Bioavailable vitamin D levels in children with first episode nephrotic syndrome: A longitudinal study.

Abstract

Background

Bioavailable vitamin D levels is could be a better marker than total 25 hydroxy vitamin D levels to assess vitamin D status in children with nephrotic syndrome.

Primary objective

To assess the levels of Serum bioavailable vitamin D in children aged 1 to 12 years with idiopathic FENS and in healthy controls.

Secondary objective

1. To measure the levels of bioavailable vitamin D in FENS and after 4 weeks of standard steroid therapy induced remission.

2. To compare levels of serum and urine VDBP in FENS and after 4 weeks of standard steroid therapy induced remission.

Materials and Methods

A longitudinal study was conducted in children between age 1 to 12 years with idiopathic first episode nephrotic syndrome. After diagnosis of nephrotic syndrome as per ISPN guidelines, additional investigations like calcium, phosphorus, ALP, 25 hydroxy vitamin D, PTH, serum Vitamin D binding protein (VDBP) and urinary VDBP. Bioavailable and free vitamin D was calculated using above data. The patients were followed up after 4 weeks of remission and investigations were repeated and bioavailable vitamin D was calculated again, later results were compared and analysed. Appropriate statistical tests were applied for parametric and non-parametric data. P value of less than 0.05 was considered statistically significant.

Results:

The mean 25 hydroxy vitamin D level was 11.27(6.08) at FENS and at 4 weeks follow-up it was 13.65 (6.24). Hence children with FENS remained deficient in 25 hydroxy vitamin D, both during relapse and remission compared to healthy controls (15.97 ng/ml). The mean serum VDBP level in FENS during relapse was 242.90 (127.75). There was significant correlation of serum VDBP with serum albumin levels (p value 0.04). At 4 weeks of remission the VDBP levels increased to 550.7(219.7), this increase was significant (p value <0.001). The mean free vitamin D at FENS was 1.54 (1.70) pg/ml, on follow-up visit at 4 weeks of remission the free vitamin D levels decreased to 0.68 (0.53) pg/ml. The mean bioavailable vitamin D in FENS during relapse was 0.75 (0.84) ng/ml and on follow-up at 4 weeks of remission increased to 1.11 (0.84) ng/ml (p value = 0.015).

Conclusion

Children with FENS are deficient of vitamin D levels. The free and bioavailable vitamin D levels are reduced in children with FENS during proteinuria, as compared to healthy controls. Further studies showing correlation of bioavailable vitamin D and 25 hydroxyvitamin D with bone mineral density are required in children to validate the usage of bioavailable vitamin D in clinical practice.

Introduction

Childhood nephrotic syndrome shows prompt response to high dose glucocorticoids, in majority of the patients, however the disease frequently has a relapsing course, requiring repeated courses of corticosteroids, thereby posing the children at risk of steroid toxicity. The standard dose for management of relapses is 60mg/m²/ day, which far exceeds the daily dose of 5mg which is considered to be a risk factor for glucocorticoids induced osteoporosis in adults [1]. At physiological doses glucocorticoids are required for normal osteoblastic differentiation, while at higher doses they are found to promote apoptosis of osteoblast [2]. Hence prompt measures are required to monitor the bone health using tools like vitamin D levels and bone mineral density in children steroid sensitive nephrotic syndrome (SSNS) and do appropriate therapeutic interventions. Idiopathic nephrotic syndrome (INS) is complicated by urinary losses of low molecular weight that are necessary for the transport of metals, hormones and drugs like thyroid binding globulin, cortisol binding protein, Vitamin D binding protein in addition to albumin [3]. Vitamin D binding Protein (VDBP) is one among the plasma proteins which is a chief carrier of vitamin D, also gets excreted in urine along with 25 hydroxyvitamin D₃, resulting in low ionized calcium levels, which may lead to tetany. Hence assessment of VDBP, along with 25 hydroxyvitamin D and calculating bioavailable vitamin D gains importance. Bioavailable vitamin D represents the portion of vitamin D not bound to vitamin D binding protein (VDBP), i.e, fraction bound to albumin and the free form[4]. However in a recent study by Banerjee et al showed that there was not much difference in levels of free 25 (OH) in relapse or remission in comparison to healthy controls [5]. There is paucity of studies on bioavailable vitamin D levels in idiopathic first episode nephrotic syndrome (FENS) in children. Reports indicate that bioavailable vitamin D levels are decreased in nephrotic syndrome in adult population [3]. The primary objective of our study is to assess the levels of serum bioavailable vitamin D in children aged 1 to 12 years with First episode nephrotic syndrome (FENS) and 4 weeks of remission and compare with healthy controls.

