Abstract
Here we determined if vitamin D deficiency is more common in children with chronic kidney disease compared to healthy children. In addition we sought to identify disease-specific risk factors for this deficiency as well as its metabolic consequences. We found that nearly half of 182 patients (ages 5 to 21) with kidney disease (stages 2 to 5) and a third of age-matched 276 healthy children were 25-hydroxyvitamin D deficient (less than 20 ng/ml). The risk of deficiency was significantly greater in advanced disease. Focal segmental glomerulosclerosis and low albumin were significantly associated with lower 25-hydroxyvitamin D which, in turn, was associated with significantly higher intact parathyroid hormone levels. We found that 25-hydroxyvitamin D levels were positively associated with 1,25-dihydroxyvitamin D, the relationship being greatest in advanced disease (significant interaction), and inversely related to those of inflammatory markers C-reactive protein and IL-6. The association with C-reactive protein persisted when adjusted for the severity of kidney disease. Thus, lower 25-hydroxyvitamin D may contribute to hyperparathyroidism, inflammation and lower 1,25-dihydroxyvitamin D in children and adolescents, especially those with advanced kidney disease.
INTRODUCTION
Vitamin D deficiency is common in children and adults, and its adverse effects extend beyond bone and mineral metabolism. Numerous studies have demonstrated associations with mortality, cardiovascular disease, insulin resistance, diabetes, autoimmune disease, infection, and inflammation (1, 2). Chronic kidney disease (CKD) imposes a heavy burden of cardiovascular, metabolic and infectious complications (3), and vitamin D deficiency is likely an important modifiable risk factor in this high-risk population. Recent studies in CKD linked vitamin D deficiency with extra-skeletal comorbidities, including insulin resistance (4, 5), anemia (6, 7), and inflammation (8, 9). In prospective studies of adults with CKD, low serum 25-hydroxyvitamin D [25(OH)D] concentration was an independent predictor of CKD progression and mortality (10-12). Vitamin D deficiency may also contribute to skeletal fragility in children with CKD.
Prior studies in children with CKD reported that the prevalence of vitamin D deficiency (25(OH)D concentration <20 ng/ml) ranged from 28 to 58% (13-18). Vitamin D deficiency is also common in otherwise healthy children; approximately 14 to 25% of children and adolescents have 25(OH)D concentrations <20 ng/ml (19-22). Risk factors for vitamin D deficiency include African ancestry, older age, female sex, geographic location, and winter season. CKD may impose additional risk factors for vitamin D deficiency, including impaired cutaneous photosynthesis of calciferol due to uremia (23), urinary losses of vitamin D-binding protein (DBP) and albumin, and decreased dairy intake due to phosphate restriction. With the exception of our recent study in pediatric kidney transplant recipients (18), none of the studies of vitamin D status in children with CKD included a concurrent healthy reference group; therefore, the independent effect of CKD severity is not known. Furthermore, the impact of the underlying cause of CKD has not been addressed.
The objectives of this study were to determine whether vitamin D deficiency is more common among children with CKD compared to healthy children, and to identify CKD-specific risk factors for vitamin D deficiency, independent of CKD severity. Secondary objectives were to determine the effect of vitamin D status on serum concentrations of parathyroid hormone (PTH), 1,25 dihydroxyvitamin D [1,25(OH)2D], and inflammatory markers among children with CKD, independent of CKD severity.
RESULTS
Participant Characteristics
We evaluated 182 children and adolescents with CKD, ages 5 to 21 years, and 276 healthy children at the Children’s Hospital of Philadelphia (CHOP) and Cincinnati Children’s Hospital Medical Center (CCHMC). Characteristics of healthy children and CKD participants, categorized according to CKD stage (24): stage 2-3 (estimated glomerular filtration rate (eGFR) 30-89 ml/min/1.73m2; n=74), CKD stage 4-5 (eGFR < 30 ml/min/1.73m2; n=54), and stage 5D (maintenance dialysis; n=54) are given in Table 1. CKD participants were of similar age to healthy participants except for those with CKD stage 5D who were older. CKD participants were more likely to be male than healthy children, as expected in childhood CKD. There were slight differences between groups in the season in which participants were studied, with CKD participants more likely to be studied in winter months (November-April) than healthy children. Similar proportions of CKD participants and healthy children were taking a multivitamin supplement containing vitamin D. Only 1 participant in the dialysis group was taking a medication known to affect vitamin D (phenytoin).
Table 1.
