The Effect of Vitamin D3 Supplementation on Hepcidin and Ferritin Serum Levels in Children With Chronic Kidney Disease
Phase: PHASE2
Conditions: Chronic Kidney Disease Stage 1, Chronic Kidney Disease Stage 2
Sponsor: Hasanuddin University

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EFFECT OF VITAMIN D3 SUPPLEMENTATION ON HEPSIDIN AND FERRITIN SERUM 
LEVELS IN CHILDREN WITH CHRONIC KIDNEY DISEASE 
May 1st 2023 
 
 
 
 
 
 
 
 
 
 
 
 
 

EFFECT OF VITAMIN D3 SUPPLEMENTATION ON HEPSIDIN AND FERRITIN SERUM 
LEVELS IN CHILDREN WITH CHRONIC KIDNEY DISEASE 
 
Protocol 
 
Responsible Personnel 
Principal Investigator Contact Information 
Name: Andi Muldiana Dwi Rachmayani 
Title: MD 
Institution: Department of Pediatrics, Faculty of Medicine, Hasanuddin University 
Address: Jln. Perintis Kemerdekaan KM 11 Makassar, South Sulawesi, Indonesia, 90245 
Telephone: +62(411)584461 
Email: winda_muldiana@gmail.com 
 
 
Summary:  
 
This study aim to assess the effect of Vitamin D administration on Hepcidin and Ferritin 
serum level in children with chronic kidney diseases. It is a clinical trial involving 43 chronic kidney 
disease patients at Dr. Wahidin Sudirohusodo Hospital in Makassar, Indonesia. Subjects are 
enrolled into two different groups, one with glomerulus filtration rate (GFR) more than 90 
mL/min/1,73 m2 and the other group consist of children with GFR less than 90 mL/min/1,73 m2. 
Both group will be given the same dose of Vitamin D, which is 2000 IU for 6 weeks. Measurements 
of vitamin D, hepcidin and ferritin level will be before and after Vitamin D supplementation. 
 
Introduction:  
 
Chronic kidney disease (CKD) is a significant global health issue characterized by a gradual 
decline in kidney function over time. Defined by the Kidney Disease: Improving Global Outcomes 
(KDIGO) guidelines, CKD involves abnormalities in kidney structure or function lasting more than 
three months, which can lead to complications such as anemia. In children, CKD can adversely 
affect growth patterns and is closely linked to anemia due to reduced erythropoietin production, 
which disrupts iron homeostasis essential for oxygen transport in the body. 
Anemia in CKD patients is primarily driven by iron deficiency, with studies indicating that 
the incidence ranges from 7.2 to 13.96 per 1,000 people annually, contributing to approximately 
800,000 deaths globally. In pediatric populations, the prevalence of anemia among children with 
CKD is notably high, affecting 36-58% of patients in regions like the United States and Germany. 
The increasing incidence of CKD in children is likely related to longer life expectancy and 
improved treatment options, although data on early-stage CKD remain limited. 
Vitamin D deficiency is another critical issue associated with CKD, particularly in children. 
Research shows that a significant percentage of patients undergoing hemodialysis exhibit low 
vitamin D levels, which can exacerbate anemia by lowering hemoglobin levels. Vitamin D plays a 
vital role in erythropoiesis, and its deficiency correlates with increased serum hepcidin levels—a 
hormone that regulates iron metabolism and can hinder iron absorption and release from stores. 
Given the interconnectedness of vitamin D levels, hepcidin concentration, and anemia in 
children with CKD, research is warranted to explore the effects of vitamin D supplementation on 
serum hepcidin and ferritin levels. There is currently a lack of studies addressing this relationship, 

especially in Indonesia. This research aims to fill the gap in knowledge regarding vitamin D's 
potential benefits for managing anemia and iron homeostasis disorders in pediatric CKD patients. 
 