Materials and Methods

We performed a longitudinal study in children with idiopathic FENS age 1 to 12 years, between January 2021 to June 2022, in a Pediatric nephrology Division of tertiary care teaching hospital in New Delhi, India. Institute Ethics Committee (IEC), (dated LHMC/IEC/2020/PG thesis/100, dated 28.12.2020) and written and informed consent was obtained prior to study. Children with clinical evidence of rickets and associated other hypoalbuminemic states ( liver disease and severe acute malnutrition) were excluded from the study. Healthy age and sex matched population were enrolled in control group. Patients were managed as per standard guidelines of Indian Society of Pediatric Nephrology [6]. At enrolment visit hemogram, kidney function test, serum albumin, cholesterol, urine microscopy, urine protein creatinine ratio was done. Additional investigations like serum calcium, phosphorus, alkaline phosphatase, 25 hydroxyvitamin D, serum PTH, serum and urinary VDBP was performed. Bioavailable vitamin D was calculated using serum VBDP, albumin and 25 hydroxy vitamin D levels using following formula. The patient was followed up after 4 weeks of steroid induced remission and bioavailable vitamin D was again calculated at remission.

Free and bioavailable 25 (OH) vitamin D levels were calculated by formula suggested by Powe et al. is summarized below [7],

Free 25(OH)D = [− b+√(b2 − 4ac)] / 2a

a = Kdbp∗ Kalb∗ albumin + Kdbp

b= (Kdbp∗ DBP)−(Kdbp∗ 25(OH)D) + (Kalb∗ albumin) + 1

c=− [25(OH)D]

Kdbp = affinity constant between 25(OH)D and DBP

Kalb = affinity constant between 25(OH)D and albumin

Bioavailable 25(OH)D = (Kalbumin + 1)[free 25(OH)D]

The affinity constants for both the equations where same, which was 7 * 10⁸ M^− 1 for VDBP versus 6 * 10⁵ M^− 1 for albumin.

Concentrations of albumin, VDBP and 25(OH) vitamin D in these equations are in mol/L, and free and bioavailable 25(OH) vitamin D were then converted to pg/ml and ng/ml respectively for ease of interpretation of results.

Results

Table 1 shows baseline characteristics of cases and controls. The mean age of children in our study was 41.97 (28.93) months, with a male to female distribution of 56.7% and 43.3% respectively. The baseline kidney function tests were normal at the onset of disease, except for mild elevations in urea attributing in prerenal AKI. The mean 25 hydroxy vitamin D levels was 11.27 ± 6.06 ng/ml as compared to healthy controls of 15.97 ± 7.01 ng/ml, which was significantly low (p < 0.05). The mean vitamin D levels improved to 13.65 ± 6.24 ng/ml at 4 weeks of remission (p < 0.05 ), however it did not reach the control levels ( 15.97 ± 7.01 ng/ml. The PTH levels decreased at remission, and fall was significant ( p 0.024). The mean serum VDBP levels were 247.9 ± 127.75 µg/ml which improved to 550.7 ± 219.7 µg/ml at remission (p value < 0.001). Simultaneously urinary VDBP showed a significant fall remission, correlating with urinary proteinuria. The free 25 hydroxy vitamin D was calculated to be 1.54 ± 1.70 pg/ml which reduced to 0.68 ± 0.53 pg/ml at 4 weeks of remission (p value < 0.05), while the bioavailable vitamin D levels rise from 0.75 ± 0.84 ng/ml to 1.11 ± 0.84 ng/ml at remission and the rise was significant. The bioavailable vitamin D levels across control group was 0.94 ± 0.25 which was significantly higher than controls (p value < 0.001). Table 2 shows change in baseline parameters after 4 weeks of remission.

Discussion

The mean 25 hydroxy vitamin D levels in our study at enrolment was 11.27(6.08) ng/ml which falls under the definition of deficiency of vitamin D according to IAP guidelines [8], and the mean vitamin D during remission was 13.65(6.24) suggestive of vitamin D insufficiency. In a study by Cetin N et al the mean Vitamin D level during remission was 16.4 ( 9.09) in children with infrequently relapsing nephrotic syndrome after a mean steroid intake of 1 year [9]. Lower levels of ionized calcium, high PTH and high ALP in a setting of vitamin D deficiency suggests biochemical abnormalities related to abnormal bone mineral metabolism and ill effects of bone health. They demonstrated that even 12 weeks of glucocorticoid therapy acts as a risk factor for osteoporosis. The risk of osteoporosis was not dependent on cumulative steroid dose. The management of FENS consist of 12 weeks of glucocorticoid therapy, hence it is ideal to get vitamin D testing at baseline and supplement children with deficiency to prevent its ill effects on bone health. Choudhary et al, emphasized on routine calcium and vitamin D supplementation irrespective of vitamin D status for osteoprotection in children with nephrotic syndrome[10] .We found that healthy controls had insufficient 25 OH vitamin D (15.97 ± 7.01) ng/ml. Children with FENS failed to achieve levels equal to healthy controls and remained 25 OH vitamin D deficient ( 13.65 ± 6.24). India despite being a tropical country, with good sunlight exposure, our healthy children were also vitamin D insufficient.