Characteristics of healthy reference and chronic kidney disease participants.
| Healthy Reference | Chronic Kidney Disease Stage Groups | P value1 | |||
|---|---|---|---|---|---|
| 2-3 | 4-5 | 5 dialysis | |||
| N | 276 | 74 | 54 | 54 | |
| Estimated GFR (ml/min/1,73m2) | -- | 46 (30, 78)2 | 18 (7, 29) | -- | -- |
| Age (y) | 13.4 ± 4.5 3 | 13.1 ± 4.2 | 14.0 ± 4.0 | 15.4 ± 3.8 | 0.02 |
| Sex: male | 139 (50%) 4 | 39 (53%) | 38 (70%) | 30 (56%) | 0.06 |
| African ancestry | 59 (21%) | 15 (20%) | 14 (26%) | 19 (35%) | 0.15 |
| Height Z-score | 0.37 ± 0.89 | -0.39 ± 1.3 | -1.15 ± 1.22 | -1.10 ± 1.25 | 0.0001 |
| BMI Z-score | 0.28 ± 0.99 | 0.26 ± 1.12 | 0.33 ± 1.26 | 0.10 ± 1.4 | 0.70 |
| Tanner stage: | 0.05 | ||||
| 1 | 75 (27%) | 22 (31%) | 14 (26%) | 6 (11%) | |
| 2-3 | 56 (20%) | 21 (29%) | 14 (26%) | 18 (34%) | |
| 4-5 | 144 (52%) | 29 (40%) | 26 (48%) | 29 (54%) | |
| Season: Nov-Apr | 121 (44%) | 47 (63%) | 24 (44%) | 30 (56%) | 0.01 |
| Vitamin D supplement | |||||
| Usage ≥ 400 IU/d | 43 (16%) | 16 (22%) | 6 (11%) | 9 (17%) | 0.44 |
| Underlying renal disease: | 0.0001 | ||||
| CAKUT | -- | 50 (48%) | 35 (34%) | 19 (18%) | |
| Systemic inflammatory & Glomerulonephritis | -- | 8 (33%) | 4 (17%) | 12 (50%) | |
| FSGS | -- | 3 (9%) | 10 (18%) | 20 (61%) | |
| Other | -- | 13 (62%) | 5 (24%) | 3 (14%) | |
| Receiving calcitriol therapy | -- | 17 (23%) | 40 (74%) | 46 (85%) | 0.0001 |
| Receiving glucocorticoids | -- | 5 (7%) | 2 (4%) | 14 (26%) | 0.0008 |
| Study site: | 0.03 | ||||
| CCHMC | 68 (25) | 29 (39%) | 20 (37%) | 19 (35%) | |
| CHOP | 208 (75%) | 45 (61%) | 34 (63%) | 35 (65%) | |
ANOVA for continuous variables and chi-squared test for categorical variables.
Median; range in parentheses (all such values)
Mean ± SD (all such values)
n; percentage in parentheses (all such values)
Underlying cause of renal disease was categorized as congenital anomalies of the kidney and urinary tract (CAKUT, n=104), including aplastic/hypoplastic/dysplastic/cystic kidneys and obstructive uropathy; systemic inflammatory disease (systemic lupus erythematosis and Wegener’s granulomatosis) and glomerulonephritis (n=24); focal segmental glomerulosclerosis (FSGS, n=33); and other (n=21). Median age at diagnosis of CKD was 4.5 years (range, birth to 19.1 y), and median interval from CKD diagnosis to study visit was 7.3 years (range, 0.1 to 20.8 y). Of the CKD participants on maintenance dialysis, 35 were on hemodialysis and 19 were on peritoneal dialysis.
Laboratory Measurements
Abnormalities in mineral metabolism were more pronounced in participants with greater severity of CKD (Table 2); intact parathyroid hormone (iPTH) and phosphorus were significantly higher and serum 25(OH)D, calcium, and 1,25(OH)2D concentrations were significantly lower with greater CKD severity. Serum albumin concentrations were lower, and the proportion of participants with low albumin concentrations (<3.0 g/dl) was higher, with greater CKD severity. Serum markers of inflammation, C-reactive protein (CRP) and IL-6 concentrations, were significantly higher with greater disease severity.
Table 2.
