Mineral bone disorder in children with chronic kidney disease: Data from the KNOW-Ped CKD (Korean cohort study for outcome in patients with pediatric chronic kidney disease) study

Background Children with chronic kidney disease (CKD) are at high risk of mineral bone disorder (MBD), which leads to fractures, growth retardation, and cardiovascular disease. We aimed to comprehensively understand the relationship between renal function and factors related to MBD and evaluate the prevalence and distribution characteristics of MBD, specifically among Korean patients from the KNOW-PedCKD cohort. Methods From the baseline data of the KNOW-PedCKD cohort, we examined the prevalence and distribution of MBD in 431 Korean pediatric CKD patients, including the level of corrected total calcium, serum phosphate, serum alkaline phosphatase, serum intact parathyroid hormone (iPTH), fibroblast growth factor 23 (FGF-23), serum vitamin D, fractional excretion of phosphate (FEP), and bone densitometry Z-scores. Results The median serum calcium level remained relatively normal regardless of the CKD stage. The levels of 1,25-dihydroxy vitamin D, urine calcium-to-creatinine ratio, and bone densitometry Z-score significantly decreased with advancing CKD stage, while those of serum phosphate, FGF-23, and FEP significantly increased with CKD stage. The prevalence of hyperphosphatemia (17.4%, 23.7%, and 41.2% from CKD stages 3b, 4, and 5, respectively) and hyperparathyroidism (37.3%, 57.4%, 55.3%, and 52.9% from CKD stages 3a, 3b, 4, and 5, respectively) significantly increased with the CKD stage. Prescriptions of medications, such as calcium supplements (39.1%, 42.1%, 82.4%), phosphate binders (39.1%, 43.4%, 82.4%), and active vitamin D (21.7%, 44.7%, and 64.7%) significantly increased with CKD stage 3b, 4, and 5, respectively. Conclusions The results demonstrated the prevalence and relationship of abnormal mineral metabolism and bone growth according to CKD stage in Korean pediatric CKD patients for the first time.


Introduction
Mineral bone disorder (MBD) represents a systemic disorder manifested by either one or a combination of abnormal mineral metabolisms, abnormal bone growth or strength, making bone more vulnerable to fracture and extra-skeletal calcification, all of which are closely interrelated and contribute to the morbidity and mortality of patients with chronic kidney disease (CKD), including those with cardiovascular disease (1). In 2006, the Kidney Disease: Improving Global Outcomes (KDIGO) working group addressed this revised terminology, replacing the previous concept of renal osteodystrophy, which described only the bone pathology in patients with CKD, stressing the systemic effects, including cardiovascular implications (2).
Mineral metabolism exerts a more profound influence in children with CKD as the development and maturation of the skeletal and vascular systems occur during childhood. CKD-MBD in children causes bone deformity and growth failure and may be accompanied by extra-skeletal cardiovascular calcifications with serious outcomes (3)(4)(5). Thus, monitoring and managing abnormal mineral metabolism from childhood throughout life is crucial. Although many studies have investigated CKD-MBD in adult patients, investigations solely into pediatric CKD-MBD are lacking. Studies from the United States conducted with the CKD in Children (CKiD) cohort reported the prevalence of abnormal mineral metabolism. They described fibroblast growth factor 23 (FGF-23) as the first factor to rise, followed by hyperphosphatemia and hyperparathyroidism across the CKD stages (6,7). While their findings were comparable with the known adult studies regarding the prevalence of MBD, the study population mainly included Caucasian, African-American, and Hispanic ethinicities and the analysis was cross-sectional. Some studies have evaluated the bone markers and fracture risk in pediatric CKD patients according to race and ethnicity, showing African-American or Hispanic patients with lower 25-hidroxy vitamin D levels and a lower risk of fracture compared with Caucasian patients, although the data may be inaccurate and subjective because selfreporting was used to determine fracture history (8). To the best of our knowledge, few studies in Asia have evaluated CKD-MBD in pediatric CKD, including objective measurements regarding bone mass. Park et al. recently demonstrated the overall baseline characteristics of the Korean cohort study for Outcome in patients with Pediatric CKD (KNOW-Ped CKD) cohort featuring some of the characteristics associated with CKD-MBD, including hyperphosphatemia and hyperparathyroidism, but not focusing on many other factors associated with CKD-MBD (9). Therefore, the objectives of this study were to comprehensively understand the relationship between renal function and factors related to MBD and change in bone mass and evaluate the prevalence and distribution characteristics of MBD, specifically among Korean patients from the KNOW-PedCKD cohort.