Surve et al, assessed the influence of VDBP levels on vitamin D status of under-five healthy children and showed that physiologic variations in PTH and VDBP are to be considered in children before deciding treatment strategies. PTH and VDBP were considered as non-modifiable risk factors for vitamin D deficiency in children especially those under the age of 5 years. The above study also emphasised on the free hormone hypothesis and importance of measuring free and bioavailable vitamin D, rather than total 25 hydroxy vitamin D, to prevent underestimation and overestimation of vitamin D status in children[11]. The mean serum VDBP level in our children with FENS during relapse, was 242.90 ( 127.75) µg/ml. This was comparable to the mean value of 210.0 (137.40 ) µg/ml in a study by Aggarwal et al, in adults with idiopathic nephrotic syndrome [3]. In children explanation for significantly lower levels compared to controls could be higher levels of proteinuria, beyond the compensatory capacity of liver and immaturity of liver’s ability to produce VDBP at higher concentrations On the follow up visit at 4 weeks of remission the VDBP levels increased to a mean value of 550.70 (219.7)µg/ml, which was fairly comparable with mean of 616 (255.03)µg/ml seen in controls. The plausible explanation for preserved VDBP as with few previous studies could be, compensatory increase in VDBP synthesis by liver and genetic variation in gene coding for VDBP. Assessment of VDBP in nephrotic syndrome gets paramount importance in children with SRNS, FRNS and SDNS, and continue to have proteinuria or remain in partial remission for prolonged periods. There was not much difference in the levels of VDBP in male and female gender. Waldron et al, showed that VDBP acts a negative acute phase reactant, but we did not find any difference in VDBP levels in children with or without infection at the onset of disease [12]. In our study serum VDBP and serum albumin showed statistically significant positive correlation (r = 0.37, p < 0.05). For every one unit decrease in albumin, VDBP decreases by 117.9 units. This is similar to the study by Aggarwal et al, showing similar correlation ( r = 0.264, p = 0.008) [3]. Hence, in comparison with healthy children, children with FENS, were hypoalbuminemic, and had reduced VDBP, therefore its assessment for knowing exact bioavailable vitamin D status gains importance. Our study also gives the normative values of bioavailable vitamin D levels in healthy Indian children were data is lacking.

According to free hormone hypothesis, it is the free form of the hormone which is active rather than the total 25 hydroxy vitamin D. Therefore we calculated the free hydroxy vitamin D from serum albumin, VDBP and 25 hydroxy vitamin D. As the serum albumin level increased during remission, the free form of vitamin D decreased. Hence it would be ideal to measure free vitamin D levels during FENS and relapse to know the exact vitamin D status in children with FENS. The fraction of 25 OH vitamin D which is loosely bound to albumin is also available for action at target sites, therefore bioavailable vitamin D levels should be measured in children with nephrotic syndrome. It was observed in our study that the mean bioavailable vitamin D increases with increase in age. This was in conjunction to decrease in VDBP with increase in age. The bioavailable vitamin D levels of healthy controls (0.95 ± 0.25 ng/ml), were comparable to children with FENS at 4 weeks of remission (1.11 ± 0.84 ng/ml). This shows that bioavailable vitamin D is especially useful to study vitamin D status in children during relapse. In our study bioavailable vitamin D showed a significant correlation with serum albumin, VDBP and 25 OH vitamin D as with the study by Aggarwal et al [3]. However bioavailable vitamin D did not show better correlation with PTH than total 25 hydroxy vitamin D.

In a study by Denburg et al, in pediatric CKD secondary to glomerular disorders having nephrotic range proteinuria had lower levels of free and bioavailable vitamin D compared to other aetiology of CKD, supporting the evidence that bioavailable vitamin D is a very good marker of vitamin D status in children with proteinuria [4]. They found mean bioavailable vitamin D in children with CKD secondary to FSGS, with significant proteinuria to be 0.8 ng/ml and those secondary to CAKUT was 3.4 ng/ml. This was in correlation with mean bioavailable vitamin D level of 0.75 ng/ml in children with FENS, as with our study.

The important limitation of first known study in our country by Aggarwal et al, was that followup at remission was not done. In our study we followed patients in remission which showed near normalization of bioavailable vitamin D levels like healthy children [3]. In the study by Aggarwal et al, it was found that bone mineral density in patients with nephrotic syndrome correlated well with bioavailable vitamin D ( r = 0.358, p = 0.0002) than total 25 hydroxy vitamin D ( r = 0.174, p value = 0.079 ), We did not perform DEXA scan in children with FENS due to logistic issues which was a limitation. Other limitations were smaller sample size and shorter period of follow-up.

Conclusion

We report that children with FENS are deficient of vitamin D levels, mandating the need for measurement of vitamin D levels and treating if deficient. The free and bioavailable vitamin D levels are reduced in children with FENS during proteinuria, as compared to healthy controls. Further studies showing correlation of bioavailable vitamin D and 25 hydroxyvitamin D with bone mineral density are required in children to validate the usage of bioavailable vitamin D in children with SSNS and SRNS.

Declarations

Author Contributions: A.B.C collected data, B, G analyzed the data, F did the dietary management, D, E did the biochemical analysis.  All the authors helped in drafting the manuscript. 

Ethics Approval: Institute Ethics Committee (IEC) approved the study and it was carried according to the ICMR National Ethical Guidelines for Biomedical and Health Research Involving Human Participants. 

Funding: None

Conflict of Interest: None reported.

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