Participants were included in the study consecutively from among the patients after considering inclusion and exclusion criteria to exclude selection bias. The flowchart of the patients included in the study group is presented in Figure 1.

**Figure 1.** Flowchart of the patients' recruitment (CKD—chronic kidney disease, G—Grade, rhGH recombinant human growth hormone).

#### *2.2. Ethical Issues*

All procedures were in accordance with the ethical standards of the institutional review board (approval no. KB/89/2013) and with the 1964 Helsinki declaration and its later amendments. Informed consent was obtained from all legal representatives and individuals (≥16 years).

#### *2.3. Clinical Parameters*

The following clinical data were collected: age (years), gender, CKD etiology [14], and stage [13] based on estimated glomerular filtration rate (GFR) [15], method of renal replacement therapy, body mass (kg), height [m] and body mass index (BMI) (kg/m2), Z-score [16], presence of arterial hypertension, and medications used.

#### *2.4. NT-proBNP and Biochemical Parameters*

Concentration of NT-proBNP (pg/mL) was determined in serum using the VITROS 5600 Integrated System (Ortho Clinical Diagnostics, Raritan, NJ, USA) with the upper limit taken from the manufacturer's normative values (125 (pg/mL)). The following biochemical parameters were evaluated: creatinine (mg/dL), urea (mg/dL), uric acid (mg/dL), hemoglobin (g/dL), albumins (g/dL), calcium (mg/dL), inorganic phosphate (mg/dL), alkaline phosphatase (IU/mL), intact parathormone (pg/mL), 25-hydroxy-vitamin D (25(OH)D) (ng/mL), total, low-density (LDL) and high-density lipoprotein (HDL) cholesterol (mg/dL), triglycerides (mg/dL), and parameters of acid base balance from arterialized capillary blood: pH, and HCO3 − (mmol/L). All biochemical parameters were measured in the morning, on fasting, simultaneously. Normal values of hemoglobin and calcium– phosphorus metabolism parameters were taken from the Kidney Disease: Improving Global Outcome (KDIGO) guidelines [17,18], and the normal value of cholesterol and triglycerides from Stewart et al. [19]; hyperuricemia was recognized when uric acid was ≥5.5 (mg/dL) [20].

#### *2.5. Blood Pressure and Parameters of Cardiovascular Damage*

Peripheral office arterial blood pressure was measured with Welch Allyn VSM 300 Patient Monitor (Welch Allyn Inc., Skaneateles Falls, NY, USA) and expressed in (mmHg) and Z-score values [21]. Common carotid artery intima-media thickness (cIMT) was evaluated with a 13-MHz linear transducer (Aloka Prosound Alpha 6, Hitachi Aloka Medical, Mitaka, Japan), using methods described previously [22] and expressed in (mm) and Z-score [23]. Central blood pressure, arterial pulse waveform, and aortal pulse wave velocity (PWV) were assessed with SphygmoCor (AtCor Medical Pty Ltd., Sydney, Australia) using methods described in detail in our previous study [22]. The following parameters were analyzed: aortic (central) office systolic, diastolic, mean, and pulse pressure (AoSBP, AoDBP, AoMAP, AoPP (mm Hg)), augmentation pressure (AP = P2 − P1, where P2 is the amplitude of late, i.e., returning systolic peak pressure, and P1 is early systolic peak pressure (mm Hg)), augmentation index (AIx) expressed as AP divided by pulse pressure (AP/PP (%)), and P2/P1 ratio (%), as well as AIx (AP/PP) normalized to heart rate of 75 beats per minute (AIx75HR (%)), and aortic (carotid–femoral) pulse wave velocity. PWV was presented as (m/s) and (Z-score) based on normative pediatric data [24].

All children underwent transthoracic two-dimensional (2D), conventional Doppler, and tissue Doppler (TD) echocardiography (ECHO) with M-mode assessment of left ventricular parameters and simultaneous recording of ECG in the second limb lead with Philips iE33, transducer S5-1 (Philips, Amsterdam, The Netherlands). The following parameters were evaluated using a classical echocardiography and conventional Doppler technique. In the end-diastolic phase: the interventricular septum transverse diameter (IVSDd) (mm), left ventricular diastolic diameter (LVDd) (mm), left ventricular posterior wall diameter (LVPWd) (mm)), left atrial transverse diameter (LAD) (mm), relative wall thickness (RWT) calculated as 2 × LVPWd divided by LVDd, left ventricular mass calculated from the Deveraux equation, and left ventricular mass (LVMI) were indexed according to DeSimone [g/m2.7] [25], as well as shortening and ejection fraction (SF, EF) (%), peak wave velocity in early and late diastole caused by atrial contraction (the E and A waves) (cm/s), and E deceleration time (Edt) [s]. The TD was used to assess the mean value of peak medial and

lateral mitral annular velocity during early filling (E') (m/s), the mean value of peak medial and lateral mitral annular velocity during late filling (A') (m/s), E/E' ratio, isovolumetric relaxation time (IVRT) [s], isovolumetric contraction time (IVCT) [s], and a maximum speed of the systolic wave (C') (m/s). Left ventricular hypertrophy (LVH) was defined as LVMI ≥ 95c. for age and sex [26], abnormal RWT was defined as >0.42. Mildly reduced and reduced ejection fraction was defined as EF between 41% and 49% and EF ≤ 40%, respectively, according to the 2021 European Society of Cardiology guidelines [27].

#### *2.6. Statistical Analysis*

Statistica 13.0 PL software (TIBCO Software Inc., Palo Alto, CA, USA) was used for calculations. The normality of the distribution of the analyzed variables was assessed using the Shapiro–Wilk test. Normally distributed data were presented as mean ± standard deviation (SD) and non-normally distributed variables as median and interquartile range (Q1–Q3). Differences between data were tested using the U Mann–Whitney test. The relationship between two variables was analyzed using Pearson's linear correlation or Spearman's correlation rank, depending on the distribution. Multivariate analysis was performed using forward stepwise regression analysis. Parameters that correlated with arterial and heart damage markers with *p* < 0.100 in univariate analysis were included in the final model. Parameters that correlated with each other with r > 0.650 were excluded from regression models to avoid collinearity. Logarithmic transformation of non-normally distributed data was performed prior to the analysis. As NT-proBNP and cardiovascular complications of CKD are significantly correlated to GFR, the latter variable was forced into the final model. A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Clinical Characteristics*

Clinical characteristics of children included in the study are summarized in Table 1. Most of the subjects were in CKD grades 2 and 3, and congenital anomalies of the kidney and urinary tract (CAKUT) were the leading primary kidney pathology. Among seven patients in grade G5, six were chronically dialyzed: five were treated with peritoneal dialysis (PD), one was treated with hemodialysis (HD), and one child with eGFR of 13.45 mL/min/1.73 m2 did not receive renal replacement therapy yet. Arterial hypertension was present in 26 patients, usually treated with one antihypertensive drug, most commonly calcium channel blockers or angiotensin-converting enzyme inhibitors.

**Table 1.** Clinical and biochemical data of the studied children.

**Table 1.** *Cont.*

SD—standard deviation, Q1—the first quartile, Q3—the third quartile, CKD—chronic kidney disease, G—grade, CAKUT—congenital anomalies of kidney and urinary tract, BMI—body mass index. 1 number of patients.

#### *3.2. NT-proBNP and Biochemical Parameters*

The concentration of NT-pro BNP and remaining biochemical parameters are depicted in Table 2. The median value of NT-proBNP in patients with CKD was 95 (pg/mL) and varied from 15 up to 28,382 (pg/mL). NT-proBNP value above the norm (i.e., >125 pg/mL) was found in 16 (42.1%) children with CKD. NT-proBNP values did not differ significantly among children with CKD G2, G3, and G4. The highest values of NT-proBNP, significantly higher than children with CKD G2–G4, were found in children with CKD in stage G5 (Figure 2). In three of them, the NT-proBNP value exceeded 1000 (pg/mL). NT-proBNP at the concentration of 1579 (pg/mL) was detected in a 16.5-year-old boy with CKD and membranoproliferative glomerulonephritis, treated with hemodialysis. The boy had arterial hypertension treated with two drugs and left ventricular hypertrophy (LVH)—his LVMI was 40.2 g/m2.7. NT-proBNP at 5146 (pg/mL) was noted in a 14.5-year-old girl with CKD and steroid-resistant nephrotic syndrome treated with PD, with arterial hypertension treated with three drugs and LVH—her LVMI was 50.0 g/m2.7. The highest NT-proBNP concentration (28,382 (pg/mL)) was found in a 7.5-year-old female patient with unknown etiology of kidney disease, treated with PD, with arterial hypertension treated with two drugs, and LVH (LVMI—46.5 g/m2.7).

**Table 2.** NT-pro BNP and biochemical characteristics of the study group (NT-proBNP—N-terminal pro-brain natriuretic peptide).

SD—standard deviation, Q1—the first quartile, Q3—the third quartile, NT-proBNP—N-terminal pro-brain natriuretic peptide, GFR—glomerular filtration rate according to Schwartz formula, LDL—low-density lipoprotein, HDL—high-density lipoprotein, 25(OH)D—25-hydroxy-vitamin D, pH—power of hydrogen, HCO3 −—bicarbonate, BE—base excess. 1 *p* = 0.010, 2 *p* = 0.004, 3 *p* = 0.018.

**Figure 2.** NT-proBNP in children with chronic kidney disease (median value and range) (G—chronic kidney disease grade).

Relevant biochemical disturbances were found in the following numbers of CKD children—anemia in 13 (34%), hypercholesterolemia in 11 (29%), hypertriglyceridemia in 14 (37%), hypercalcemia in 1 (2.6%), hyperphosphatemia in 2 (5.2%), and elevated iPTH in 8 (21.1%) patients.

#### *3.3. Blood Pressure and Markers of Arterial and Heart Damage*

Blood pressure and markers of arterial and heart damage are shown in Table 3. At the time of the study, elevated office systolic blood pressure was found in 6 (15.8%) and elevated DBP in 4 (10.5%) children. Abnormal (i.e., ≥95c.) PWV was found in 1 (2.6%), and abnormal cIMT in 12 (31.6%) CKD children. LVH was found in 4 (10.5%) and abnormal RWT in none of the subjects. None of the children had mildly reduced or reduced EF.