Serum concentrations of calcitropic hormones, minerals and inflammatory markers among healthy reference and chronic kidney disease participants.
| Healthy Reference | Chronic kidney disease stage | P value1 | |||
|---|---|---|---|---|---|
| 2-3 | 4-5 | 5 dialysis | |||
| 25(OH)D (ng/ml) | 25.2 ± 10.52 (n=276) |
29.5 ± 12.8 (n=74) |
21.4 ± 12.8 (n=54) |
15.6 ± 9.9 (n=54) |
<0.0001 |
| Vitamin D deficient | |||||
| (25(OH)D <20 ng/ml) | 31% (86/276) | 26% (19/74) | 50% (27/54) | 74% (40/54) | < 0.0005 |
| iPTH (pg/ml) | 26 (9, 61)3 [19, 34] 3 (n=112) |
48 (2, 521) [27, 68] (n=74) |
148 (8, 1046) [74, 404] (n=54) |
251 (8, 1416) [108, 607] (n=54) |
<0.0001 |
| 1,25(OH)2 D (pg/ml) | |||||
| No calcitriol therapy | 39 (11, 101) [33, 46] (n=276) |
38 (15, 78) [29, 47] (n=57) |
21 (5, 40) [17, 36] (n=14) |
14 (8, 21) [9, 20] (n=8) |
<0.0001 |
| On calcitriol therapy | -- | 34 (15, 51) [29, 41] (n=17) |
33 (18, 149) [26, 45] (n=40) |
19 (5, 78) [13, 24] (n=45) |
<0.0001 |
| Calcium (mg/dl) 4 | -- | 9.4 ± 0.5 (n=74) |
9.1 ± 0.8 (n=53) |
8.7 ± 1.3 (n=54) |
<0.0001 |
| Corrected calcium (mg/dl) 5 | -- | 9.5 ± 0.4 (n=74) |
9.3 ± 0.5 (n=52) |
9.1 ± 1.1 (n=54) |
0.04 |
| Phosphorus (mg/dl) 4 | -- | 4.4 ± 0.9 (n=73) |
5.4 ± 1.4 (n=53) |
5.8 ± 1.9 (n=54) |
<0.0001 |
| Albumin (g/dl) 4 | -- | 4.1 (1.7, 4.8) [3.8, 4.3] |
3.9 (1.4, 4.8) [3.4, 4.2] |
3.5 (1.4, 5.1) [3.1, 3.8] |
0.0001 |
| <3 mg/dl | -- | 5 (7%) 6 (n=74) |
6 (12%) (n=52) |
8 (15%) (n=54) |
0.33 |
| CRP (mg/dl) 4 | -- | 0.3 (0.2, 33.0) [0.2, 0.6] (n=74) |
0.6 (0.2, 1.9) [0.3, 0.6] (n=52) |
0.6 (0.2, 4.4) [0.3, 1.0] (n=54) |
0.02 |
| IL-6 (pg/ml) 4 | -- | 0.95 (0.30, 9.93) [0.63, 1.68] (n=68) |
1.21 (0.30, 14.88) [0.76, 2.19] (n=46) |
2.55 (0.46,60.55) [1.61, 4.97] (n=48) |
<0.0001 |
ANOVA or Wilcoxon rank sums test among groups
Mean ± SD (all such values)
Median; range in parentheses; inter quartile range in brackets (all such values)
Not measured on healthy reference participants
Corrected for serum albumin concentrations = total calcium (mg/dl) + 0.8 × [4-serum albumin (g/dl)]
Number; percentage in parentheses (all such values)
Predictors of Vitamin D Status in CKD and Healthy Participants
Table 3 summarizes the final multivariable linear and logistic regression models that include significant predictors of 25(OH)D concentrations in CKD and healthy children. Mean 25(OH)D concentration adjusted for covariates was lower, and the risk of vitamin D deficiency (<20 ng/ml) was higher in late stage renal disease (i.e., stages 4-5D) CKD; there was no apparent difference in 25(OH)D concentrations between early stage CKD and healthy children. Enrollment at CHOP, older age, African ancestry, and winter season were statistically significant independent predictors of lower 25(OH)D concentrations, whereas sex, Tanner stage, vitamin D supplement usage, and body mass index (BMI) Z-score were not associated with 25(OH)D concentrations after adjusting for other covariates (p>0.05). There was no interaction between season and CKD stage in predicting 25(OH)D concentrations or vitamin D deficiency, even when restricting the sample to those with late stage renal disease. Likewise, there was no interaction between CKD stage and African ancestry or study site in predicting 25(OH)D concentrations p>0.05.
Table 3.