Study design and population
From April 2011 to April 2016, a total of 469 patients < 20 years of age with CKD stages 1-5 were enrolled in the KNOW-PedCKD study, a nationwide, 10-year prospective and observational cohort study from seven major pediatric nephrology centers in Korea. Patients undergoing kidney replacement therapy or those who had cancer or had been treated for cancer within the previous five years were excluded from this cohort. The KNOW-PedCKD study is registered at ClinicalTrials.gov with the designation number NCT02165878 (submitted on June 11, 2014) and was approved by the Institutional Review Boards of all participating centers.
The detailed study protocol for comprehensive assessment of clinical findings and structured follow-up has been reported previously (10). The authors used the baseline data when the patients were enrolled in the cohort. Clinical data was also collected, such as chronological age, bone age at baseline, serum corrected total calcium, serum phosphate, serum alkaline phosphatase (ALP), serum intact fibroblast growth factor 23 (FGF-23), serum intact parathyroid hormone (iPTH), serum vitamin D (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D), urine calciumto-creatinine ratio, fractional excretion of phosphate (FEP), and bone densitometry Z-scores, as well as the intake of calcium supplements, non-calcium phosphate binders, and active vitamin D. Among the patients initially recruited, 431 were finally included in this study; patients aged > 18 years and those with missing data of interest were excluded.

Statistical analyses
Continuous variables are presented as the mean ± standard deviation and median (range). We compared the level of each variable according to the CKD stage using the Kruskal-Wallis test. The incidence of disordered mineral metabolism and distribution of medication intake were compared using the Chi-square test or Fisher's exact test. The relationship between variables and the eGFR was analyzed using linear, inverse, and compound models. The correlation between MBD and prescribed medications for MBD was analyzed using a multivariate analysis of covariance (MANCOVA). A P-value < 0.05 was considered to be statistically significant. We performed all statistical analyses using SAS version 9.4 (SAS Institute Inc., Cary, NC) and IBM SPSS Statistics for Windows, version 25.0 (IBM Corp., Armonk, N.Y., United States).

Results
The patterns of mineral metabolism according to CKD stages The patterns of mineral metabolism according to the CKD stages and the distribution of patients are described in Table 1. The median serum calcium level was within the normal range, regardless of the CKD stage, with no significant difference between different stages. The serum phosphate, serum iPTH, serum FGF-23, and FEP levels significantly increased (P < 0.001) with advanced CKD stages; the median serum phosphate level (mg/dl) was 4.60, 4.90, 5.10, and 5.80 in CKD stages 3a, 3b, 4, and 5, respectively, and the levels of iPTH, FGF-23, and FEP constantly increased as the CKD stage advanced from stage 1 to 5 (P < 0.001). In contrast, the levels of 1,25-dihydroxyvitamin D, urine calcium-to-creatinine ratio, and bone densitometry Z-scores decreased with advancing CKD stages; the median 1,25-dihydroxyvitamin D level decreased as the CKD stage advanced from stage 1 to 4 (P < 0.001). The urine calcium-tocreatinine ratio and bone densitometry Z-scores significantly decreased (P < 0.001) as the CKD stage advanced from stage 1 to 3. The ΔBone age % consistently showed negative values across all CKD stages, with no statistically significant difference between stages.