**Table 3.** Blood pressure, arterial, and heart parameters in the study group.

SD—standard deviation, Q1—the first quartile, Q3—the third quartile, SBP—systolic blood pressure, DBP—diastolic blood pressure, MAP—mean arterial pressure, PP—pulse pressure, cIMT—common carotid artery intima media thickness, AP—augmentation pressure, P—peak pressure, Aix—augmentation index, AIx75HR—augmentation index normalized to heart rate 75 beats per minute, SEVR—subendocardial viability ratio, PWV—aortic pulse wave velocity, d—end-diastolic phase, IVS—interventricular septum transverse diameter, LVD—left ventricular diastolic diameter, LVPW—left ventricular posterior wall diameter, LAD—left atrial transverse diameter, RWT—relative wall thickness, LVM—left ventricular mass, LVMI—left ventricular mass index, SF—shortening fraction, EF—ejection fraction, E—peak wave velocity in early diastole, A—peak wave velocity in late diastole caused by atrial contraction, Edt—E deceleration time, E'—mean value of peak medial and lateral mitral annular velocity during early filling, A'—mean value of peak medial and lateral mitral annular velocity during late filling, IVRT—isovolumetric relaxation time, IVCT—isovolumetric contraction time, C'—maximum speed of systolic wave.

#### *3.4. Correlations of NT-proBNP and Markers of Arterial and Heart Damage*

Significant correlations of NT-proBNP are depicted in Table 4. In CKD children, NT-proBNP correlated positively with markers of arterial damage: AP/PP, P2/P1, cIMT Z-score, and with the marker of diastolic dysfunction—E/E'. NT-proBNP concentration correlated negatively with the alfacalcidol dose and GFR. No significant relations were

found among NT-proBNP and blood pressure, PWV ((m/s) and Z-score), AIx75HR, LVMI, RWT, SF, EF, and E/A.

**Table 4.** Significant correlations of NT-proBNP with analyzed clinical, biochemical, and cardiovascular parameters in children with CKD (Spearman's rank correlations).

GFR—glomerular filtration rate, AP—augmentation pressure, PP—pulse pressure, Aix—augmentation index, P—peak pressure, cIMT—carotid intima-media thickness, E—peak wave velocity in early diastole, E'—mean value of peak medial and lateral mitral annular velocity during early filling by tissue Doppler.

PWV correlated significantly with peripheral and central DBP (r = 0.417, *p* = 0.034 and r = 0.406, *p* = 0.04, respectively), LVPWd (r = 0.506, *p* = 0.010), LVM (r = 0.482, *p* = 0.015), and A' (r = 0.467, *p* = 0.038); PWV Z-score correlated with heart rate (r = 0.519, *p* = 0.013), A (r = 0.600, *p* = 0.011), and E/A (r = −0.578, *p* = 0.015); there was also trend towards a positive correlation between PWV Z-score and triglycerides (r = 0.404, *p* = 0.503); AP/PP correlated positively with PTH (r = 0.383, *p* = 0.048), cIMT Z-score (r = 0.533, *p* = 0.016), and A (r = 0.495, *p* = 0.031); P2/P1 correlated positively with calcium (r = 0.489, *p* = 0.010), alkaline phosphatase (r = 0.452, *p* = 0.020), PTH (r = 0.423, *p* = 0.028), cIMT Z-score (r = 0.510, *p* = 0.022), A (r = 0.616, *p* = 0.005), and negatively with E/A (r = −0.511, *p* = 0.026); cIMT correlated also positively with triglycerides (r = 0.461, *p* = 0.016) and negatively with calcium (r = −0.392, *p* = 0.043); cIMT Z-score correlated positively with triglycerides (r = 0.546, *p* = 0.011) and with RWT (r = 0.573, *p* = 0.008), negatively with E' (r = −0.543, *p* = 0.030) and C' (r = −0.733, *p* = 0.001).

LVMI correlated positively with triglycerides (r = 0.377, *p* = 0.030) and uric acid (r = 0.370, *p* = 0.031); RWT with C' (r = −0.466, *p* = 0.011); E/A with calcium (r = −0.38, *p* = 0.043), alkaline phosphatase (r = −0.623, *p* < 0.001), and triglycerides (r = −0.450, *p* = 0.016); A' with number of antihypertensive medications (r = 0.508, *p* = 0.013); E/E' with calcium (r = 0.466, *p* = 0.011); and C' correlated positively with hemoglobin (r = 0.413, *p* = 0.026), and negatively with AP/PP (r = −0.455, *p* = 0.044) and P2/P1 (r = −0.489, *p* = 0.029).

The correlations of the analyzed parameters with age are presented in Supplementary Materials Tables S1 and S2. In the studied children, there was no significant association between age and NT-proBNP (r = −0.166, *p* = 0.320). Age correlated negatively with serum calcium, inorganic phosphate, alkaline phosphatase, and pH (r = −0.336, *p* = 0.039; r = −0.397, *p* = 0.014; r = −0.590, *p* = 0.001, and r = −0.459, *p* = 0.001, respectively), and positively with both peripheral and central systolic and diastolic blood pressures expressed in (mm Hg) (r = 0.388–0.493, *p* = 0.046–0.009); no significant associations were found between age and blood pressure Z-scores. Age correlated also positively with PWV (m/s) (r = 0.490, *p* = 0.011), but not with PWV Z-score (r = 0.090, *p* = 0.677), and negatively with augmentation indices (r = −0.396–−0.521, *p* = 0.040–0.005). Moreover, numerous cardiac dimensions (IVSDd, LVDd, LVPWd, LAD) correlated positively with age (r = 0.459–0.727, *p* = 0.012–< 0.001); LV mass was positively related to age, too (r = 0.703, *p* < 0.001). In addition, age correlated negatively with A and positively with E/A and E' (r = −0.393, *p* = 0.035; r = 0.464, *p* = 0.011 and r = 0.438, *p* = 0.017, respectively).

In the multivariate analysis, logNT-proBNP was the only significant predictor of the cIMT Z-score (beta = 0.402, 95CI (0.082–0.721), *p* = 0.014), and P2/P1 (beta = 0.130, 95CI (0.082–0.721), *p* = 0.014).

#### **4. Discussion**

Chronic kidney disease and cardiovascular disease are conditions that inter-influence. In CKD patients, a gradual decline in GFR leads to overhydration and accumulation of uremic toxins. Besides fluid overload, CKD patients are exposed to numerous other traditional (hyperlipidemia, volume-independent arterial hypertension) and non-traditional, i.e., "uremia-specific" risk factors, such as malnutrition, calcium–phosphorus disturbances, anemia, and hyperhomocysteinemia. Together, they contribute to cardiovascular damage and significant shortening of estimated lifespan [1,28]. Thus, it is crucial to establish the individual CVD risk to stratify patients to particular risk groups, diagnose the disease early, improve the treatment process, and initiate cardio- and renoprotective measures. NT-proBNP is one possible biomarker of increased cardiovascular risk.

In our cohort of 38 children, we observed an abnormally elevated value of NT-proBNP in almost half of the individuals. As there is no final consensus on normal pediatric NT-proBNP values, we used the manufacturer's range. Nir and Lam proposed slightly higher normal values of the marker in the pediatric population, but they used a different kit [29,30]. NT-proBNP accumulates during CKD because of impaired renal clearance [31,32]. In our study group, NT-proBNP correlated negatively with GFR, and a gradual increase in NT-proBNP following CKD grades was found. A high concentration of NT-proBNP may contribute to cardiac strain in CKD, indicating vascular system overload. NT-proBNP provided essential prognostic and diagnostic information on fluid overload and cardiovascular damage in adults with CKD [9,33,34], despite its strong relation to kidney function. It was proven that elevated NT-proBNP concentration is correlated twofold with mortality risk [33]. NT-proBNP level indicating increased CVD risk in CKD population seems to be substantially higher in comparison to healthy people [9].

There are limited data on the usefulness of NT-proBNP as a marker of cardiovascular damage in pediatric CKD patients. A positive correlation between NT-proBNP concentration and E/A, left atrial diameter, and left ventricle hypertrophy (LVH) was reported in small pediatric CKD cohorts [11,12]. We have observed a positive correlation between NT-proBNP and the degree of diastolic dysfunction measured by tissue Doppler echocardiography. Similarly, Kim et al. outlined the correlation between NT-proBNP and E/E' in adults, suggesting that NT-proBNP might be an early marker of diastolic dysfunction in CKD patients [34]. No correlation among LV mass, LV ejection fraction, and NT-proBNP was found in our children. We hypothesize that this might be a derivative of a relatively good kidney function (66% of the studied subjects were in CKD grade G2 or G3) and a low prevalence of LV hypertrophy in the analyzed cohort. Of note, none of the patients had even mildly reduced left ventricular ejection fraction. In turn, mild heart damage in our cohort could be explained by a low grade of renal impairment and good control of arterial hypertension. This is a significant difference compared to studies in adult patients with CKD and might explain the failure to demonstrate a statistically significant relationship between left ventricular mass, systolic function, and NT-proBNP in the studied children.

Arterial damage is the earliest indicator of cardiovascular disease in CKD children. Unique, uremia-related biochemical milieu leads to Mönckeberg's arteriosclerosis characterized by intramural calcium–phosphorus deposition, the osteoblast-like transformation of fibroblast, and a high risk of stroke or myocardial infarction [35]. We found numerous correlations among arterial damage and heart dimensions and function parameters, suggesting a strict interplay between arterial and cardiac dysfunctions in these patients.

We have found positive correlations among NT-proBNP, vascular stiffness indicators (AP/PP, P2/P1), and cIMT. NT-proBNP concentrations (expressed as decimal logarithms) were also significant predictors of cIMT and P2/P1 in multivariate analysis. While cIMT is a well-established marker of cardiovascular disease, P2/P1 and its derivative—the augmentation index shows a weaker correlation with hard-end points than the gold standard of arterial stiffness—aortic pulse wave velocity [36,37]. Little is known about pathophysiological relations between intimal and medial thickening and the heart. Sasaki found no significant associations between cIMT or the presence of atherosclerotic plaques and NT-proBNP level [38]. On the other hand, Asian authors found a positive correlation between cIMT and the concentration of this biomarker in adults with CKD [10,39,40].

NT-proBNP influences adipocyte function and was found to be negatively related to total and LDL-cholesterol concentrations [41,42]. These data suggest that NT-proBNP may have protective actions against arteriosclerosis and atherosclerotic plaque formation. It is possible that this compensatory mechanism is ineffective in CKD despite NT-proBNP accumulation. Our results suggest that NT-proBNP might serve in CKD pediatric patients as a valuable tool assessing the risk of arterial damage. Because of the cross-sectional study design, a causal relationship between cIMT thickening and NT-proBNP rise cannot be established. There is a need for prospective studies to establish its position as a biomarker of cIMT in this and other high-risk pediatric populations.

Numerous associations between NT-proBNP and calcium–phosphate metabolism parameters were observed in our study group, suggesting cardiovascular damage induced by these metabolic derangements. Similar relations were revealed in research conducted among pediatric CKD G3–G5 patients by Rinat [11]. In both adult [43] and pediatric [11,12] studies, NT-proBNP correlated positively with blood pressure. Despite evaluation of both peripheral and central blood pressure, no such relation was revealed in our cohort. We think that relatively mild kidney impairment and the common use of antihypertensive medications could mask this relationship. Furthermore, we evaluated blood pressure based on individual office measurements, which could be a source of potential bias.

