*2.3. Biochemical Parameters*

Parameters, others than calcium, creatinine and 25(OH)D, examined during Stage 1 has been used to confirm diagnosis of idiopathic hypercalciuria.

Blood was collected from cubital vein to biochemical tubes (BD Falcon, Warsaw, Poland), and serum was obtained by the centrifugation method (20 min., 4 ◦C, 2000× *g*).

24-h and second morning urine samples were collected to the sterile container, and urinalysis and urine culture were performed. The urine samples were centrifuged (20 min., 4 ◦C, 2000× *g*) and the supernatant was collected for biochemical analysis. The concentration of calcium, phosphorus, magnesium, and sodium was measured by the photometric absorption method in Cobas 6000 analyzer (Roche Diagnostics, Warsaw, Poland). Creatinine and urea concentration in serum and urine were evaluated by the enzymatic method in Cobas 6000 analyzer. eGFR (estimated glomerular filtration rate was calculated using the Schwartz formula (0.413 × height (cm)/serum creatinine (mg/dL)). A result above 90 mL/min/1.73 m<sup>2</sup> was considered as normal.

The Dia-Sorin LIAISON® analyzer (Dia-Sorin, Saluggia, Italy) and chemiluminescent immunoassays (CLIA) were used to determine the concentration of total 25-hydroxy-vitamin D in serum and plasma. The values below 30 ng/mL indicated 25(OH)D deficiency.

Calciuria was assessed on the basis of calcium excretion in a 24-h urine sample (urine calcium concentration (mg/dL) × body weight (kg)/collection volume (dL)) and calcium/creatinine ratio (Ca/Cr) calculated from the second morning urine sample (urine calcium (mg/dL)/ urine creatinine (mg/dL). Calciuria was defined as calcium excretion above 4 mg/kg/day, the values of calcium-creatinine index indicating hypercalciuria depending on age (Table 1).


**Table 1.** Values of the calcium-creatinine index in children, indicative of hypercalciuria.

#### *2.4. Ultrasonography and Densitometry*

Abdominal ultrasound examinations were performed with the use of GE Logiq 5 Expert (Warsaw, Poland) and Philips EPIQ 5G (Warsaw, Poland) equipment. In all children, the abdominal ultrasound examination was performed according to the same examination protocol assessing the length and echogenicity of the kidneys, the presence/absence of dilatation of the calico-pelvic systems, and the presence/absence of urinary tract stones.

The densitometry was performed with the HOLOGIC model Delphi W (S/N 70608) in the Whole Body projection.

### *2.5. Statistical Analysis*

Statistical evaluation of the results was performed using T-tests and one-way ANOVA with Bonferroni correction (in the case of a normal distribution) or non-parametric Kruskal–Wallis and Mann–Whitney U tests (in the case of an abnormal distribution). The data distribution was evaluated using the Shapiro–Wilk test. The percentile data were analyzed by the χ<sup>2</sup> tests with modifications or the Fischer test, depending on the size of the subgroups. Correlation analysis and the related regression analysis were performed for variables whose relationships could have medical significance. The Statistica software (version 13.1; StatSoft, Cracov, Poland)) was used. The *p* < 0.05 was considered as statistically significant.

#### **3. Results**

#### *3.1. Study Population*

36 children (18 boys, 18 girls) aged 5–16 (mean 10.47 ± 3.41) were examined. Seven patients were followed for one year, the rest (29 children) for 2 years. Vitamin D deficiency (concentration < 30 ng/mL) was observed in all children (Table 2). At the beginning of the study, 13 children had stones in the urinary tract visible on ultrasound.


**Table 2.** Mean values of the assessed parameters at the beginning of the study.

### *3.2. Vitamin D Level*

The vitamin D 25 (OH) D concentration in the serum determined after 3, 6, 9, 12, 15, 18, 21, and 24 months showed a statistically significant increase. We did not observe significant changes in the serum calcium concentrations or urinary calcium excretion (measured in mg/kg/24 h and with the use of calcium-creatinine ratio) (Figure 2 and Table 3). There were no significant changes in bones density measured by the Z-score after 12 or 24 months of supplementation (Figure 3).

**Figure 2.** Time-dependent serum concentrations of vitamin D (**a**), calcium (**b**), and Ca/Creatinine (**c**), determined in the second morning urine sample, converted to creatinine ratio, and calciuria (**d**) determined in 24 h urine collection, in patients receiving vitamin D supplementation.

**Table 3.** The mean values of the assessed parameters after 24 months of vitamin D supplementation.


**Figure 3.** Z-score values in patients who received 12 or 24 months of vitamin D supplementation.

#### *3.3. Correlations*

A statistically significant correlation was found between vitamin D concentration and calcium concentration in the serum after 3 and 21 months of vitamin D supplementation. The average calcium concentration increased from 9.72 ± 1.19 to 10.06 ± 0.38 mg/dLwhich was still in the normal range), and the vitamin D concentration increased from 20.02 ± 8.52 to 27.89 ± 6.24 ng/mL. There was no statistically significant correlation between vitamin D and calcium concentration in the serum at other time points (Table 4, Figure 4). There was also no statistically significant correlation between vitamin D concentration and calcium excretion (in daily urine collection and in second morning urine sample, assessed by creatinine level) as well as between vitamin D concentration and Z-score assessed by densitometry (Table 4). The formation of new stones in the urinary system or enlargement of existing stones was not observed in the ultrasound examination performed during each follow-up visit (every 3 months) in children supplemented with vitamin D (Table 5).