Use of medications according to CKD stages and their impact
The use of medications for CKD-MBD according to the CKD stage is shown in Table 3. The prevalence of calcium supplements (4.0%, 39.13%, 42.11%, and 82.35% among the CKD stages 3a, 3b, 4, and 5, respectively) and non-calcium phosphate binder (sevelamer or lamthanum carbonate) prescriptions (4.0%, 39.13%, 43.42%, and 82.35% among the CKD stages 3a, 3b, 4, and 5, respectively) showed a significant increase according to the CKD stages (P < 0.001). The prevalence of active vitamin D supplements (alfacalcidiol or calcitriol) also showed significant increase (P < 0.001) through all CKD stages (0.0%, 4.27%, 10.67%, 21.74%, 44.74%, and 64.71% among CKD stages 1-5, respectively). Multivariate analysis of covariance (MANCOVA) was performed to determine whether there was a statistically significant difference between the patients who received medications (calcium and noncalcium phosphorus binder and active vitamin D supplements) and non-users on CKD-MBD variables controlling for GFR ( Table 4). Patients taking phosphorus binders had a significantly higher serum iPTH level than non-users. Those taking active vitamin D supplements showed a higher serum calcium level and a lower phosphorus level than those not taking them.

Relationship between the parameters of MBD and eGFR
We further investigated the relationship between each variable of mineral metabolism and the eGFR (Figure 1). The urine calcium-to-   The statistical significance level was set at P < 0.05.