Objectives of Trial: 
1. Primary Objective 
a. To assess the effect of Vitamin D supplementation on hepcidin and ferritin serum 
level in children with CKD 
2. Secondary Objective 
a. To assess vitamin D, hepcidin, and ferritin level in children with CKD and GFR less 
than 90 ml/min/1,73 m2 before vitamin D3 supplementation 
b. To assess vitamin D, hepcidin, and ferritin level in children with CKD and GFR 
more than 90 ml/min/1,73 m2 before vitamin D3 supplementation 
c. To assess vitamin D, hepcidin, and ferritin level in children with CKD and GFR less 
than 90 ml/min/1,73 m2 after vitamin D3 supplementation 
d. To assess vitamin D, hepcidin, and ferritin level in children with CKD and GFR 
more than 90 ml/min/1,73 m2 after vitamin D3 supplementation 
e. To compare vitamin D, hepcidin, and ferritin level in children with CKD and GFR 
less than 90 ml/min/1,73 m2 before and after vitamin D3 supplementation 
f. To compare vitamin D, hepcidin, and ferritin level in children with CKD and GFR 
more than 90 ml/min/1,73 m2 before and after vitamin D3 supplementation 
g. To compare vitamin D, hepcidin, and ferritin level between children with GFR less 
than 90 ml/min/1,73 m2 and children with GR more than 90 ml/min/1,73 m2 before 
vitamin D3 supplementation 
h. To compare vitamin D, hepcidin, and ferritin level between children with GFR less 
than 90 ml/min/1,73 m2 and children with GR more than 90 ml/min/1,73 m2 after 
vitamin D3 supplementation 
 
Hypothesis: 
1. Serum hepcidin level is lower after vitamin D3 supplementation in children with CKD 
2. Serum ferritin level is lower after vitamin D3 supplementation in children with CKD 
3. Serum hepcidin level is lower in pediatric CKD patients with GFR more than 90 
ml/min/1,73 m2 compare to GFR less than 90 ml/min/1,73 m2 
4. Serum ferritin level is lower in pediatric CKD patients with GFR more than 90 ml/min/1,73 
m2 compare to GFR less than 90 ml/min/1,73 m2  
 
Study Design: 
 
Interventional study 
 
Study Population: 
 
Number of samples: 
 
43 subjects 
 
Inclusion criteria: 

• 
Patientsa aged 2 to 18 years diagnosed with chronic kidney diseases at Dr. 
Wahidin Sudirohusodo hospital 
• 
Parents have signed their child's consent to participate in the study 
Exclusion criteria: 
• 
History of blood transfusion 3 months prior 
• 
Liver dysfunction 
• 
Sepsis 
• 
Routine consumption of Vitamin D 
• 
Routine consumption of iron supplement 
• 
Patients diagnosed with iron deficiency anemia 
 
 
Product Description: 
Vitamin D3 with the dose of 2,000 IU 
 
Outcomes:  
The primary outcomes being measured in this study are serum vitamin D levels, serum 
hepcidin levels and serum ferritin levels. These outcomes will be measured in children with CKD 
before and after 6 weeks of vitamin D3 supplementation. We plan to compare these levels in two 
groups of CKD patients - those with glomerular filtration rate (GFR) <90 mL/min/1.73 m2 and those 
with GFR ≥90 mL/min/1.73 m2 . Measurement will be done by collecting 3 ml of blood to measure 
the level of vitamin D, hepcidin and serum ferritin.  
Statistical analysis will utilize unpaired T test or Mann-Whitney test will be used to compare 
vitamin D, hepcidin and ferritin serum level in subjects according to the distribution of the data. 
Paired T Test or McNemar test will also be use to compare proportion of individually matched 
event for the two groups before and after vitamin D3 supplementation. Lastly, Pearson or 
Spearman test will be done to assess the correlation between vitamin D level and hepcidin and 
ferritin serum level.  
 
 
Conduct of Trial: 
This study will be conducted as a clinical trial involving children with chronic kidney disease 
(CKD). Participants will be divided into two groups based on their glomerular filtration rate (GFR): 
those with GFR <90 mL/min/1.73 m2and those with GFR ≥90 mL/min/1.73 m2. All participants will 
undergo initial blood tests to measure serum levels of vitamin D, hepcidin, and ferritin. Following 
these baseline measurements, the children will receive vitamin D3 supplementation for a specified 
period. After the supplementation period, blood tests will be repeated to measure the same 
parameters. We will then compare the pre- and post-supplementation levels of vitamin D, 
hepcidin, and ferritin within each GFR group, as well as between the two GFR groups. This 
approach will allow us to assess the effect of vitamin D3 supplementation on serum hepcidin and 
ferritin levels in children with CKD, and to determine if the effect differs based on the severity of 
kidney dysfunction as indicated by GFR.  