Moreover, one should remember that, according to literature data, other factors may influence NT-proBNP levels, such as anemia, BMI (especially obesity), and gender [44–46]. None of these variables influenced NT-proBNP in our cohort, except for BMI. Nevertheless, this marker ought to be carefully interpreted in CKD patients considering factors that might affect it.

In our cohort of CKD patients, we revealed numerous significant correlations among biochemical parameters, heart dimensions, arterial stiffness parameters, and age. Of note, there was no significant association between age and NT-proBNP. A negative association between age and parameters of calcium–phosphorus metabolism reflects normal bone metabolism, varying with age, observed in both healthy [47] and CKD children [48]. As age and body size are crucial determinants of blood pressure and cardiac dimensions, proper indexation and comparison of the measured value with population-based norms is necessary in pediatric patients [21,26]. Noteworthy, age-normalized blood pressure and left ventricular mass index did not show any significant correlations with age.

Progressive stiffening of the arteries (measured as aortic PWV) is a well-known phenomenon, responsible, e.g., for isolated systolic hypertension in the elderly [49]. Agedependent increase in PWV is seen already in the first two decades of life and was confirmed in large cohorts of healthy children [24,50]. On the other hand, the inverse relationship among age, body dimensions, and augmentation index was observed in the general population, similar to our cohort. In younger (and therefore shorter) patients, the pulse wave reflected from the peripheral arteries reaches back to the aorta more quickly due to its shorter pathway, resulting in an increase in the augmentation index in the youngest children, as revealed by Hidvegi et al. [51].

Some limitations of our study may be identified. We reported only a single-center study with a limited number of CKD patients, and broader research should be carried out, including a comparison of the NT-proBNP level in a control group. In addition, the vast majority of the subjects were in CKD G2 and G3 with minor biochemical and cardiovascular abnormalities, which might have influenced the number of NT-proBNP correlations. Finally, the study's cross-sectional nature precludes drawing final casual relationships between NT-proBNP and the measured parameters.

#### **5. Conclusions**

Our cross-sectional analysis revealed numerous correlations between NT-proBNP and arterial and heart damage indices in children with chronic kidney disease. As NT-proBNP was a significant determinant of cIMT and P2/P1 in the multivariate analysis, we conclude that NT-proBNP may serve as a possible marker of thickening of the carotid artery wall in pediatric patients with kidney function impairment. NT-proBNP could be used in everyday clinical practice to assess cardiovascular risk in these subjects as evaluation of its serum concentration is easy accessible, relatively cheap, and repeatable. Conversely, due to our study's limitations, the final role of NT-proBNP as a biomarker of arterial damage, left ventricular hypertrophy or diastolic cardiac dysfunction in children with CKD needs confirmation in prospective studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jcm10194344/s1, Table S1: Correlations of age with NT-proBNP, clinical, and biochemical parameters in the study group. Table S2: Correlations of age with blood pressure, arterial, and heart parameters in the study group.

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

**Funding:** This research was funded from the statutory funds of the Department of Pediatrics and Nephrology, Medical University of Warsaw.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Local Bioethics Committee of the Medical University of Warsaw (approval no. KB/89/2013).

**Informed Consent Statement:** Informed consent was obtained from all subjects (≥16 years) and their representatives involved in the study.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Article* **Urinary Levels of Cathepsin B in Preterm Newborns**

**Monika Kamianowska 1,\*, Marek Szczepa ´nski 1, Anna Krukowska 1, Aleksandra Kamianowska 2 and Anna Wasilewska 2**

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +48-85-746-84-98; Fax: +48-85-746-86-63

**Abstract:** Increased investment in perinatal health in developing countries has improved the survival of preterm newborns, but their significant multiorgan immaturity is associated with short and longterm adverse consequences. Cathepsin B, as a protease with angiogenic properties, may be related to the process of nephrogenesis. A total of 88 neonates (60 premature children, 28 healthy term children) were included in this prospective study. We collected urine samples on the first or second day of life. In order to determine the concentration of cathepsin B in the urine, the commercially available enzyme immunoassay was used. The urinary concentrations of cathepsin B normalized with the urinary concentrations of creatinine (cathepsin B/Cr.) in newborns born at 30–34, 35–36, and 37–41 (the control group) weeks of pregnancy were (median, Q1–Q3) 4.00 (2.82–5.12), 3.07 (1.95–3.90), and 2.51 (2.00–3.48) ng/mg Cr, respectively. Statistically significant differences were found between the group of newborns born at 30–34 weeks of pregnancy and the control group (*p* < 0.01), and between early and late preterm babies (PTB) (*p* < 0.05). The group of children born at 35–36 weeks of pregnancy and the control group did not differ significantly. This result suggests that the elevated urinary cathepsin B/Cr. level may be the result of the kidneys' immaturity in preterm newborns.

**Keywords:** cathepsin B; tubular damage; premature neonates; immaturity

#### **1. Introduction**

Preterm birth (before 37 completed weeks of gestation) accounts for 11% of births worldwide [1]. Increased investment in perinatal health in developing countries and interventions such as antenatal steroids have improved survival in this group of children, but significant multiorgan immaturity is associated with short and long-term adverse consequences [2]. The first kidney glomeruli form at 9–10 weeks of gestation [3]. During the late second and third trimester, there is an exponential increase in the number of nephrons between 18 and 32 weeks [4,5]. Nephrogenesis in humans ends by approximately 34–36 weeks of gestation, with over 60% of nephrons being formed during the last trimester [6]. Hence, in premature neonates, normal nephrogenesis is interrupted, and both nephron number and kidney size are reduced [7]. While nephrogenesis may continue in premature neonates for up to 40 days following birth, these nephrons are not normal and age at an increased rate [8]. However, despite this postnatal development of the kidneys, premature children are still left with a lower number of nephrons. For example, a premature neonate born at 26 weeks of gestation, despite 40 additional days of nephrogenesis, will only have nephron development until 32 weeks as opposed to continuing nephrogenesis to 36 weeks in term gestation [9,10]. Kidney injury from hypoperfusion and drug nephrotoxicity leads to further frequently unnoticed changes in kidney structure and function. Thus, the glomerular and tubular maturation of the kidneys of preterm newborns may be confounded by the nephropathy of prematurity and acute kidney injury (AKI), whose incidence in neonates is estimated at 8–24% of children hospitalised in the Intensive Neonatal Care Units. One-third of this group are premature babies [7,11–13].

**Citation:** Kamianowska, M.; Szczepa ´nski, M.; Krukowska, A.; Kamianowska, A.; Wasilewska, A. Urinary Levels of Cathepsin B in Preterm Newborns. *J. Clin. Med.* **2021**, *10*, 4254. https://doi.org/10.3390/ jcm10184254

Academic Editor: Andreas Skolarikos

Received: 23 July 2021 Accepted: 14 September 2021 Published: 19 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Cathepsin B is a lysosomal cysteine protease synthesized on the rough endoplasmic reticulum as a pre-proenzyme; cathepsin B in its mature, double-chain form comprises a heavy chain and a light chain [14–17]. Cathepsin B is a protein belonging to hydrolases and is involved in the processing of hormones and proteins, regulation of the cell cycle, autophagy, and cell death [18,19]. In kidneys, cathepsin B is found in proximal tubule cells. It is involved in the digestion of proteins reabsorbed from the tubular fluid following glomerular filtration. A small amount of this protease can be detected in urine under physiological conditions. Its levels increase because of tubular damage or renal dysfunction [18,20]. During pregnancy, cathepsin B is predominantly found in placental and decidual macrophages, which may be important in the mediation of villous angiogenesis and decidual apoptosis [21]. With tumours, the expression of cathepsin B correlates with angiogenesis and is thought to promote the remodeling of the extracellular matrix to permit neovascularization [22,23].

This study assessed whether cathepsin B may be involved in the maturation of the foetus' kidneys by evaluating the effect of prematurity on the concentration of cathepsin B in the urine of preterm newborns.

#### **2. Patients and Methods**

#### *2.1. Patient Recruitment and Sample Collection*

A total of 88 neonates were included in this prospective study. Sixty of them were born prematurely at 30–36 weeks of pregnancy. The neonates were hospitalized in the Department of Neonatology and Intensive Neonatal Care at the Medical University of Bialystok, Poland between December 2017 and December 2018. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Bioethics Committee of The Medical University of Bialystok (protocol code: R-I-002/360/2016, date of approval: 17 October 2016). Prior to the study written informed consent has been obtained from the parents of all the neonates. The clinical condition of the neonates was good or average; they were appropriate for gestational age (AGA), with the weight between the 10th and 90th centile of birth weight for their gestational age using normalized growth curves [24]. The group of premature babies born at 30–36 weeks of pregnancy were divided into two subgroups: 28 children born between 30 and 34 weeks of pregnancy (hospitalized in the ward for preterm babies) and 32 children born between 35 and 36 weeks of pregnancy (hospitalized in a rooming-in ward). The reference group comprised 28 healthy babies. These neonates developed through normal pregnancies, with no prenatal and perinatal complications.

The inclusion criteria for this study were normal prenatal and postnatal ultrasound examination results of the kidneys, and a good or average clinical condition. The exclusion criteria were: 1-min Apgar scores < 4; congenital abnormalities, including urinary tract defects (polycystic kidney disease, hydronephrosis, duplex kidneys/ureters, renal agenesis, or other anatomical abnormalities); inborn error of metabolism; heart disease; abnormal ultrasound examination of the kidneys and the central nervous system (hyper-echoic zones around the lateral ventricles of the brain and intraventricular hemorrhage grade I and II were accepted); abnormal laboratory tests (including elevated levels of inflammatory markers); treatment with catecholamines, antibiotics, diuretics, or mechanical ventilation. Additionally, the children of mothers with a burdened medical history were excluded from the study.

Collection of urine samples in the study group was performed using single-use sterile bags (Medres, Zabrze, Poland). Urine was collected once on the first or second day of life. The urine samples obtained after centrifugation were kept in the refrigerator (4 ◦C) for no longer than 2 h and then frozen at −80 ◦C. Repeated freeze–thaw cycles were not used. Collection of the blood samples was conducted during the first or second day of the neonate's life during routine practice in the Unit. S-Monovette 1.2 mL, Clotting Activator/Serum test tubes (Sarstedt AG & Co., Nümbrecht, Germany) for venous blood

sampling were used. A blood cell morphology test and blood biochemistry tests were performed right after taking the blood samples.

