed to a uremic environment,  and that IL-10 transcript levels increased in parallel by about five-times with respect to the values observed in a group of age-matched healthy controls. 
We classified group-A subjects into two subgroups; A1 with vitamin D insufficiency defined as serum concentration of 25(OH)D3 between 15 and 30 ng/mL and group-A2 with vitamin D deficiency defined as 25(OH)D3 levels <15 ng/mL. 
We found lower levels of serum IL-10 in group-A2 (3 ± 1.5 pg/mL) than in group-A1 (5.3 ± 1.6 pg/mL), and there was a highly significant positive correlation between the serum levels of vitamin D and IL-10. Also, we found in this study lower levels of vitamin D in patients with positive C-reactive protein (CRP) than those with negative CRP. This can be explained by the inihibitory effects of 1,25(OH) 2D3 on T helper-1 cell differentiation, IFN-γ, IL-6 and TNF-α production, together with the stimulatory effects on IL-10 release. These factors have been proposed as mechanisms that contribute to the protective effects of vitamin D on the cardiovascular system. Additionally, the persistent systemic inflammatory response observed in CKD contributes to a further loss of renal function, and vitamin D is believed to play a significant role in this process as well. Vitamin D deficiency was reported to be associated with increased renal inflammation in a variety of renal diseases, , whereas the anti-inflammatory effects of a 1,25(OH)2D3 analog, paricalcitol, could be demonstrated by inhibition of renal inflammatory infiltration in a mouse model of obstructive nephropathy. ,
There were significant lower levels of serum IL-10 in group-A2 than in group-A1 subjects. This finding reflects that vitamin D promotes the activity of IL-10-secreting cell, suggesting that vitamin D enhanced the development of T-helper 2 cells  and the release of anti-inflammatory IL-10. Thus, 1,25(OH)2D3 indirectly shifts CD4+ T-cell polarization from an inflammatory T-helper 1 and T-helper 17 to a protective T-helper 2 and regulatory T-cell phenotype.  In this study, there was no correlation between vitamin D and SIL-2R levels when the study subjects were analyzed as a whole. However, there was a negative correlation between these two parameters when applied to CRF cases only, which means that in CRF, the low levels of vitamin D lead to increase in SIL-2R levels; this is explained by the role of 1,25 (OH)2D3 in inhibiting production of T-helper 1-related cytokines such as IL-2. 
We did not find any significant difference in the age, duration of the disease, duration on dialysis, hemoglobin level, urea, creatinine, iPTH and SIL-2R levels between groups-A1 and -A2. Also, we did not find any correlation between vitamin D levels and age and duration on dialysis. Patients with CKD not only have low serum levels of 1,25(OH)2D3 but also have low circulating 25(OH)D3 levels irrespective of disease progression, which in turn further contributes to low 1,25(OH)2D3 levels. 
As expected, we found a highly significant positive correlation between vitamin D levels and serum calcium, while there was a negative correlation between vitamin D levels and blood urea, phosphorus and iPTH levels; this is explained by the well known established role of vitamin D in regulating bone metabolism in CKD. 
The limitations of the present study include a small sample size. Also, many other factors may affect immunity in these patients and further studies are recommended to evaluate the role of vitamin D supplementation in ESRD patients on pro- and anti-inflammatory cytokines. In conclusion, we found that children with ESRD on HD have low levels of vitamin D, and this has a strong positive correlation with IL-10 levels and a negative correlation with SIL-2R levels. This leads to an imbalance between synthetic capacity of pro- and anti-inflammatory cytokines and, thus, we recommend careful monitoring and correction of 25(OH)D3 levels not only for correction of bone mineralization but also for controlling immunity and inflammatory complications, which are leading causes of death in these patients.
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Doaa M Youssef
Pediatric Department, Zagazig University, Zagazig
[Figure 1], [Figure 2]
[Table 1], [Table 2]