Predictors of vitamin D concentrations and vitamin D deficiency in healthy reference and CKD participants: results from multiple linear regression and logistic regression modeling.
| Deficient | Severely deficient | ||||
|---|---|---|---|---|---|
| 25(OH)D (ng/ml) | 25(OH)D < 20 ng/ml | 25(OH)D < 10 ng/ml | |||
| CKD stage: | p < 0.0001 | p < 0.0001 | p = 0.0002 | ||
| Healthy Reference (n=276) | 24.2 ± 0.71 | 1.00 | 1.00 | ||
| 2-3 (n=74) | 26.6 ± 1.1 | 1.12 | (0.54, 2.32) | 1.56 | (0.53, 4.59) |
| 4-5 (n=54) | 20.9 ± 1.3 | 2.77 | (1.32, 5.81) | 4.24 | (1.68, 10.71) |
| 5D (n=54) | 16.1 ± 1.3 | 10.28 | (4.48, 23.61) | 6.38 | (2.59, 15.60) |
| Season: | p < 0.0001 | p < 0.0001 | p = 0.0009 | ||
| May – Oct (n=222) | 24.9 ± 0.7 | 1.00 | 1.00 | ||
| Nov – Apr (n=236) | 19.0 ± 0.8 | 4.55 | (2.64, 7.86) | 3.22 | (1.56, 6.64) |
| Age (y): | p < 0.0001 | p < 0.0001 | p = 0.02 | ||
| 5-8.9 (n=79) | 25.9 ± 1.2 | 1.00 | 1.00 | ||
| 9-11.9 (n=86) | 22.8 ± 1.1 | 1.29 | (0.51, 3.26) | 3.27 | (0.61, 17.57) |
| 12-14.9 (n=108) | 20.1 ± 1.0 | 5.21 | (2.24, 12.12) | 4.62 | (0.94, 22.72) |
| 15-21.9 (n=185) | 19.0 ± 0.8 | 6.59 | (2.93, 14.82) | 7.18 | (1.55, 33.09) |
| African ancestry: | p < 0.0001 | p < 0.0001 | p < 0.0001 | ||
| No (n=350) | 27.4 ± 0.6 | 1.00 | 1.00 | ||
| Yes (n=107) | 16.4 ± 1.0 | 16.66 | (8.61, 32.24) | 8.78 | (4.41, 17.45) |
| Study site: | p < 0.0001 | p = 0.007 | p = 0.03 | ||
| CCHMC (n=136) | 24.2 ± 0.9 1 | 1.002 | 1.00 | ||
| CHOP (n=321) | 19.6 ± 0.7 | 2.28 | (1.26, 4.15) | 2.54 | (1.06, 6.08) |
Least squares means ± SE (all such values)
Odds ratio; 95% CI in parentheses (all such values)
CKD-Specific Predictors of Vitamin D Status
Among CKD participants, underlying cause of CKD and low serum albumin concentration were associated with lower 25(OH)D concentrations. Mean (± SE) 25(OH)D concentrations, adjusted for age, African ancestry, location, season, and CKD stage, by underlying renal disease category were: CAKUT 24.8 ± 1.1 ng/ml; systemic inflammatory disease and glomerulonephritis 19.2 ± 2.1 ng/ml; FSGS 14.6 ± 1.8 ng/ml; and other 25.5 ± 2.2 ng/ml (p<0.0001). Adjusted mean 25(OH)D concentration among participants with a low albumin (<3.0 g/dl) was 11.1 ± 2.2 ng/ml compared to 23.5 ± 0.9 ng/ml among participants with albumin ≥3.0 g/dl (p<0.0001). Group differences persisted when both diagnostic category and low albumin were included in the same model. Of note, 58% (11/19) of children with albumin <3.0 g/dl had 25(OH)D concentrations <10 ng/ml, and 95% (18/19) had 25(OH)D concentrations <20 ng/ml. Serum 25(OH)D concentrations were positively associated (r=0.50, p<0.0001) with albumin concentrations. Among dialysis patients, there was no difference in mean 25(OH)D concentrations between those who had received a prior transplant and those who had not (14.7 vs. 16.0 ng/ml, p=0.60). Current use of glucocorticoids was not associated with 25(OH)D concentrations (p=0.78). The dialysis participant taking phenytoin had a 25(OH)D concentration of 38 ng/ml, which was notably higher than that of the other dialysis participants.
Predictors of iPTH Concentrations in CKD Participants
Multiple regression analyses revealed that ln iPTH concentrations were positively associated with serum phosphorus concentrations and CKD stage and negatively associated with corrected serum calcium and 25(OH)D concentrations (Table 4). There was no interaction between albumin or low albumin concentration (<3.0 g/dl) and 25(OHD) concentrations for predicting iPTH concentrations, whether or not calcitriol treatment was included in the regression model (all p>0.28). Likewise there was no interaction between CKD stage and 25(OH)D concentrations for predicting iPTH concentrations, whether or not calcitriol treatment was included in the regression model (p>0.12).