#### *2.2. Determination of Urinary Cathepsin*

The levels of urinary cathepsin B were determined using a commercially available ELISA kit (Cloud–Clone Corp., Katy, TX, USA), according to manufacturer's instructions, and were expressed in nanograms per milliliter. The detection range was 0.312–20 ng/mL, according to specifications of the kit. The mean intra-assay and inter-assay coefficients of variation (CV) for cathepsin B were <10% and <12%, respectively.

In order to eliminate the potential confounding effect of urinary dilution, we normalized the urinary cathepsin B concentrations for the urinary concentration of creatinine; this was expressed in nanograms per milligram of creatinine (ng/mg Cr). The urinary concentration of creatinine was determined using Jaffé's method.

Calculation of estimated GFR was performed using the Schwartz formula (for the preterm babies, eGFR = 0.33 × L/Scr.; for term babies, eGFR = 0.45 × L/Scr., where L is the length in centimeters and Scr. is serum creatinine in milligrams per deciliter).

The determination of urinary cathepsin B was performed in the Department's Laboratory of Pediatrics and Nephrology, at the Medical University of Bialystok. The morphology and serum biochemistry tests were performed in the Department of Laboratory Diagnostics at the University Clinical Hospital in Bialystok.

#### *2.3. Statistical Analysis*

The statistical analysis was completed using Statistica 13.3 package (StatSoft, Cracow, Poland). It expressed discrete variables as counts (percentage), continuous variables as median and quartiles (Q1–Q3). In order to determine normal distribution, the Shapiro– Wilk test was used. The Mann–Whitney U test was used for intergroup comparisons of continuous variables because the data were not normally distributed. To establish the direction and power of association between urinary cathepsin B concentrations and other variables, Spearman's rank correlation coefficients were used. The results were significant at *p* < 0.05.

#### **3. Results**

Eighty-eight neonates were included in the study. Twenty-eight of them were healthy neonates, and sixty were born prematurely at 30–36 weeks of pregnancy. Both groups were sex-matched (*p* > 0.05). Birth weight, length, and head and chest circuits were significantly lower in premature neonates than in term children; however, all the children were appropriate for gestational age. Table 1 presents the characteristics of premature babies.

In both examined groups of premature babies (born at 30–34 and at 35–36 weeks of pregnancy), there were no statistically significant differences in the type of delivery (vaginal delivery or caesarean delivery). Both groups were sex-matched (*p* > 0.05). Birth weight was statistically significantly lower in neonates born at 30–34 weeks of pregnancy than in babies born at 35–36 weeks of pregnancy. All premature babies were appropriate for gestational age, and when we divided both groups of preterm neonates according to the percentile of birth weight (10–50 percentile and 51–90 percentile), they were matched according to their percentile of birth weight. Both 5-min and 10-min Apgar scores were ≥8 in all neonates, and lower 1-min and 3-min Apgar scores characterized younger children.

**Table 1.** Characteristics of premature babies.

*p*—comparison of children born at 30–34 weeks and 35–36 weeks of pregnancy; NS—non statistical; nCPAP—nasal continuous positive airway pressure.

In both groups of preterm neonates, blood morphology and biochemical tests were normal. Statistically significantly higher number of leucocytes, urea, and alanine aminotransferase concentrations were found in younger children (Table 2).

**Table 2.** Basic laboratory results of premature neonates.

*p*—comparison of children born at 30–34 weeks and 35–36 weeks; NS–non statistical.

All neonates had normal renal function parameters (serum and urinary concentration of creatinine, estimated GFR, and urine output). The serum concentration of creatinine did not show a statistically significant difference between the groups. The serum concentration of creatinine was higher in neonates born by cesarean delivery when compared to children born by vaginal delivery. The difference was close to being statistically significant (*p* = 0.053). Urinary creatinine was significantly lower in premature babies when compared to the reference group (*p* < 0.01). The lowest concentration of urinary creatinine was found in children born at 30–34 weeks. Similarly, eGFR was significantly lower in premature children compared to healthy controls (*p* < 0.01); however, we did not notice any difference in eGFR between children born at 30–34 weeks and 35–36 weeks (*p* > 0.05) (Table 3).

**Table 3.** Examined parameters in premature neonates and the reference group—median values and interquartile range (IQR).

*p*1—comparison of children born at 30–36 weeks and the reference group; *p*2—comparison of children born at 30–34 weeks and the reference group; *p*3—comparison of children born at 35–36 weeks and the reference group; *p*4—comparison of children born at 30–34 weeks and 35–36 weeks. Cr.—creatinine; eGFR—estimated glomerular filtration rate; NS—non statistical.

> In the entire group of premature neonates, the urinary level of cathepsin B/Cr. was higher compared to the reference group (*p* < 0.05). The highest urinary excretion of cathepsin B/Cr. was found in babies born at 30–34 weeks of pregnancy, and the difference was statistically significant when compared to the reference group (*p* < 0.01) and when compared to babies born at 35–36 weeks.(*p* < 0.05). However, no statistically significant difference in the urinary level of cathepsin B/Cr. was observed between children born at 35–36 weeks of pregnancy and the reference group.

> The analysis did not show any relationship between the concentration of urinary cathepsin B/Cr. and the way of delivery (vaginal delivery or caesarean delivery), Apgar score, prenatal steroid therapy, use of parenteral nutrition, use of nCPAP, or oxygen therapy. No statistically significant differences in the concentrations of urinary cathepsin B/Cr. were found between the groups of boys and girls.

#### **4. Discussion**

Kidney immaturity as a consequence of preterm delivery and perinatal problems connected to prematurity such as respiratory and circulatory failure, asphyxia, and antibiotic therapy are risk factors of renal tubular injury in premature neonates. The aim of this prospective study was to determine the values of urinary cathepsin B in premature neonates. It was hypothesized that cathepsin B may be involved in the processes of kidney maturation in the foetus.

Normally, cathepsin B is highly expressed in the proximal tubule. It is the main enzyme involved in the lysosomal digestion of proteins, which are reabsorbed via endocytosis from the glomerular ultrafiltrate. This protease has long been taken under consideration as a biomarker of tubular damage because in response to damages, its levels are lower in tubules and higher in urine [18]. It has recently been shown that the excessive reabsorption of ultrafiltered proteins by proximal tubular cells induces tubular damage and apoptosis/necrosis through the exhaustion of the lysosomal degradation pathway and the leakage of lysosomal enzymes such as cathepsin B into the cytoplasm and urine [20]. The prominent cytoplasmic release of cathepsin B in tubular epithelial cells is crucial for tubular cell injury and the activation of a cytoplasmic macromolecular complex involved in the progression of kidney diseases [25]. Studies conducted on animals showed that cathepsin B is involved in kidney diseases [26–29]. Wang et al. showed that in human proximal tubular epithelial cells undergoing apoptosis, expression levels and the activation of cathepsin B are increased, and that the serum cathepsin B level was associated with aging-related cardiovascularrenal parameters even in healthy people [30–32]. In the study conducted by Piwowar et al., cathepsin B activity increased significantly (*p* < 0.001) in the urine of diabetic patients as compared to the control group, and they concluded that it may be useful as a non-invasive surrogate marker of incipient nephropathy [33].

Knowing that many factors can contribute to tubular damage, we identified a group of sixty children with birth weights appropriate for the gestational age and clinical conditions assessed as good or medium. The children included in this study did not need drugs and mechanical ventilation. Their prenatal and postnatal ultrasound examination of kidneys was normal, and no deviations in laboratory tests and in parameters of renal function were found.

The significantly highest urinary level of cathepsin B/Cr. was observed in neonates born at 30–34 weeks of pregnancy. The lack of statistically significant differences between the urinary level of cathepsin B/Cr. in children born at 35–36 weeks of pregnancy and the reference group was caused by the fact that these neonates were born close to the term of delivery. Thus, it may be supposed that the stages of development of the kidneys in both groups were similar. The significantly highest urinary level of cathepsin B/Cr. was found in neonates born at 30–34 weeks. This may have resulted from the fact that cathepsin B may take part in the process of nephrogenesis as a protease with angiogenic properties [21–23,34].

Aisa et al. studied cathepsin B activity in the urine of neonates with intrauterine growth-restricted (IUGR) (median gestational age—36 weeks) and preterm neonates (median gestational age—35 weeks), both at 30–40 days of the corrected age. They found that cathepsin B activity in the urine was statistically significantly increased in preterm children, and even more increased in neonates with IUGR, when compared to the control group (median, (Q1–Q3)): 2.303, (1.7–2.582); 3.633, (2.146–4.848); 1.044, (0.8335–1.372) IU/min mmol, respectively. Taking into consideration significantly lower total renal volume, cortical volume, and observed proteinuria in the IUGR and preterm neonates, they suggested that cathepsin B activity may be useful in the early prediction of renal susceptibility to damage in this group of children [35].

In order to determine if other factors besides immaturity could affect the renal tubules' functions, the relationship of the concentration of urinary cathepsin B and urinary cathepsin B/Cr. with gender, way of delivery, birth weight, centile of birth weight, Apgar score, prenatal steroid therapy, respiratory disorders (use of nCPAP, oxygen therapy), and the use of parenteral nutrition was examined. However, no correlations were found between them. This result suggests that elevated urinary cathepsin B/Cr. level may be the result of the kidneys' immaturity in preterm newborns.

The main limitation for this study was the sample size of recruited children, which was quite small, as it was very difficult to get study consent from some parents.

#### **5. Conclusions**

In conclusion, the results showed that preterm neonates born at 30–34 weeks of gestation had elevated urinary cathepsin B/Cr. levels, which may be a result of the immaturity of kidneys. This preliminary observation should be confirmed in a multicenter study, which would consider not only preterm neonates with birth weights appropriate for gestational age and clinical conditions assessed as good or medium, but also those with clinical conditions assessed as bad, with or without AKI. Certainly, extending the study to the following weeks of life of the studied children, performing them both during hospitalization and after discharge from the hospital, would provide valuable information, confirming the role of cathepsin B as a biomarker of kidney maturity.

**Author Contributions:** M.K.—had primary responsibility for protocol development, patient screening, enrolment, outcome assessment, preliminary data analysis, and the writing of the manuscript. M.S.—took part in the protocol's development and contributed to the writing of the manuscript. A.K. (Anna Krukowska)—had primary responsibility for collecting a specimen. A.K. (Aleksandra Kamianowska)—took part in outcome assessment and the writing of the manuscript. A.W.—took part in the protocol's development and the analytical framework for the study, and contributed to the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The grant of the Medical University of Bialystok funded this study.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Bioethics Committee of The Medical University of Bialystok (protocol code: R-I-002/360/2016, date of approval: 17 October 2016).

**Informed Consent Statement:** Prior to the study written informed consent has been obtained from the parents of all the neonates to publish this paper.

**Acknowledgments:** We thank the employees of the Laboratory of the Department of Pediatrics and Nephrology, Medical University of Bialystok, Poland for participating in the study.