Discussion
MBD is a crucial complication that should be closely monitored in pediatric patients with CKD. This study first demonstrated the prevalence of disordered mineral metabolism and its relationship with the eGFR in Korean pediatric patients with CKD for the first time in Asia.
The median serum calcium concentration remained relatively normal regardless of the CKD stage ( Table 1). The stable serum calcium level until the eGFR fell below 20 ml/min/1.73 m 2 (Figure 1) was in accordance with the course of serum calcium level in a previous study on adults with CKD (16). However, looking at the proportion of patients with hypercalcemia and hypocalcemia separately, both show an increasing tendency across the CKD stages, which is more predominant in those with hypercalcemia ( Table 1). This finding was in accordance with the findings from adult CKD, in which hypocalcemia does not appear until an advanced stage of 4-5, at the expense of high PTH levels from stage 3. The increasing prevalence of hypercalcemia might be attributed to the increasing prescription of calcium supplements and active vitamin D in patients, or to tertiary hyperparathyroidism in long-standing CKD patients. In this regard, the recent 2017 KDIGO guidelines highlighting caution against hypercalcemia have profound significance due to the accumulating evidence suggesting that hypercalcemia is associated with increased mortality and nonfatal cardiovascular events (17)(18)(19). Therefore, maintaining serum calcium levels within the normal range for adults is being emphasized in clinical practice. However, owing to the lack of controlled studies and high-grade guidelines for pediatric CKD patients, and due to the negative effect on bone mass in children and adolescents, the strict criteria that are cautious in adults seem to be applied a little weakly in children to maintain calcium levels within the normal range in the age group. In this context, analysis of the follow-up data of our study might aid in evaluating calcium control in pediatric patients with CKD.
The inverse relationship between the serum phosphate level and eGFR was highlighted in our study cohort. The rapid rise in the serum phosphate level in stage 4-5 CKD (Table 1, Figure 1) was earlier than shown in previous adult CKD data from the SEEK study, while the CKiD cohort showed relatively stable phosphate levels until the eGFR fell to < 20 ml/min/1.73 m 2 (6,16). Higher median phosphate levels in earlier CKD stages might be associated with differences in age distribution, leading to the baseline difference in normal phosphate levels and dietary consumption. In our study, the phosphate binder prescription increased according to the increasing CKD stage, as did the rising serum phosphate Frontiers in Pediatrics level across the CKD stages. However, the lack of demonstrated efficacy of phosphate binders for lowering serum phosphate in CKD stages 3a-4, the unproven safety of phosphate binders, and the uncertainty regarding the improvement in prognosis with dietary phosphate restriction in adult studies led the treatment goal to change from "maintaining phosphate within a normal range" to "treating patients with progressive hyperphosphatemia" (19-22). This revised recommendation may also have significance in children with CKD regarding the management target of the serum phosphate level and dietary modification. Although prospective studies with pediatric patients with CKD are lacking, the choice of phosphate binders and the level of dietary restriction should be prudently titrated in pediatric patients based on the trend of phosphate levels during follow-up. The significant increase in the iPTH level with increasing CKD stage was especially remarkable among advanced CKD stages (CKD stage 4-5) ( Table 1, Figure 1), corresponding with the established adult data of hyperparathyroidism in CKD stage 3-5 (16, 23). However, the prevalence of hyperparathyroidism increased with CKD stages 1 through 3b and remained stable throughout CKD stages 4 and 5 ( Table 2). This result may be explained by the increase in the reference range of the serum iPTH level in advanced CKD stages and the increased administration rate of active vitamin D supplements according to increasing CKD stages. While Japan, a neighboring nation from Asia, sets the iPTH target level slightly lower at 60-240 pg/ml and adjusts it strictly, the Korean society aims to control iPTH based on the target suggested by the K/DOQI guidelines that are universally applied to both adults and children (14,24). Because active vitamin D, phosphate binders, and calcimimetics are relatively accessible in Korea with reimbursement, the results from this study regarding the iPTH trend can be generally applied worldwide.
The serum 25-hydroxy vitamin D deficiency and serum 1,25dihydroxy vitamin D deficiency remained high regardless of the CKD stage (> 70.0% in all stages) ( Table 1), and only the serum 1,25-dihydroxy vitamin D level showed a significant decrement with increasing CKD stage in an exponential manner. This phenomenon may be caused by a decrease in the degree of 1-α-hydroxylation of 25-hydroxy vitamin D owing to progressive damage to the proximal renal tubule with advancing renal deterioration, leading to a decline in the 1,25-dihydroxy vitamin D level (25). In contrast, the median level of 1,25-dihydroxy vitamin D showed a slightly higher value in stage 5 than in stage 4 CKD. This slight rebound of the 1,25dihydroxy vitamin D level at CKD stage 5 from our study was in agreement with the results from the CKid study (6), where the 1,25dihydroxy vitamin D level significantly decreased with decreasing eGFR but slightly increased with an eGFR < 20 ml/min/1.73 m 2 . These findings can be explained by the higher prescription rate of active vitamin D supplements in patients with an eGFR < 20 ml/ min/1.73 m 2 and those with CKD stage 5 in the CKid and our study.
MANCOVA results showing the correlation between phosphorus binders or vitamin D and CKD-MBD variables are confusing in Table 4. In this analysis, phosphorus binders and active vitamin D did not show a significant effect on serum phosphorus and iPTH levels. These findings might be due to a limitation of this crosssectional study, which only reveals simultaneous clinical situations without defining a time sequence between the medication and laboratory findings. For example, when patients with hyperphosphatemia are prescribed a phosphorus binder, they might have normal phosphorus levels at the sampling time, while those with normal phosphorus levels not receiving phosphorus binders would continue to have normal phosphorus levels, thereby disturbing the causal relationship.