**Figure 4.** Dependence of serum calcium concentration on vitamin D concentration at 3 (**a**) and 21 (**b**) months after starting vitamin D administration..


**Table 4.** Correlations between vitamin D and calcium concentration in the serum, and calcium excretion (in the 24-h and the second morning urine sample, assessed by creatinine level) at different time points. The statistically significant values are marked in bold.

**Table 5.** The relationship between vitamin D concentration and urolithiasis activity after 3, 12, and 24 months of vitamin D supplementation.


### **4. Discussion**

Interest in vitamin D and its properties has increased significantly over the past two decades. Numerous studies conducted in various regions of the world indicate that vitamin D deficiencies occur in all countries, regardless of the latitude and age [15–18]. This is also true for Polish children. In a multicentre study of the pediatric population, a clear deficiency of vitamin D was found in 75–80% of children [19].

Similar observations were made at the Department of Paediatrics, Nephrology, and Paediatric Allergology of Military Institute of Medicine, especially in children with idiopathic hypercalciuria and urolithiasis. These observations were the basis of the presented study. Vitamin D was administered to children with urolithiasis in the course of idiopathic hypercalciuria. This is one of the few studies on the use of vitamin D treatment in such group of patients. There is controversy on the effect of vitamin D administration on the urolithiasis activity. Earlier reports suggested the possibility of negative impact of vitamin D supplementation on the activity of urolithiasis, and vitamin D treatment in these patients was, therefore, contraindicated [20,21].

Despite physiological bases some authors have already demonstrated that vitamin D supplementation does not increase calciuria. Increased calcium reabsorption in the renal tubules, associated with increased serum vitamin D levels, should reduce urinary calcium excretion [1,4]. Therefore, we decided to use vitamin D in the doses lower than those normally used in the deficient population (ie. 400 IU/day or 800 IU/day). In case of a significant calciuria increase and/or increase in urolithiasis activity, the vitamin D administration was planned to be stopped. However, this problem did not occur in any of our study patients. The dose of vitamin D was determined based on serum 25(OH)D concentration. The dose of 400 IU/day of vitamin D was administered to patients with serum 25(OH)D concentrations > 20 ng/mL and a dose of 800 IU/day in patients with serum 25(OH)D < 20 ng/mL concentration. There are no clear recommendations in the world literature regarding the administration of vitamin D in patients with low bone density and idiopathic hypercalciuria [4,9,20]. All known studies were performed in adult population and results are often contradictory. The observations from our study confirmed the observations of Ticinesi et al. [22] showing the decreased serum vitamin D concentration in patients with urolithiasis. The authors reported the patients with urolithiasis had a lower concentration of vitamin D than the patients from the control group (without urinary problems). They even concluded that vitamin D deficiency (<20 ng/mL) increases the risk of calcium stones.

Many studies have been conducted to assess the safety of vitamin D supplements in healthy people. Most studies have shown no adverse effects, either with constant use or with single high dose of vitamin D [23–28]. However, most of the analyzed cases were postmenopausal women or people with accompanying chronic diseases, including lupus, renal failure, diabetes, multiple sclerosis, or respiratory diseases, the course of which may affect the results. None of such analyzes were performed in children. In the present study, we showed, that vitamin D administration correlated with a statistically significant increase in the serum concentration of the hepatic metabolite of vitamin D. This result was consistent with other studies [29–34]. We also showed that low doses of vitamin D (400 IU/day) in patients with D hypovitaminosis caused a significant increase in the concentration of 25(OH)D in the serum. We also showed that there was no significant effect of vitamin D administration on calciuria and there was no correlation between concentration of vitamin D and calciuria, which is in agreement with the study of Eisner et al. [31]. Similar results were also observed by other authors. Penntiston et al. [34] showed that supplementation with high doses of ergocalciferol (vitamin D2) in healthy postmenopausal women, including four postmenopausal women with hypercalciuria, did not increase calcium excretion in urine. Similar results were obtained by Leaf et al. (29) who showed that in adult population with urolithiasis there was no significant increase in calcium excretion, despite the use of high doses of ergocalciferol (50,000 IU per week) for 2 months. Also, Johri et al. (30) found no statistically significant increase in calcium excretion in urine after 2 months supplementation with vitamin D (20,000 IU per week) in adults with urolithiasis, and vitamin D deficiency (<12 ng/mL in serum). However, in the group of patients with a low level of calciuria, there was an increase of calcium excretion in urine, which could have been associated with latent idiopathic hypercalciuria, and mutation or polymorphism in CYP24A1 gene. Also, Letaverier et al. [33] found a higher level of calcium extraction in the urine of rats supplemented with vitamin D. The calciuria was enhanced by the administration of calcium [33].