**Conflicts of Interest:** The authors have no conflict of interest to declare.

#### **References**

## *Article* **Alpha-1 Acid Glycoprotein and Podocin mRNA as Novel Biomarkers for Early Glomerular Injury in Obese Children**

**Anna Medy ´nska 1,\*, Joanna Chrzanowska 2, Katarzyna Ko´scielska-Kasprzak 3, Dorota Bartoszek 3, Marcelina Zabi ´ ˙ nska 3 and Danuta Zwoli ´nska 1**

**Abstract:** Introduction: Obesity, which is a serious problem in children, has a negative impact on many organs, including kidneys, and obesity-related glomerulopathy (ORG) is an increasingly common cause of ESKD (end-stage kidney disease) in adults. Early-detected and -treated glomerular lesions are reversible, so it is important to find a useful marker of early damage. The study aimed to evaluate the albumin-to-creatinine ratio (ACR), urinary alpha-1-acid glycoprotein (α1-AGP), and mRNA of podocyte-specific proteins as indicators of glomerular injury and their relationship with the degree of obesity and metabolic disorders. Materials and Methods: A total of 125 obese children and 33 healthy peers were enrolled. Patients were divided into two groups, depending on SDS BMI values. ACR, α1-AGP, mRNA expression of nephrin, synaptopodin, podocin, and C2AP protein in urine sediment were measured. Results: ACR values did not differ between groups and were within the normal range. α1-AGP and mRNA expression were significantly higher in obese children compared with controls. mRNA expression of the remaining podocyte proteins was similar in both groups. No significant differences concerning all examined parameters were found depending on the degree of obesity. There was a positive significant correlation between α1-AGP and ACR. Conclusions: Increased α1-AGP before the onset of albuminuria suggests its usefulness as a biomarker of early glomerular damage in obese children. An increased podocin mRNA expression also indicates podocyte damage and may be linked to ORG development. The lack of increase in expression of other podocyte proteins suggests that podocin mRNA may be a more specific and sensitive biomarker. The degree of obesity has no impact on the tested parameters, but further studies are needed to confirm it.

**Keywords:** obesity; glomerular injury; alpha-1 acid glycoprotein; urinary mRNA expression of podocyte-associated proteins

#### **1. Introduction**

Over the past few decades, overweight and obesity have been recognized as one of the most serious public health problems worldwide, affecting not only adults but also children. It is estimated that in the United States obesity affects approximately one-fifth of the school-age population [1]. According to the World Obesity Forum, 20–35% of European children are overweight or obese [2]. Similar results were obtained by the Polish authors examining school-aged children [3]. Obesity and obesity-related metabolic diseases may lead to hypertension and diabetes, which are associated with an increased risk of chronic kidney disease (CKD).

**Citation:** Medy ´nska, A.; Chrzanowska, J.; Ko´scielska Kasprzak, K.; Bartoszek, D.; Zabi ´ ˙ nska, M.; Zwoli ´nska, D. Alpha-1 Acid Glycoprotein and Podocin mRNA as Novel Biomarkers for Early Glomerular Injury in Obese Children. *J. Clin. Med.* **2021**, *10*, 4129. https:// doi.org/10.3390/jcm10184129

Academic Editor: Katarzyna Taranta-Janusz

Received: 13 July 2021 Accepted: 8 September 2021 Published: 13 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The pathogenetic role of obesity in the development of adult CKD has been confirmed in 15–30% of patients, and a large meta-analysis showed that obesity is an independent risk factor for the development of CKD [4,5]. Moreover, it has been found that overweight and obesity are closely related to an increased risk of end-stage kidney disease (ESKD) [6]. Experimental and clinical studies have demonstrated that obesity promotes structural, hemodynamic, and metabolic alterations in the kidney, damaging different nephron segments, particularly the glomerulus. In 1974, an association was first reported between massive obesity and glomerulopathy, which is morphologically similar to focal segmental glomerulosclerosis (FSGS) [7]. Nowadays, obesity-related glomerulopathy (ORG) is an increasingly recognized cause of ESKD in the adult population.

Sodium reabsorption in the proximal tubules and in the loops of Henle plays a key pathogenic role in the development of ORG. Low sodium concentration in the macula densa dilatates afferent arterioles and leads to hyperfiltration, which increases renal plasma flow (RPF) and the glomerular filtration rate.

Abitbol et al., who studied a group of children with kidney disease and proteinuria, found that the glomerular volume was significantly greater in obese children than in the non-obese control [8]. It should be emphasized that reduced kidney function is for a long time asymptomatic and detected only incidentally.

Given that obese children often remain obese in adulthood, and obesity-related complications, including the kidney, begin in childhood, efforts should be made to detect them as soon as possible. In the era of the obesity epidemic, this should be a special health challenge for pediatric nephrologists.

It has long been known that albuminuria, not detectable in standard urinalysis, is a useful early marker of glomerular damage, as it precedes the development of overt proteinuria. Recent studies indicate that alpha-1-acid glycoprotein (α1-AGP) is a better indicator of the permeability of the glomerular filtration barrier than albuminuria [9]. Alpha-1-acid glycoprotein is an acute-phase protein synthesized primarily in the liver. It has immunomodulatory properties and participates in pro- and anti-inflammatory activities by affecting the release or inhibition of cytokines (TNF-α, IL-1, IL-6) [10]. Experimental studies in mice have shown that increased α1-AGP levels help maintain metabolic balance by suppressing inflammation [11]. In humans, α1-AGP mRNA expression correlates with mRNA expression of both pro- and anti-inflammatory cytokines released by adipose tissue (TNF-α, IL-6, adiponectin) [12]. Authors suggest that α1-AGP attenuates the inflammatory process in human adipose tissue. However, El-Beblawy et al., who studied children and adolescents with type 1 diabetes, found that α1-AGP is an independent factor for diabetic microvascular complications and that its urinary concentration increases with overt nephropathy [13].

Podocytes are terminally differentiated and highly specialized cells of the visceral epithelium, which play a major role in maintaining the structure and function of the glomerular filtration barrier. A slit diaphragm, consisting of structural and signaling proteins, is a junction connecting foot processes of neighboring podocytes. In many glomerulopathies, injured podocytes detach from the glomerular basement membrane into the urine and cause filtration barrier leakage [14–16]. This process has been used to assess the activity and progression of glomerulonephritis in adults and children [15–17]. Alternatively, urinary mRNA expression of podocyte-associated proteins (synaptopodin, nephrin, podocin, podocalyxin, and CD2AP) can be assessed.

#### **2. Objectives**

The study aimed to evaluate the urinary excretion of albumin, α1-AGP, and mRNA of podocyte-specific proteins as indicators of glomerular injury in obese children and their association with the degree of obesity SDS BMI reference ranges and metabolic disorders.

#### **3. Materials**

We examined 125 children (68 girls) aged 8–17.9 years (mean 13.7 ± 2.84) treated for obesity in the outpatient clinic at the Department of Pediatric Endocrinology and Diabetology (Wroclaw Medical University, Wrocaw, Poland). We enrolled only children with simple obesity or without concomitant chronic or acute infectious diseases. We excluded patients with secondary obesity associated with endocrine disorders, genetic syndromes, or central nervous system diseases. The control group included 33 healthy peers (18 girls) aged 7.6–17.8 years (mean 12.9 ± 3) with normal body weight. Patients were divided into two groups, depending on their SDS BMI values. Patients' characteristics are presented in Table 1.

**Table 1.** Patients' characteristics according to the SDS BMI values.

NS—insignificant statistical difference, SD—standard deviation.

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Bioethics Committee of the Wroclaw Medical University (No. KB-376/2016). Informed consent was obtained from the parents and subjects above 16 years old.

#### **4. Methods**

Anthropometric parameters in all enrolled patients were measured: height with Harpenden Stadiometer with 0.1 cm precision and weight with an electronic weight scale (SECA, Hamburg, Germany) with an accuracy of 0.05 kg. Body mass index was calculated according to the following formula: BMI = body weight (kg)/(height (m))2. According to the WHO recommendations, obesity was defined as 2 standard deviations (SD) above the reference median, and overweight as 1 SD above the reference median. BMI charts for Polish children population were used [3].

Serum and urine samples were tested to measure baseline serum lipid parameters (total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides), carbohydrate parameters (fasting glucose), renal function (creatinine), and hsCRP. Estimated creatinine clearance (eGFR), using the Schwartz formula [18], and insulin resistance index were calculated. In all patients, albuminuria, α1-AGP, and urinary creatinine levels and the glucose loading test were performed. Additionally, mRNA expression of podocyte-associated molecules (nephrin, synaptopodin, podocin, and C2AP protein) in urinary sediments were assessed, as markers for the presence of detachment podocyte in the urine.

Blood samples were drawn from cubital veins after an overnight fast, during routinely performed laboratory tests. Samples were clotted for 30 min, and centrifuged for 15 min at 3000 rpm. After separation, the serum was frozen at −80 ◦C until assayed. Serum creatinine, total cholesterol, HDL cholesterol, LDL cholesterol, glucose, insulin levels, and blood were assessed on the same day.

Urine was collected from the first morning void (50–150 mL), which was then centrifuged at 800× *g* for 30 min at 4 ◦C. The supernatant was frozen at <−80 ◦C until assayed. The pellet was washed in 50 mL PBS and centrifuged at 800× *g* for 10 min at 4 ◦C. Then, the pellet was analyzed for mRNA expression of structural genes, and stabilized in RNA later fluid (24 h, 4 ◦C). Samples were stored at <−70 ◦C until assayed. Creatinine and α1-AGP were measured by ELISA kits supplied by R&D Systems, Inc. (Minneapolis, MN, USA). Urinary albumin was measured by ELISA kits supplied by Wuhan (EIAab Science Co., Ltd., Wuhan, China).

All assays were performed according to the manufacturers' instructions. The concentration of parameters was expressed per milligram of creatinine in urine, except for albuminuria, which was expressed in milligrams per gram of urinary creatinine (mg/g).

#### **5. Gene Expression**

The research involved a real-time PCR analysis of urine sediment gene expression of NPHS1, NPHS2, CD2AP, and SYNPO genes, referenced to 18S rRNA or GAPDH. Total RNA was isolated from urine sediments with a QIAamp RNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer protocol, including genomic DNA removal with RNase-free DNase (Qiagen, Germany). The samples were reversely transcribed with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA) according to the manufacturer protocol. The reaction mixes containing 0.9 μL of reverse transcription product in 10 μL of Taqman Fast Advanced Master Mix were applied to each well of custom-designed plates (Taqman), including the following assays in triplicate: Hs00190446\_m1 (NPHS1), Hs00387817\_m1 (NPHS2), Hs00961458\_m1 (CD2AP), Hs00200768\_m1 (SYNPO), Hs99999905\_m1 (GAPDH), and Hs99999901\_s1 (18S rRNA). Amplification reaction was performed on Taqman 7900 HT instrument with a fast protocol and analyzed with SDS 2.2.2 software. The results are presented as ΔCT = CTtarget − CTreference, or as ΔΔCT = ΔCTmean control sample − ΔCTtest sample. Mean control sample was the mean value of ΔCTs in the healthy control group.