 
Figure 1. Study Procedure 
 
Statistical Aspects and Considerations: 
This study employs a comprehensive statistical approach to analyze the effect of vitamin 
D3 supplementation on serum hepcidin and ferritin levels in children with chronic kidney disease 
(CKD). We will utilize both descriptive and inferential statistics to examine their data. Descriptive 
statistics will be used to summarize the categorical data as counts and ratios, while quantitative 
Patients aged 2 to 18 years admitted to Dr. Wahidin Sudirohusodo Hospital 
Patients who meet inclusion criteria and do not meet exclusion criteria and 
have signed the informed consent form 
Group I 
GFR >= 90 mL/min/1,73 m2 
Group 2 
GFR < 90 mL/min/1,73 m2 
2,000 IU of Vitamin D supplementation for 6 weeks  
25OHD3, hepcidin and ferritin measurement 
Data collection 
Data analysis 
Physical examination and laboratory examination will be done in 24 hours 
Measurement of ureum, creatinine, serum hepcidin, ferritin and 25OHD3 level 
in blood 

data will be presented as mean values with standard deviation. For inferential statistics, the study 
will employ Pearson's correlation test to differentiate between groups and assess statistical 
significance. The significance level is set at P < 0.05 for statistical significance and P < 0.01 for 
high significance. The statistical analysis will be conducted using SPSS version 20.0. Important 
considerations for this study include ensuring an adequate sample size to detect clinically 
meaningful differences, which will be determined through a sample size calculation. We will 
account for potential confounding variables, assess whether assumptions are met for the planned 
statistical tests, handle any missing data appropriately, and conduct sensitivity analyses to 
evaluate the robustness of results. By addressing these statistical aspects and considerations, 
our study aims to produce valid and reliable results regarding the effects of vitamin D3 
supplementation on serum hepcidin and ferritin levels in children with CKD, comparing outcomes 
between groups with different glomerular filtration rates (GFR < 90 mL/min/1.73 m2and GFR ≥ 90 
mL/min/1.73 m2). 
 
 
References 
 
Altemose, K. E., Kumar, J., Portale, A. A., Warady, B. A., Furth, S. L., Fadrowski, J. J., & Atkinson, 
M. A. (2018). Vitamin D insufficiency, hemoglobin, and anemia in children with chronic 
kidney disease. Pediatric Nephrology, 33(11), 2131–2136. https://doi.org/10.1007/s00467-
018-4020-5 
Amanullah, F., Malik, A. A., & Zaidi, Z. (2022). Chronic kidney disease causes and outcomes in 
children: 
Perspective 
from 
a 
LMIC 
setting. 
PLoS 
ONE, 
17(6 
June). 
https://doi.org/10.1371/journal.pone.0269632 
Avner, E. D., Harmon, W. E., Niaudet, P., Yoshikawa, N., Emma, F., & Goldstein, S. L. (Eds.). 
(2016a). Pediatric Nephrology. Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-
662-43596-0 
Avner, E. D., Harmon, W. E., Niaudet, P., Yoshikawa, N., Emma, F., & Goldstein, S. L. (Eds.). 
(2016b). Pediatric Nephrology. Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-
662-43596-0 
Becherucci, F., Roperto, R. M., Materassi, M., & Romagnani, P. (2016). Chronic kidney disease 
in children. In Clinical Kidney Journal (Vol. 9, Issue 4, pp. 583–591). Oxford University 
Press. https://doi.org/10.1093/ckj/sfw047 
Chen, T. K., Knicely, D. H., & Grams, M. E. (2019). Chronic Kidney Disease Diagnosis and 
Management: A Review. In JAMA - Journal of the American Medical Association (Vol. 322, 
Issue 
13, 
pp. 
1294–1304). 
American 
Medical 
Association. 
https://doi.org/10.1001/jama.2019.14745 
Ganz, T., & Nemeth, E. (2016). Iron Balance and the Role of Hepcidin in Chronic Kidney Disease. 
In Seminars in Nephrology (Vol. 36, Issue 2, pp. 87–93). W.B. Saunders. 
https://doi.org/10.1016/j.semnephrol.2016.02.001 
Gois, P. H. F., Wolley, M., Ranganathan, D., & Seguro, A. C. (2018). Vitamin D deficiency in 
chronic kidney disease: Recent evidence and controversies. In International Journal of 
Environmental 
Research 
and 
Public 
Health 
(Vol. 
15, 
Issue8). 
MDPI 
AG. 
https://doi.org/10.3390/ijerph15081773 
Iyengar, A., Kamath, N., Reddy, H. v., Sharma, J., Singhal, J., Uthup, S., Ekambaram, S., Selvam, 
S., Rahn, A., Fischer, D. C., Wan, M., & Shroff, R. (2022). Determining the optimal 
cholecalciferol dosing regimen in children with CKD: A randomized controlled trial. 
Nephrology Dialysis Transplantation, 37(2), 326–334. https://doi.org/10.1093/ndt/gfaa369 