Table 4.
Predictors of serum iPTH concentrations among CKD participants.
| Estimate1 | P value | |
|---|---|---|
| Serum 25(OH)D (ng/ml) | 0.04 | |
| < 10 | 148 (108, 203) | |
| 10-20 | 127 (98, 165) | |
| > 20 | 93 (76, 113) | |
| Serum phosphorus (mg/dl) | 25.8 % | < 0.0001 |
| Serum corrected calcium (mg/dl) | -24.5 % | < 0.003 |
| CKD stage: | <0.0001 | |
| 2/3 | 77 (52, 115) | |
| 4/5 | 196 (134, 285) | |
| 5D | 213 (149, 304) |
The least squares mean (95% confidence interval) or the percentage change in iPTH concentrations per unit change in predictor variable
Predictors of 1,25(OH)2D Concentrations in CKD and Healthy Participants
Among healthy children and CKD participants not receiving calcitriol, there was a significant interaction between 25(OH)D concentration and CKD stage for predicting 1,25(OH)2D concentrations (p<0.0001), with the relationship being greater in more advanced CKD. For a 1 ng/ml increase in 25(OH)D concentration, 1,25(OH)2D concentrations increased as follows: healthy children 0.4% (n=275, p=0.01); CKD stage 2/3 0.7% (n=57, p=0.04); CKD stage 4/5 4.2% (n=14, p=0.006); CKD stage 5D 2.3% (n=8, p=0.15). When accounting for 25(OH)D concentration and CKD stage, ln iPTH concentration was positively associated (β=0.10, p=0.02) with ln 1,25(OH)2D concentration, consistent with iPTH effects to up-regulate the renal 1-α–hydroxylase.
Association between Vitamin D Status and Inflammation in CKD Participants
In bi-variate analyses, ln CRP concentrations were inversely associated with 25(OH)D concentrations (r=-0.31, p<0.0001, excluding two extreme outliers), and CRP concentrations were positively associated with greater CKD severity (p=0.02, Table 2). These relationships persisted when including both in a multiple regression model [25(OH)D concentration p=0.009, CKD stage p=0.01]. In bi-variate analyses, ln Interleukin-6 (IL-6) concentrations were inversely associated with 25(OH)D concentrations (r= -0.22, p=0.008), and with CKD stage (p<0.0001) (Table 2). When both were fitted in a multiple regression model, 25(OH)D concentrations were no longer associated with IL-6 concentrations (p=0.88), whereas CKD stage remained significant (p<0.001); geometric means (95% CI) were: CKD stage 2/3 1.09 (0.87, 1.36), stage 4/5 1.31 (1.02, 1.69), and stage 5D 2.79 (2.14, 3.63). Serum 1,25(OH)2D concentrations were not associated with IL-6 or CRP, adjusted for CKD severity.
DISCUSSION
This is the first study to compare the vitamin D status of a large sample of children and adolescents with CKD stages 2-5D with healthy children. Inclusion of healthy children is an important strength given the high background prevalence of vitamin D deficiency in the general pediatric U.S. population (19, 20). Analyses confirmed that 25(OH)D deficiency was common among healthy children (31% <20 ng/ml) and demonstrated that late stage CKD was associated with significantly greater odds of 25(OH)D deficiency, compared with healthy children, even when adjusted for age, African ancestry, location, and season. Furthermore, these analyses demonstrated that the association of season with 25(OH)D deficiency did not vary according to CKD stage – suggesting that impaired dermal photosynthesis of vitamin D did not contribute substantially to the lower 25(OH)D concentrations in advanced CKD.
This study advances our understanding of the independent effects of hypoalbuminemia and the underlying cause of renal disease on vitamin D status. Serum 25(OH)D concentrations among participants with a low serum albumin (<3.0 g/dl) were profoundly lower than concentrations in participants with albumin ≥3.0 g/dl: 11.1 vs. 23.5 ng/ml, independent of CKD severity and underlying renal disease. Prior studies in CKD reported that 25(OH)D concentrations were positively associated with serum albumin (25, 26), and inversely associated with degree of proteinuria (16, 27, 28) and albuminuria (8). Patients with significant proteinuria represent a unique population with respect to vitamin D status due to the urinary losses of DBP and albumin, to which >99% of circulating 25(OH)D and 1,25(OH)2D are bound (29). Total serum 25(OH)D concentrations may not reflect bioavailable or free vitamin D when serum DBP and albumin are reduced, and the clinical significance of low 25(OH)D concentrations in these individuals is not known. However, we did not detect an interaction between 25(OH)D and serum albumin for predicting iPTH concentrations; CKD participants with low 25(OH)D and hypoalbuminemia had comparable compensatory increases in iPTH concentrations suggesting a 25(OH)D deficiency state.