#### **6. Statistical Analyses**

The results are expressed as mean (x), median (M), range (min–max), lower and upper quartiles (25–75 Q), and standard deviations (SD). The equality of means in independent groups was tested with ANOVA analysis of variance and in heterogeneous groups with a Mann-Whitney non-parametric U-test; homogeneity of variance was assessed by Bartlett's test. For discrete parameters, we analyzed the frequency of trait occurrence using the c2df test with Yates' correction with an appropriate number of degrees of freedom df (df = (m − 1) \* (*n* − 1), where m—the number of rows, *n*—the number of columns). For correlation analysis of selected variables, we used the Pearson or Spearman's correlation coefficient. A value of *p* ≤ 0.05 was considered statistically significant. Statistical analysis was performed with EPIINFO v. <IP_ADDRESS> statistical software package.

#### **7. Results**

The parameters of lipid metabolism differed significantly between the study group and the control group in terms of triglycerides and HDL cholesterol values. Total cholesterol and LDL cholesterol levels in both groups were comparable. Estimated creatinine clearance values also differed between the groups and were significantly higher in the obese child population. Fasting glucose was above normal in only four children in the study group. Detailed data are presented in Table 2.

**Table 2.** Biochemical parameters in study and control group.

**Table 2.** *Cont.*

For a distribution deviating from the normal distribution, no mean was calculated. \* analysis with the nonparametric Mann-Whitney U test.

Albumin excretion rate did not differ between the study and control groups, with values within normal range in all children (<30 mg/g creatinine). The excretion of α1-AGP was significantly higher in obese children compared with the healthy control. Detailed results are presented in Table 3.

**Table 3.** ACR, α-1AGP, and hsCRP in the study and control groups.

\* analysis with the non-parametric Mann-Whitney U test.

However, no differences were shown between the degree of obesity and albumin, and α-1-AGP levels (Table 4).

**Table 4.** Glomerular damage indicators between groups of children depending on the degree of obesity.

Analysis with the non-parametric Mann-Whitney U test.

The mRNA expression of nephrin, synaptopodin, and CD2AP in urine sediment was not significantly different between the groups, irrespective of the degree of obesity. Podocin mRNA expression was significantly higher in the group of obese patients compared with the non-obese group. Unexpectedly, it was significantly higher in children with a lower degree of obesity than in those with a higher degree of obesity. The results are shown in Table 5.

**Table 5.** The level of mRNA expression of the podocin gene between groups of children depending on the degree of obesity, expressed as ΔΔCt.

For a distribution deviating from the normal distribution, no mean was calculated. \* analysis with the nonparametric Mann-Whitney U test.

We found a positive correlation only between α1-AGP and albuminuria (*r* = 0.95; *p* = 0.000), and between α1-AGP and hsCRP (*r* = 0.54; *p* = 0.000).

#### **8. Discussion**

The study showed for the first time significantly increased α1-AGP excretion in the urine of obese children as compared with non-obese peers in the absence of overt albuminuria, considered so far as an early marker of glomerular damage. Burgert et al., among others, reported microalbuminuria in more than 10% of obese American children without other comorbidities [19]. According to the authors, the appearance of albuminuria depends primarily on hyperglycaemia, which was found in our study only in four children, which may have had an impact on the ACR result.

In the study by Lurbe et al., the prevalence of microalbuminuria in obese children was only 2.4%. These authors showed no relationship between the degree of obesity and increased albumin-to-creatinine ratio (ACR), which is consistent with our observations [20]. Sawamura et al. observed albuminuria in 21% of 64 overweight and obese children and adolescents, which did not correlate with eGFR, indicating that hyperfiltration is not the only factor that damages the glomerular filtration barrier in obesity [21].

Recent studies indicate that α1-AGP is a more sensitive marker of glomerular filtration barrier injury than albuminuria. This was proved, among others, by Talks et al., who studied the urinary α1-AGP and albumin leakage in healthy volunteers in an altitude-induced hypoxia (5023 m). These authors reported a significant increase in urinary leakage of albumin and α1-AGP, but the percentage of increase was significantly higher for α1-AGP. They observed no increase for positively charged dimeric lambda free light chains (λ-FLCs) [9]. It should be emphasized that the molecule of α1-AGP is smaller than the molecule of albumin (43 kDA vs. 66 kDA) and similar in size to the podocyte slit diaphragm pore. Similar observations were reported in adult patients with type 2 diabetes. Both Christiansen et al. [22] and Narita et al. [23] have shown increased urinary α1-AGP excretion in diabetic patients with normoalbuminuria. Similarly, in a study of 60 children with type 1 diabetes, α1-AGP excretion preceded the onset of microalbuminuria and significantly increased with its onset [13]. Moreover, these authors reported a significant positive correlation between α1-AGP excretion and albuminuria, which is in line with our observations. Therefore, it seems that urinary α1-AGP may be a useful marker for assessing glomerular injury in obese children. However, in our study we did not show any significant differences in urinary α1-AGP according to the degree of obesity.

The mechanism of glomerular injury in obese patients is complex and not fully understood. Certainly, hemodynamic and metabolic as well as endocrine disturbances are interconnected. For this reason, the effect of damage to individual kidney structures may not be the same in all obese patients. Although, according to Tsuboi et al., severe forms of ORG affect mainly morbidly obese patients, the severity of kidney damage is not necessarily related to the severity of obesity [24].

Alpha-1-acid glycoprotein, a major acute-phase protein, displays a number of antioxidative and anti-inflammatory properties [25]. For this reason, its increased levels in serum and urine have been linked to vascular inflammation and subclinical atherosclerosis. This has been confirmed by El-Beblawy et al., who found a significant correlation of serum and urinary α1-AGP with hsCRP and cartoid intima-media thickness (IMT) in children with type 1 diabetes [13].

It is very likely that in obese children, α1-AGP may be a good marker not only of glomerular injury but also of very early atherosclerotic lesions. In this study, we found also a significant positive correlation between the α1-AGP and hsCRP.

Yang et al. have shown that in patients with cardiovascular disease, elevated levels of high-sensitivity CRP increase the risk of developing microalbuminuria [26]. It cannot be excluded that we would observe a similar association with longer duration of obesity. In fact, we found significantly higher hsCRP concentrations in obese children compared with non-obese children.

Many studies have demonstrated an association of podocyturia and mRNA expression of podocyte proteins in urinary sediment with an activity and progression of different glomerulopathies, including adult diabetic nephropathy [27] and lupus nephropathy [28].

However, only a few studies have demonstrated this association with ORG and the "silent" glomerular damage in obesity. For instance, Chen et al. reported a significantly lower number of podocyte density in 46 adults with clinically overt ORG compared with healthy controls [29]. Moreover, these changes were strongly associated with proteinuria and abnormalities in fasting glucose and insulin levels [29]. However, Pereira et al., who studied urinary expression of podocyte-associated mRNAs, showed higher expression levels of podocin and nephrin mRNAs in obese and overweight adults as compared with healthy controls. Moreover, it was associated with the severity of obesity and metabolic syndrome. What is, however, worth emphasizing is that they did not detect albuminuria [30].

However, higher serum insulin levels were related to podocyturia, regardless of obesity. Our study, which is, to our knowledge, the first one conducted in a population of obese children, is consistent with the previous results only with regard to podocin mRNA, because the mRNA expression of other podocyte proteins did not differ between the obese

children and the healthy control. Moreover, our study did not confirm the association of podocin mRNA with weight gain or metabolic disorders, which may be due to the fact that our population was younger as compared with the obese population in other studies. The above results confirm that glomerular lesions assessed by mRNA expression of podocyte proteins occur even before the onset of albuminuria. The significantly higher mRNA expression levels of podocin, and not of other proteins, are difficult to explain. Some suggest that podocin may be a more specific and sensitive marker in podocyte damage than nephrin, podocalyxin, or synaptopodin. This was confirmed, among others, by Garovic et al., who conducted investigations in pregnant women with preeclampsia [31]. This is also suggested by the results of experimental studies on rats with diphtheriainduced kidney damage with a subsequent increase in urine podocin and nephrin mRNA expression [32]. After 8 days of observation, the presence of podocin mRNA was still detected, and no expression of nephrin mRNA was found. Given the fact that obese patients may develop ORG, podocyturia measured with urinary mRNA expression levels of podocyte-assosiated proteins may be one of the first indicators of ORG development.

#### **9. Conclusions**

Increased α1-AGP before the onset of albuminuria suggests its usefulness as a biomarker of early glomerular damage in obese children. An increased podocin mRNA expression also indicates podocyte damage and may be linked to ORG development. The lack of increase in expression of other podocyte proteins suggests that podocin mRNA may be a more specific and sensitive biomarker. The degree of obesity has no impact on the tested parameters, but further studies are needed to confirm it.

**Author Contributions:** Conceptualization, A.M. and D.Z.; Data curation, A.M., J.C., K.K.-K., D.B. and M.Z.; Investigation, A.M., J.C.; Methodology, A.M., K.K.-K., D.B. and M. ˙ Z.; Writing—original ˙ draft, A.M., K.K.-K. and D.Z.; Writing—review and editing, A.M. and D.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financed by a grant from Wroclaw Medical University, Poland (grant number ST-C210.16.082).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Bioethics Committee of the Wroclaw Medical University (No. KB-376/2016).

**Informed Consent Statement:** Informed consent was obtained from all subjects (≥16 years) and their representatives involved in the study.