KDIGO 2012. (2012). Clinical Practice Guideline for the Evaluation and Management of 
Chronic Kidney Disease. www.publicationethics.org 
Lv, J. C., & Zhang, L. X. (2019). Prevalence and Disease Burden of Chronic Kidney Disease. In 
Advances in Experimental Medicine and Biology (Vol. 1165, pp. 3–15). Springer New York 
LLC. https://doi.org/10.1007/978-981-13-8871-2 
Nalado, A. M., Olorunfemi, G., Dix-Peek, T., Dickens, C., Khambule, L., Snyman, T., Paget, G., 
Mahlangu, J., Duarte, R., George, J., & Naicker, S. (2020). Hepcidin and GDF-15 are 
potential biomarkers of iron deficiency anaemia in chronic kidney disease patients in South 
Africa. BMC Nephrology, 21(1). https://doi.org/10.1186/s12882-020-02046-7 
Roemhild, K., von Maltzahn, F., Weiskirchen, R., Knüchel, R., von Stillfried, S., & Lammers, T. 
(2021). Iron metabolism: pathophysiology and pharmacology. In Trends in Pharmacological 
Sciences 
(Vol. 
42, 
Issue 
8, 
pp. 
640–656). 
Elsevier 
Ltd. 
https://doi.org/10.1016/j.tips.2021.05.001 
Romagnani, P., Remuzzi, G., Glassock, R., Levin, A., Jager, K. J., Tonelli, M., Massy, Z., Wanner, 
C., & Anders, H. J. (2017). Chronic kidney disease. In Nature Reviews Disease Primers 
(Vol. 3). Nature Publishing Group. https://doi.org/10.1038/nrdp.2017.88 
Ueda, N., & Takasawa, K. (2018). Impact of inflammation on ferritin, hepcidin and the 
management of iron deficiency anemia in chronic kidney disease. In Nutrients (Vol. 10, 
Issue 9). MDPI AG. https://doi.org/10.3390/nu10091173 USTUNER, B., BEK, S., EREN, 
N., BAKİRDOGEN, S., & KALENDER, B. (2021). Hepcidin and Vitamin D Levels: Mutual 
Effects in Anemia of Chronic Kidney Disease. Kocaeli Üniversitesi Sağlık Bilimleri Dergisi. 
https://doi.org/10.30934/kusbed.661316 
Vahdat, S. (2020). Vitamin D and Kidney Diseases: A Narrative Review. International Journal of 
Preventive Medicine, 11, 195. https://doi.org/10.4103/ijpvm.IJPVM 
Whyte, D. A., & Fine, R. N. (2008). Chronic kidney disease in Children. Pediatrics in Review, 
29(10), 335–341. https://doi.org/10.1542/pir.29-10-335 
Wong, M. M. Y., Tu, C., Li, Y., Perlman, R. L., Pecoits-Filho, R., Lopes, A. A., Narita, I., Reichel, 
H., Port, F. K., Sukul, N., Stengel, B., Robinson, B. M., Massy, Z. A., & Pisoni, R. L. (2021). 
Anemia and iron deficiency among chronic kidney disease Stages 3-5ND patients in the 
chronic kidney disease outcomes and practice patterns study: Often unmeasured, variably 
treated. Clinical Kidney Journal, 13, 613–624. https://doi.org/10.1093/CKJ/SFZ091