Underlying cause of renal disease, especially FSGS, was a significant risk factor for vitamin D deficiency, independent of hypoalbuminemia and CKD severity. FSGS is often associated with significant or nephrotic-range proteinuria. While serum albumin is affected by the magnitude of proteinuria, it does not necessarily reflect the duration or selectivity of proteinuria. Therefore, the comparatively worse vitamin D status of subjects with FSGS could be attributable in part to protracted urinary losses of albumin and DBP. In addition, subjects with FSGS may have a disproportionate burden of proximal tubular dysfunction or injury. In the kidney, vitamin D-DBP complexes are freely filtered by the glomerulus and reabsorbed via megalin/cubilin-mediated endocytosis in the proximal tubule (30, 31).
We found an association between 25(OH)D deficiency and higher iPTH concentrations in children and adolescents with CKD, independent of serum phosphorus and calcium concentrations and CKD severity. There was no evidence of a CKD – 25(OH)D interaction, indicating that 25(OH)D deficiency is associated with a comparable increase in iPTH across all CKD stages. As eGFR declines, 1-α-hydroxylation of 25(OH)D to 1,25(OH)2D is impaired, leading to a compensatory rise in iPTH. Prior studies of 25(OH)D and iPTH concentrations in children with CKD demonstrated conflicting results and did not adjust for the confounding effects of CKD severity (16, 17). We recently demonstrated that higher 25(OH)D at time of transplantation and greater increases in 25(OH)D following transplantation were independently associated with greater decreases in iPTH in pediatric renal transplant recipients (18).
Prior studies in adults with CKD have documented a positive association between 25(OH)D and 1,25(OH)2D concentrations (25, 27, 32, 33). However, this is the first study to demonstrate that this association differed significantly according to CKD severity; the slope between 25(OH)D and 1,25(OH)2D was steeper as CKD severity progressed. This suggests a greater substrate dependence in more advanced CKD, and that 25(OH)D adequacy is important in CKD for renal production of circulating 1,25(OH)2D in advanced CKD when 1α-hydroxylase activity is most compromised.
Recent studies in patients with CKD have demonstrated an inhibitory effect of vitamin D on renal and systemic inflammation. Zehnder et al. (34) demonstrated an inverse association between urinary concentrations of the chemokine, macrophage chemoattractant protein-1, and serum 25(OH)D and 1,25(OH)2D concentrations in CKD patients. 1,25(OH)2D concentrations were also inversely correlated with renal macrophage infiltration. Stubbs et al. (9) treated 25(OH)D deficient end stage renal disease patients with cholecalciferol and demonstrated decreased circulating tumor necrosis factor-α, IL-6 and IL-8 concentrations. To our knowledge, our study is the first to report an association between CRP and 25(OH)D in children and adolescents with CKD that persisted when adjusting for CKD stage.
This study has some limitations. We do not have information on sun exposure. It is possible that children and adolescents with CKD spend less time outdoors and that their vitamin D deficiency is attributable, in part, to sun exposure. However, the association of season with 25(OH)D status was comparable in CKD and healthy participants, suggesting that difference in sun exposure did not confound the analyses adjusted for season. We do not have information on dietary vitamin D intake, although we accounted for vitamin D supplement usage. Phosphate restriction in advanced CKD may have contributed to lower 25(OH)D concentrations via reduced dairy intake. We do not have data on urine protein excretion nor protein intake. Serum albumin is influenced by nutritional status as well as proteinuria; therefore, some of the associations between 25(OH)D and albumin concentrations may relate to nutritional status rather than proteinuria. However, in our recent study in this cohort, lean mass deficits were not associated with lower serum albumin concentrations (35). Use of non-fasting blood samples may have increased the variability in serum calcium and phosphorus concentrations and thereby iPTH concentrations making it more difficult to detect subtle effects. Lastly, the cross-sectional design limits our ability to make causal inferences.