**Data Availability Statement:** Data associated with the paper are not publicly available but are available from the corresponding author at reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Differences between Obese and Non-Obese Children and Adolescents Regarding Their Oral Status and Blood Markers of Kidney Diseases**

**Katarzyna Ma´ckowiak-Lewandowicz 1,\*, Danuta Ostalska-Nowicka 1, Jacek Zachwieja 1,† and Elzbieta Paszy ´ ˙ nska 2,†**

**Abstract:** (1) Background: A rarely discussed effect of obesity-related glomerulopathy (ORG) may slowly lead to irreversible glomerular damage and the development of chronic kidney disease. These patients need to undertake medical care, but whether they should be included in intensive oral care is still not mandatory. The study aimed to assess a relationship between renal, metabolic, and oral health indicators among pediatric patients affected by simple obesity. (2) Methods: 45 children and adolescents with simple obesity hospitalized (BMI 34.1 ± 4.8 kg/m2, age 15.4 ± 2.3) and compared with 41 aged-matched healthy controls (BMI 16.4 ± 2.4 kg/m2, age 15.4 ± 2.7). Echocardiography, 24-h ambulatory blood pressure monitoring, ultrasound exam with Doppler, and laboratory tests including kidney and metabolic markers were performed. Oral status was examined regarding the occurrence of carious lesions using decay missing filling teeth (DMFT), gingivitis as bleeding on probing (BOP), and bacterial colonization as plaque control record (PCR). (3) Results: The strongest correlation was revealed between BMI and concentration of uric acid, cystatin C, GFR estimated by the Filler formula (r = 0.74; r = 0.48; r = −0.52), and between oral variables such as PCR and BOP (r = 0.54; r = 0.58). Children and adolescents with obesity demonstrated untreated dental caries, less efficient in plaque control and gingivitis. (4) Conclusions: No specific relation to markers of kidney disease were found; however, more frequent gingivitis/bacterial colonization and significant differences in oral status between obese and non-obese patients were revealed. Susceptibility to inflammation may be conducive to developing metabolic syndrome and kidney damage in the form of obesity-related glomerulopathy and contribute to future dental caries. Uric acid seems to indicate metabolic syndrome and cardiovascular complications (LVMI > 95 percentiles). Cystatin C and uric acid might aspire to be early markers of kidney damage leading to obesity-related glomerulopathy.

**Keywords:** obesity; childhood; dental caries; gingivitis; kidney injury; glomerulopathy; uric acid; cystatin C

#### **1. Introduction**

The COVID-19 outbreak, which contributed to a decrease in physical activity and strengthening of harmful eating habits resulting from remote learning and lockdown, has significantly accelerated an increase in the number of obese patients in Poland. It seems to be a necessary task of the medical environment to address a high risk of obesity complications among pediatric patients with obesity. Typical complications of obesity in children and adolescents, for example, arterial hypertension and dyslipidemia, are well known. A rarely discussed effect is obesity-related glomerulopathy (ORG). Despite its initial asymptomatic course, it slowly leads to irreversible glomerular damage and

**Citation:** Ma´ckowiak-Lewandowicz, K.; Ostalska-Nowicka, D.; Zachwieja, J.; Paszy ´nska, E. Differences between Obese and Non-Obese Children and Adolescents Regarding Their Oral Status and Blood Markers of Kidney Diseases. *J. Clin. Med.* **2021**, *10*, 3723. https://doi.org/10.3390/jcm10163723

Academic Editors: Kinga Musiał, Katarzyna Taranta-Janusz and Alberto Martínez-Castelao

Received: 28 June 2021 Accepted: 17 August 2021 Published: 21 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

development of chronic kidney disease, with end-stage renal failure in 1 per 3 patients. In the first period of ORG, obesity leads to hyperfiltration. An increased tubular flow decreases the contact time between proteins and tubular epithelium and produces an increased radial gradient of albumin concentration, resulting in reduced protein reabsorption that may occur without evidence for glomerular dysfunction. The excessive reabsorption of proteins generates apoptosis, renal oxidative stress, inflammation, hypoxia, and lipid accumulation, leading to fibrosis [1,2]. Clinically, ORG is manifested with proteinuria of various severity, depending on the extension of glomerular structural changes: from permanent, mild proteinuria to nephrotic proteinuria [3]. A final diagnosis of obesity-related glomerulopathy is based on biopsy, which carries a higher risk of complications than standard diagnosis due to the patient's obesity and more difficult access to the material collected during a biopsy [4]. It is not a routine procedure and selecting a group of ORG patients without this type of invasive diagnostic procedure is a big challenge for physicians [5]. ORG is suspected in obese patients with proteinuria and concomitant arterial hypertension and lipid metabolism disorders [6,7]. Significant underestimation of ORG patients stems from the fact that markers of this disease revealed by thin-needle biopsy are also found in patients without proteinuria [8]; therefore, new, early markers of kidney damage that would find use in diagnosing ORG are searched for, such as urine megalin and expression of connexin 43 [9,10]. Pediatric patients affected by obesity should be encouraged to undertake medical care, but whether they should be included in oral care because of dental or gingival diseases remains still an uncertain requirement; however, more and more information concerning overlapping relationships with oral cavity homeostasis is available in the literature. Proinflammatory cytokines, produced by adipose cells, lead to low-grade chronic inflammation in the salivary glands [11]. The cause of the imbalance of salivary substances is a significant increase in susceptibility to caries, even in very young patients [12]. A diet rich in carbohydrates favors bacterial colonization, creating favorable conditions for caries promotion. Another oral manifestation of obesity may take the form of gingival inflammation due to poor oral hygiene. Then gingivitis may generate periodontal tissues response and intensify metabolic disorders or even promote weight gain. Combining the above-mentioned dental complications of obesity, such as dental caries, microbial imbalance, and defective salivary composition stimulates potential risk for systemic diseases [11]. It will most likely be connected with an inflammatory response to bacterial pathogens and the generation of reactive oxygen species. Oxidative imbalance contributes to generating lipid peroxide, which can diffuse into the bloodstream [12]. The components of the oxidative stress cascade interact with glomeruli and contribute to obesity-related glomerulopathy [12].

The aim of the study was to assess a correlation between renal, metabolic, and oral health indicators among pediatric patients affected by simple obesity.

#### **2. Materials and Methods**

#### *2.1. Study and Control Groups*

The study group finally consisted of 45 children and adolescents with simple obesity, admitted to the Department of Pediatric Nephrology and Hypertension in Pozna ´n between 1 January 2019 and 31 December 2020. A reason for hospitalization was the verification of primary hypertension. The control group consisted of 41 age-matched healthy children and adolescents. The flow chart of the study is presented as Supplemental Material in Figure S1.

Echocardiography was performed on all patients with the assessment of left ventricular hypertrophy marker (left ventricular mass index—LVMI > 95 centile), as were abdominal ultrasound exam with Doppler test to evaluate the blood flow through kidneys' arteries and blood laboratory analysis to exclude comorbidities [13]:

All patients were fasting for twelve hours for an ultrasound exam and laboratory tests. During next twenty four hours, urine collection was carried out by collecting every urine sample in a container, starting with the second urinating on the first day of hospitalization, ending the next day with the first portion of urine. Laboratory tests were performed using a standardized method, according to the manufacturer's instructions. Glomerular filtration rate was estimated by Schwartz and Filler formula [14]. Based on the Filler formula, hyperfiltration was defined as GFR > 130 mL/min/1.73 m2 and hypofiltration when GFR was less than 90 mL/min/1.73 m2. Arterial blood pressure was assessed by 24-h ambulatory blood pressure monitoring [15]. Atrial hypertension was diagnosed when average systolic or diastolic blood pressure or both, obtained in the course of over three different medical visits was at/above the 95th percentile for age, sex, and height [16]. Simple obesity (BMI > 95 percentiles, BMI Z-score > 1.4) was diagnosed when secondary obesity was excluded. BMI values were transformed into BMI Z-scores using WHO reference values for pediatric BMI [17].

Inclusion and exclusion criteria for study and control groups are described in Table 1 [18]. The protocol allowed 45 children and adolescents with simple obesity; 25 patients additionally with primary hypertension within this group; therefore, the obesity group was divided into two subgroups: suspicion of obesity-related glomerulopathy (sORG) and without ORG group (without ORG). The selection of obesity patients with sORG was based on scientific reports [4,7]: the presence of significant clinical microalbuminuria (>30 mg/day), primary hypertension (revealed during hospitalization), and lipid disorders (decreased concentration of HDL and increased concentration of triglycerides). Seven adolescents met the whole inclusion criteria for suspicion of obesity-related glomerulopathy group. Forty-one healthy children and adolescents enrolled in the control group had to fulfill the inclusion criteria presented in Table 1.

#### *2.2. Dental Examination*

A dental assessment was carried out at the dental office by one qualified dentist (E.P.) during hospitalization in the Department of Pediatric Nephrology and Hypertension, in a blind fashion for each child, before the final diagnosis. Before this study, the dental examiner was calibrated after a training course evaluation described in a previous project [19]. Dental evaluation of the occlusal, buccal, and lingual teeth surfaces was performed after the cleaning and drying (excluding the third molars) under visual and tactile examination in artificial light, without magnification, using a dental mirror and blunt probe [20]. The dental assessment included the number of milk/permanent (using lower case letter for primary teeth/capital letters for permanent teeth) decayed teeth (d/D), the number of missed teeth (m/M), the number of filled teeth (f/F), as a dmft/DMFT score [20]. Active dental caries was scored when the lesion manifested a visible cavity, undermined enamel, or softened area. A tooth rebuilt due to dental caries was recorded when a tooth had at least one final restoration applied to cure decay. The missing constituent of the DMFT index was estimated when a tooth had been removed due to dental caries failures. A manual graded periodontal explorer assessed dental plaque and gingival inflammation (LM-instruments, LM8 5050 probe, Osakeyhtiö, Parainen, Finland). Plaque deposit was estimated by the plaque control record index (PCR) [21]. Gingival inflammation was determined using the bleeding on probing index (BOP) [22], measured in six points of the gingival sulcus of all teeth (excluding the third molars). The proportion of surfaces (%) with dental plaque or bleeding-on-probing gums, respectively, were calculated for each subject as % of sites [23]. STROBE protocol was included and attached in the Supplemental Material.

#### *2.3. Statistical Analysis*

The Shapiro–Wilk test was used to assess the normality of the data. The homogeneity of variance of each variable was calculated with Levene's test. Non-parametric Mann– Whitney U-test was applied in the analyses of data with non-normal distribution. Student t-test was employed in the analyses of variables with normal distribution. Spearman's correlation rank test was performed to analyze the correlation of parameters with non-normal distribution. Pearson test was employed to test the correlation of normal distribution variables. The statistical significance level was set at *p* < 0.05. Statistical analyses were conducted using Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA).

#### **3. Results**

This section may be divided into subheadings. It should provide a concise and precise description of the experimental results, their interpretation, and the empirical conclusions that can be drawn.

#### *3.1. Anthropometric, Clinical, and Biochemical Evaluation*

The final group consisted of 86 participants (45 with obesity and 41 control subjects). The mean age of the obese patients was 15.4 ± 2.33 years, the mean age of the controls was 15.4 ± 2.74 years, with no statistically significant difference. BMI and BMI Z-score were statistically different between patients and controls (*p* = 0.001; *p* = 0.001). The clinical and biochemical characteristics of all patients are presented in Table 2.