In the present study, the children's diet contained a normal concentration of calcium, and children did not receive additional calcium supplementation. Their calcium serum concentration was within the normal range. We found a statistically significant correlation between the concentration of calcium and vitamin D in the serum after 3 and 21 months of vitamin D administration. We also found that there was no increase in the activity of urolithiasis (defined as the presence of new stones in the urinary tract), assessed by the ultrasound analysis performed every 3 months. Similar results were obtained by Ferraro et al. [35] who evaluated the influence of vitamin D treatment on the increased risk of urolithiasis in almost 200,000 medical professionals. They divided the study group based on the daily intake (from 100 to 1000 IU) of vitamin D [35]. They found that vitamin D supplementation did not increase the risk of kidney stones. However, a limitation of the study was the method of assessing the activity of urolithiasis, which was solely based on reported renal colic incidents. In contrast, Letavernier et al. [33] showed the higher activity of stone formation in the group receiving calcium with vitamin D compared to the group receiving calcium alone, vitamin D alone, or the control group.

Bone mass is built in the first three decades of life and after the reached peak, involution of bone mass begins. The size of bone peak mass is fundamental for the risk of developing osteoporosis in the future [36,37]. The correlation between hypercalciuria and urolithiasis and osteopenia and osteoporosis is well documented [31,38]. In children, it is probably associated with increased calcium resorption from bone and/or increased bone turnover [1]. However, there is still no clear opinion on the effect of vitamin D supplementation on bone density in children with urolithiasis and hypercalciuria. The studies of Garcia-Nieto et al. [39] on the population of children with idiopathic hypercalciuria found osteopenia in 30% of the respondents (22 out of 73 children). In children with reduced bone density, they observed lower excretion of citrates and higher excretion of uric acid, which could affect both the activity of urolithiasis and the bone calcification state [39]. A comparison of 88 children with idiopathic hypercalciuria with 29 healthy children by Penido et al. [40] showed reduced bone mineral density in the lumbar spine in 35% of children with idiopathic hypercalciuria. Schwaderer et al. [41] observed a higher risk of bone mineralization disorders in children with idiopathic hypercalciuria and active urolithiasis than in children with idiopathic hypercalciuria alone. They also noted that such disorders more often affected boys with an increased Body Mass Index (BMI). On the other hand, Artemiuk et al. [42] showed a correlation between low vitamin D concentrations and reduced bone density in the lumbar spine of children. They also drew attention to the more frequent occurrence of bone mass reduction in older children compared to the younger group (mean age 11 vs. 8.5 years). In the present study, there was no improvement in bone density parameters in a densitometric examination, measured with Z-score, after 12 and 24 months supplementation with vitamin D.

Adequate dietary calcium intake is important for both urolithiasis course and bone density. Both too little and too much calcium supply in the diet favors the formation of stones in the urinary tract. According to the current opinion, patients diagnosed with idiopathic hypercalciuria should be switched to the normocalcemic diet [43]. On the other hand, patients with low bone mass are recommended a high-calcium diet, which, as previously shown, may promote the greater activity of urolithiasis. In the present work, we showed that the supplementation with low doses of vitamin D for 24 months together with the normocalcium diet is insufficient to improve bone mass. The lack of improvement in bone mineral density parameters is probably related to too low doses of vitamin D or too short observation period. One of the most important conclusions from our work is that the administration of the 400 or 800 IU/day of vitamin D seems to be safe as it did not affect the severity of urolithiasis activity.

#### **5. Limitation**

We are aware, however, that our study had some limitation: 1. the size of the studied group of children was small, 2. we had not full control of the diet or 3. regular administration of vitamin D. In our opinion the study of a larger population of children with supplementation with higher doses of vitamin D should be performed. In other limitations, we assumed that a three-month biochemical evaluations of patients were sufficient to conclude that patients followed with the recommendations.

#### **6. Conclusions**

(1). Supplementation with low doses of vitamin D (400–800 IU/day) in children with idiopathic hypercalciuria significantly increases the concentration of 25OHD vitamin D in the serum and does not affect the level of calciuria. It does not increase the dynamics of stones formation in the urinary tract, but also does not improve bone density.

(2). The use of vitamin D preparations in these patients is safe, without a significant influence on the severity of disease activity.

(3). Children with idiopathic hypercalciuria should be advised to carefully monitor the parameters of calcium metabolism and the level of urolithiasis activity without giving up vitamin D supplementation.

**Author Contributions:** Conceptualization, J.M. and K.J.; methodology, A.W., K.J.; validation, J.M., A.L. and K.J.; formal analysis, M.M.-Ł., A.L.; investigation, J.M., K.J., A.W., B.K.; resources, M.M.-Ł.; data curation J.M., B.K.; writing—original draft preparation, J.M., A.L. and A.W.; writing—review and editing, A.L., K.J. and M.M.-Ł.; visualization, J.M.; supervision, K.J., B.K.; project administration, J.M.; funding acquisition, B.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was supported by the statutory grant of the Military Institute of Medicine, Warsaw, Poland—project no. 293 (1/8841).

**Conflicts of Interest:** The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