Accurate and reliable measurement of 25(OH)D concentrations is necessary for valid comparisons between groups and when assessing the impact of potential risk factors and prevalence estimates. Accuracy and reliability of 25(OH)D measurements has been an issue owing to differences in assay methods and the need for consistent skilled laboratory technique and reagents, respectfully (36). All of our samples were measured using the DiaSorin assay in a single highly experienced laboratory that achieved International Vitamin D External Quality Assessment Scheme (DEQAS) certification (37); DEQAS aims to ensure the analytical reliability of 25(OH)D and 1,25(OH)2D assays through quarterly testing of pooled specimens (38). The DiaSorin RIA assay, when performed in a highly proficient laboratory, has been shown to be similar to the HPLC reference method (36) and the NIST LC/MS assay (39). Our consistent laboratory methodology minimizes the likelihood of measurement bias affecting comparisons between groups. However, residual measurement error increases the variability in 25(OH)D concentrations thereby reducing the statistical power to detect underlying relationships. This would have resulted in failure to detect associations. In addition, assay bias, even if small, would result in over or under estimation of prevalence estimates of vitamin D deficiency.
In summary, we showed that vitamin D deficiency was more common in children and adolescents with late stage CKD. Vitamin D status was worse in the presence of hypoalbuminemia and among those with FSGS. Lower 25(OH)D concentrations were associated with lower circulating 1,25(OH)2D concentrations, especially in late stage CKD. Lower 25(OH)D concentration also was associated with higher iPTH and CRP concentrations, independent of CKD severity. Thus, vitamin D deficiency is a highly prevalent and modifiable risk factor in children and adolescents with CKD, contributing to the disordered mineral metabolism and inflammation in this high-risk population. Trials of vitamin D supplementation are needed to determine the effect of repletion on clinical outcomes.
METHODS
Study Participants
This cross-sectional study was conducted as part of a larger study of bone health in children and adolescents with CKD, ages 5 to 21 years, at CHOP and CCHMC. A total of 182 individuals with CKD stages 2 to 5 were included in this study sample (CHOP n=114, CCHMC n=68). Recruitment and data collection occurred between January 2002 and November 2006. Subjects with cognitive or developmental disorders preventing completion of the study procedures were ineligible. Prior transplantation was an exclusion criterion in children with pre-dialysis CKD, but not in dialysis patients. We recently reported that greater CKD severity was associated with greater muscle deficits in these participants (35). Changes in serum vitamin D concentrations following renal transplantation were reported in a sample of 58 children and adolescents (18); however, disease-specific risk factors for vitamin D deficiency were not assessed and the limitation of the study to end-stage renal disease precluded analyses of the impact of CKD severity
Healthy children (n=276), ages 5 to 21 years, were recruited from general pediatrics practices or community advertisements in the greater Philadelphia (n=208) and Cincinnati (n=68) areas. Healthy participants were ineligible if they had chronic illnesses or were on medications potentially affecting growth, maturation or nutritional status.
The Institutional Review Boards at CHOP and CCHMC approved the study protocol. Informed consent was obtained from study subjects ≥18 years of age, and assent along with parental consent from subjects <18 years of age.
Anthropometry, Sexual Maturity, and African Ancestry
Height was measured with a stadiometer and weight with a digital scale. Measurements were obtained in triplicate. Height and BMI (kg/m2) Z-scores were calculated using Centers for Disease Control (CDC) 2000 growth reference (40). Sexual maturity was determined by self-assessment (41) and classified according to Tanner stage criteria (42). Information on ancestry was obtained by self-report.
Laboratory Measurements
Non-fasting blood samples were collected in the CKD and healthy participants. Serum 25(OH)D and 1,25(OH)2D concentrations were measured by radioimmunoassay with I125-labeled tracer (DiaSorin Inc., Stillwater, MN) in the laboratory of Dr. Bruce Hollis (43). Intra-assay coefficient of variation (CV) for 25(OH)D was 2.2% and for 1,25(OH)2D was 7-11% (44). Dr. Hollis’ laboratory has been involved in DEQAS since 1997 and has received certification regarding the analytical reliability of 25(OH)D and 1,25(OH)2D assays performed every year (37). Intact PTH concentrations were measured by radioimmunoassay with 125I-labeled antibody with a CV of 3-5% (Scantibodies Clinical Laboratory, Santee, CA). Laboratory measurements on healthy participants were performed in the same laboratories concurrently with those from CKD participants and included 25(OH)D and 1,25(OH)2D (n=276) and iPTH (n=119). The following were obtained in CKD participants only: CRP (low-sensitivity) was measured using a fixed-point immuno-rate method (Vitros, Johnson & Johnson Co., Rochester, NY) with a CV of 3-8%. IL-6 was measured by high-sensitivity solid phase ELISA (R&D Systems, Inc., Minneapolis, MN) with a CV of 8%. Serum albumin was measured by spectrophotometric enzymatic assay (Vitros, Johnson & Johnson Co., Rochester, NY) with a CV of 1-2%. Serum calcium and phosphorus were measured in the clinical laboratories using standard methods with CV’s of 1.3% and ≤ 2.1%, respectively; calcium concentration was adjusted for albumin concentration (45). Serum creatinine concentration was measured by a spectrophotometric enzymatic assay (Vitros, Johnson & Johnson Co., Rochester, NY) with a CV of 1-5%. eGFR (ml/min/1.73m2) was calculated based on height and serum creatinine using the pediatric estimating equation generated in the Chronic Kidney Disease in Children (CKiD) Prospective Cohort Study (46). CKD participants were categorized into three groups according to CKD stage as defined by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines (24): CKD stage 2-3 (eGFR 30-89 ml/min/1.73m2; n=74), CKD stage 4-5 (eGFR < 30 ml/min/1.73m2; n=54), and stage 5D (maintenance dialysis; n=54).