The increase in the FGF-23 level with the decrease in renal function observed in our study was similar to previous findings from studies with children (6,26,27). FGF-23 is a bone-derived circulating hormone that stimulates phosphate excretion through renal proximal tubular cells and suppresses the renal synthesis of 1,25-dihydroxy vitamin D and their level, and increases to lower the levels of serum phosphate toward a normal range, which was shown from the CKid study as the earliest detectable abnormality in mineral metabolism (6). FGF-23 secretion increases as CKD progresses, and the serum phosphate level is theoretically maintained at a relatively normal level until progression to advanced CKD (28). Our study demonstrated this mechanism well with the inverse relationship between eGFR and FGF-23 and between eGFR and FEP. However, as patients progress to advanced CKD, although the excretion of phosphate is increased from each renal unit, the overall excretion of renal phosphate diminishes due to a decline in the absolute number of functioning nephrons. Therefore, hyperphosphatemia inevitably ensues. Increasing FEP with CKD progression, as shown from our study, is a sign of nephron stress, which the physiologic adaptive mechanism has become maladaptive, leading to over-excretion of phosphate and compensation for the absolute number of nephron loss as CKD progress (29). This phenomenon was also shown from previous adult studies showing increasing FEP with decreasing creatinine clearance, leading to neutral phosphorus balance in some CKD stage 3/4 stage patients (21,30). Several studies have reported that increased FGF-23 levels are associated with CKD progression, left ventricular hypertrophy, and premature death (31)(32)(33)(34). Accumulating evidence suggests that FGF-23 levels are further associated with cognitive dysfunction and even host defense and inflammation (35-37). Although there are no guidelines for reducing FGF-23 levels in patients with CKD, dietary phosphate restriction, non-calcium phosphate binders, intensified dialysis, and renal transplantation seem to be reliable options in both children and adults with CKD. As active vitamin D further stimulates FGF-23 secretion, using a minimal dosage of active vitamin D to reach the target iPTH value should be emphasized in clinical practice (38).
As CKD progresses in children, more patients are at risk of decreased bone density caused by CKD-MBD. For the first time, we demonstrated the progressive deterioration in bone density as CKD progressed from stages 1 through 3 ( Table 1). Although the bone density improves in CKD stages 4-5, it consistently falls behind the bone age compared to chronological age across all CKD stages among the KNOW-Ped CKD cohort. The decreasing bone density then rebounds according to the increasing CKD stage, which can be partly explained by the increasing interventions, including the intake of vitamin D, calcium, and phosphate binders. Bone densitometry has limitations as an indicator of the comprehensive status of bone disease. Because of the age distribution and different growth velocities among patients, it does not indicate the state of bone turnover, making it difficult to assess the presence of adynamic bone disease. Additionally, because it relies on areal density rather than volumetric density, the bone density of short statured children is usually underestimated (39-41). However, we believe understanding the current status of the bone density distribution has certain significance in the setting of limited bone biopsy due to its invasiveness and burden.
This study has several limitations that warrant discussion. First, missing data or unavailable information, a different number of patients for each CKD stage might have caused biased results, skewing the tendency of mineral concentration across the CKD stages. Second, the level of dietary restriction and compliance with each patient's medications, which may have impacted the serum mineral or hormone levels, were not included. Third, risk factor analysis was not conducted, nor the subgroup analysis according to different etiology was not performed as the cohort was composed of patients with heterogeneous etiologies (including glomerulopathy, congenital anomalies of kidney and urinary tract, cystic kidney disease, etc.) and progression rates. Therefore, we determined that it was unconvincing to demonstrate the association of a single factor with CKD-MBD progression since CKD "per se" can also influence the factor. Fourth, we did not analyze baseline cardiovascular parameters in this study due to the low prevalence of detectable extra-skeletal calcifications. Finally, owing to the multicenter study design, there may have been potentially significant inter-study variations among individual center settings. However, the strength of this study lies in that this is the first nationwide cohort study of pediatric CKD-MBD and the first prospective and most extensive pediatric CKD cohort study in Asia, demonstrating the distribution and prevalence of CKD in Asian ethnicity and presenting the relationship between eGFR change and variables associated with CKD-MBD in a pediatric population. As Korea has good access to CKD-MBDrelated medications and a relatively well-established reimbursement system, the management of Korean pediatric CKD patients closely conforms to universal guidelines, and thus the results of this study may be applicable worldwide. Therefore, the baseline study of mineral metabolism is meaningful as a cornerstone of future research. We will follow the study participants for ten years. We expect further studies regarding dynamic changes in mineral metabolism and growth, including analysis by underlying etiologies in children with CKD from the full-length cohort data to be available.

Conclusion
Among Korean pediatric patients from the baseline data of the KNOW-PedCKD cohort, the median calcium level remained relatively normal with increasing use of calcium supplements and active vitamin D across the CKD stages. Disordered mineral bone metabolism with respect to serum phosphate, serum FGF-23, hyperparathyroidism, and bone densitometry Z-score was evident with advancing CKD stages. Further study with follow-up data showing the trajectory or course of CKD-MBD stratified with underlying CKD etiologies might elucidate the progression and the impact of medical management on CKD-MBD in pediatric patients. Furthermore, a comparative study and consensus on what modality should be used for assessment of bone disease, growth, and extra-skeletal calcification are needed.

Data availability statement
The datasets generated for this study is accessible after approval by the investigators of KNOWPed-CKD study (http://www.knowpedckd.org/pedckd/main/main.html). To request datasets, contact the corresponding author.

Ethics statement
The studies involving human participants were reviewed and approved by Institutional Review Board of all the participating centers including Seoul National University Hospital, Asan Medical Center, Samsung Medical Center, Jeju National Hospital, Kangnam Hallym Sacred Heart Hospital, Severance Hospital, Kyungbook National University Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

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