Disease Characteristics and Medications
Medical charts were reviewed for the date of diagnosis of CKD, underlying etiology of renal disease, dialysis history, and current and past active vitamin D sterol treatment. Current medication use and information on vitamin D intake from multivitamins and dietary supplements was obtained by questionnaire. Among those taking a vitamin D-containing supplement, the median intake was 400 IU/day (range 14-650). For subsequent analyses vitamin D supplement use was dichotomized as ≥400 IU/day versus <400 IU/day.
Statistical analyses
Statistical analyses were performed with JMP statistical software, version 8.0 (SAS Institute Inc., Cary, NC). The frequency distributions of all variables were assessed for normality. Non-normally distributed variables [iPTH, 1,25(OH)2D, CRP and IL-6 concentrations] were log-transformed to better approximate normality. Serum 25(OH)D concentrations were categorized as deficient (<20 ng/ml), in accordance with the 2011 Report on Dietary Reference Intakes for calcium and vitamin D from the Institute of Medicine (47, 48), or severely deficient (<10 ng/ml). Season was categorized as summer (May-October) vs. winter (November-April); summer months had the highest mean 25(OH)D concentrations among white healthy participants. Descriptive characteristics of the study groups were compared by ANOVA, Kruskal-Wallis or Chi-square tests.
Multivariable linear and logistic regression analyses were conducted to compare mean 25(OH)D concentrations and risk of vitamin D deficiency between healthy and CKD groups adjusting for age, sex, African ancestry, BMI Z-score, Tanner stage, use of vitamin D supplements, season, and study site. Variables were kept in the regression models if p value was <0.05. Multiplicative interactions between CKD stage and season, African ancestry, and study site were tested. Separate multiple linear regressions models were conducted within CKD participants to assess CKD-specific predictors of 25(OH)D concentration. CKD-specific factors were underlying renal diagnosis, CKD severity, and low serum albumin concentration (<3.0 g/dl).
Multivariable regression analyses were conducted to determine the effect of 25(OH)D concentration on iPTH, 1,25(OH)2D, CRP, and IL-6 concentrations among CKD participants. Because these variables were log-transformed for analyses, we presented results for categorical predictor variables as geometric means and 95% confidence intervals (95% CI) and for continuous predictor variables as the percentage change in Y associated with a unit change in X. Serum iPTH concentration was modeled as a function of the following independent variables: serum phosphorus, corrected calcium, calcitriol use, and 25(OH)D concentration; interactions between serum 25(OH)D concentration and CKD stage, and between 25(OH)D and albumin concentrations were tested. Serum 1,25(OH)2D was modeled as a function of 25(OH)D, CKD stage, and the interaction between CKD stage and 25(OH)D concentration. CKD participants who were receiving calcitriol therapy were excluded from these analyses. Both CRP and IL-6 concentrations were modeled as a function of 25(OH)D concentration and CKD stage.
Acknowledgments
We would like to thank the study participants and their families for their time and dedication to this study. We greatly appreciate the assistance of the Clinical Research Coordinators and the staff at the Clinical Translational Research Centers at Cincinnati Children’s Hospital Medical Center and Children’s Hospital of Philadelphia in the conduct of this study.
Funding: This project was supported by NIH grants R01-DK060030, R01-HD040714, K24-DK076808, and UL1-RR-024134 and UL1-RR-026314.
Footnotes
DISCLOSURES
None of the authors have financial conflicts of interest to disclose.
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