**Table 2.** Anthropometric, biochemical, and oral data for obesity group and control individuals.

**Table 2.** *Cont.*

Results are expressed as mean ± standard deviation, median (min-max ranges). Statistical significance is given according to *p*-value (*p* ≤ 0.05) vs. non-significance (ns). Statistical tests used were Mann–Whitney *U* test, *t*-test, and Welch test. Abbreviations: BMI—body max index; BUN—blood urea nitrogen; UA—uric acid; GFR F—glomerular filtration rate estimated by the Filler formula; GFR S—glomerular filtration rate calculated by the Schwartz formula; LVMI—left ventricular mass index, D—number of decayed secondary teeth; M—number of missing secondary teeth; F—number of filled secondary teeth; DMFT—decayed, missing, filled teeth score, evaluating dental caries in permanent teeth; d—number of decayed primary teeth; m—number of missing primary teeth; f—number of filled primary teeth; dmft score—total score evaluating the number of decayed, missing, filled primary teeth; PCR [%]—plaque control record index; BOP [%]—bleeding on probe index.

> The study and control group did not differ in serum concentrations of creatinine and urea. Significantly higher concentrations of uric acid and cystatin C were observed in obesity group (*p* = 0.001; *p* = 0.028). According to the Schwartz formula, GFR values did not differ significantly between the study and control group (115.57 ± 20.54 mL/min/1.73 m2 and 118.02 ± 36.02 mL/min/1.73 m2, respectively). Mean GFR estimated by Filler formula in the obesity group was 111.26 ± 31.15 mL/min/1.73 m2 and 127.18 ± 22.54 mL/min/1.73 m2 in the control group (*p* = 0.019). The study and control group presented no proteinuria in urine analysis. Clinically significant microalbuminuria (>30 mg/d) was observed in 7 patients with obesity, while in the control group, microalbuminuria >30 mg/d was not detected (*p* = 0.183).

> In the study group, 25 patients presented primary hypertension, while AH was not observed in the control group. Obesity and control groups did not differ in serum concentrations of total cholesterol and LDL. Significantly higher concentrations of HDL,

triglycerides and glucose were observed in study group (*p* = 0.001; *p* = 0.001; *p* = 0.029). Median LVMI was significantly different between obesity and control groups (*p* = 0.001).

#### *3.2. Oral Data*

Analysis of the oral cavity revealed that obesity group presented a significantly higher number of D (*p* = 0.001) and DMFT score (*p* = 0.016), amount of dental plaque (PCR), (*p* = 0.001), and gingival inflammation (BOP), (*p* = 0.001). No differences in the results of the dental examination of the primary dentition were detected between the study and the control group (Table 2).

#### *3.3. Correlations between the Variables*

In the obesity group, Spearman analysis evidenced a significant correlation between BMI and concentrations of creatinine, urea, cystatin C, GFR estimated by the Filler formula, microalbuminuria, and the number of decayed teeth D. The strongest correlation was revealed between BMI and concentration of uric acid (r = 0.737), and oral variables such as PCR and BOP (r = 0.539; r = 0.583). Additionally, the most significant correlation was found between BMI Z-score and oral status estimated by BOP and DMFT scores (r = 0.499; r = 0.499). In the study group, statistical analysis evidenced a significant correlation between uric acid and HDL, triglycerides, and BOP. The strongest correlation was revealed between UA and LVMI (r = 0.616). There was a significant correlation between cystatin C and BOP, LVMI, and between BOP and triglycerides, glucose, HDL, and GFR estimated by the Filler formula (Table 3).

**Table 3.** Significant results of the Spearman's and Pearson's correlation rank tests regarding clinical and biochemical parameters (*p* < 0.05) for the obesity group.

**Table 3.** *Cont.*

Abbreviations: BMI—body max index; UA—uric acid; GFR F—glomerular filtration rate estimated by the Filler formula; LVMI—left ventricular mass index. D—number of decayed secondary teeth; DMFT—decayed, missing, filled teeth score, evaluating dental caries in permanent teeth; PCR [%]—plaque control record index; BOP [%]—bleeding on probe index.

#### *3.4. Clinical and Biochemical Characteristics: Suspicion of Obesity-Related Glomerulopathy Group and without ORG Group*

Individuals composing the suspicion of ORG and without ORG group did not differ in serum concentrations of creatinine, urea, cystatin C and GFR estimated by the Filler formula. Significantly higher concentrations of uric acid were observed in the suspicion ORG group (*p* = 0.002). Mean GFR calculated by the Filler formula in the suspicion of ORG group was 130.14 ± 54.88 mL/min/1.73 m2 and 106.37 ± 20.38 mL/min/1.73 m2 in the without ORG group. Median microalbuminuria was significantly different between the probable ORG group and without ORG group (*p* = 0.001) (Table 4).

**Table 4.** Anthropometric, biochemical, and oral data under suspicion of ORG (sORG) and without ORG among individuals.

**Table 4.** *Cont.*

Results are expressed as mean ± standard deviation, median (min-max ranges). Statistical significance is given according to *p*-value (*p* ≤ 0.05) vs. non-significance (ns). Abbreviations: sORG—suspicion of obesity-related glomerulopathy. BMI—body max index; BUN blood urea nitrogen; UA—uric acid; GFR F—glomerular filtration rate estimated by the Filler formula; GFR S—glomerular filtration rate calculated by the Schwartz formula; LVMI—left ventricular mass index. D—number of decayed secondary teeth; DMFT—decayed, missing, filled teeth score, evaluating dental caries in permanent teeth; PCR [%]—plaque control record index; BOP [%]—bleeding on probe index.

#### **4. Discussion**

#### *4.1. Dental Caries*

In this study, no specific relations between oral status and markers of kidney disease were found; however, more frequent gingivitis/bacterial colonization and significant differences in oral status between obese and non-obese children were revealed. Obese patients demonstrated a higher incidence of oral-related complications according to dental status and less efficient oral biofilm control and gingival inflammation than non-obese subjects.

Based on the literature review on childhood obesity, cause-and-effect relationships to oral diseases show strong associations with periodontal diseases and different relationships to caries progression, such as confirming [24–26] or denying [27–30]. It seems to depend on multiple factors contributing to the disease progress and the risk of dental caries in primary and permanent dentition among pediatric patients affected by simple obesity. This is, to the best of our knowledge, the third study related to obese children in our country [31,32]. In comparison to the first study carried out on obese children in the similar age group of 7 and 12 years old, in the group of younger children, no difference was found between healthy and obese children (82.2% versus 95.0%; ns), while in the group of children aged 12, the incidence of caries was significantly higher in the group of obese children (53.2% versus 84.2%; *p* = 0.004) [31]. In addition, obese adolescents revealed a correlation with the number of surfaces affected by caries, dental plaque, and gingivitis (*p* = 0.001). The second survey confirmed high caries incidence of obese children aged 6–12 years old, but the relationship between BMI and insufficient oral hygiene has not been demonstrated [32]. A similar study of 91 participants divided on normal and overweight children between 6–12 years old revealed a low caries experience and risk classification CAMBRA [30]. The

lack of differences in the group consisted of younger children may be explained by the very high caries prevalence among Polish children under 7 years old, estimated at 85.0% [33].

In contrast, older children aged 12–18 have easier access to dental operatory offices and school oral health education/promotion facilities. Other co-factors such as preferred diet, socioeconomic class, industrialized origin life conditions may be found as moderators [29,34,35]. Assuming the present trial's statistical differences between DMFT values at age 15, obese children revealed that increasing body weight boosts caries prevalence.

At this point, it is worth emphasizing the role of dentistry in the monitoring of childhood obesity [36,37]. Frequent contact with children and young adults in the dental setting makes it possible for them to prevent obesity. The overweight parent and child– dentist relationship in an extended period may allow that dietary advice may be given to reduce sugar consumption and meal frequency. This could be valid from the oral diseases and BMI reduction points of view. Moreover, becoming aware of the oral indicators of obesity may strengthen motivation to alter any unhealthy behaviors [36,37].

#### *4.2. Dental Plaque and Gingival Bleeding*

It is noteworthy that the relationship between obesity and periodontal inflammation is reported wider than dental caries and gingivitis experience, probably because of exposure to a number of factors [38–40]. Based on the literature mentioned above, a failure in periodontal health maintenance is due to an influence of blood proinflammatory cytokines, such as IL-1, IL-6, TNF-α, CRP, and oxidative stress activity [12]. Proinflammatory mediators secreted by adipose cells, interfering with the immune system, may modify the host's response to plaque antigens and contribute to blocking periodontal protection [41]; therefore, focusing on gingivitis observed in obese children results from metabolic disturbances, inflammatory factors, and poor oral hygiene habits. Additionally, predisposition to gingivitis may be related to circulating sex hormones as higher estrogen levels owing to androgen disturbances in adipose tissue [42,43]. Even if a clinical survey did not show significant deviations in periodontal tissues, such as the number and depth of gingival pockets or periodontal attachment loss, poor cleaning habits with gingival inflammation may be observed among obese children [44].

At such a young age, periodontal health elements such as depth and attachment level are usually not examined because of their developmental status; however, it is known that high inflammatory scores and poor oral hygiene are more likely to generate permanent periodontal inflammation in the future. Moreover, the effectiveness of periodontal surgical treatment coexisting with obesity may have an uncertain recovery prognosis [45–47]. In this study, we demonstrated the adolescent patients affected by gingivitis, defined by the BOP index. This local manifestation of systemic inflammation accompanying obesity may suggest the development of metabolic syndrome and kidney damage in the form of obesity-related glomerulopathy.

Notably, obesity in pediatric patients may promote oral colonization with pathogens, which the PCR index may assess. This is in line with current literature concerning weight gain in childhood, where it is reported how excessive bacterial plaque increases unfavorable microbiota composition in the oral cavity [48,49]. In this context, Craig et al. revealed how oral microbiota trajectories varied in childhood according to weight gain [50]. Zeigler et al. documented an increase in both *Firmicutes* and *Bacteroidetes* sp. in obese patients compared to normal-weight individuals [43]. Nagawa et al. demonstrated that some species might support progesterone and estrogen access to vitamin K production needed for bacterial growth factors [51]. It seems that the role of oral bacteria in the formation and maintenance of inflammation in obesity may help to understand not only quantity but quality effects on microbial colonization or abundance in the oral cavity [50]. Changes leading to oral microbial dysbiosis need further microbiota investigations.

#### *4.3. BMI and Obesity-Related Glomerulopathy*

The study revealed significantly higher concentrations of uric acid, cystatin C, and mean GFR estimated by Filler formula in the obesity group than in the control group. These laboratory results showed the early, asymptomatic kidney injury without clinically important microalbuminuria in obese patients. The initial hypothesis could be that serum cystatin C and uric acid might aspire to be early markers of kidney damage in obesity, getting ahead of an increase in the concentration of creatinine, urea, and hypofiltration. Moreover, GFR calculated with the Filler formula based on serum concentration of cystatin C represents a more precise index of hyperfiltration, which is characteristic of ORG [52].

Additionally, our study presented the markers of metabolic syndrome in the study group and no hypertension, hypertriglyceridemia, low concentrations of HDL in the control group, which confirms the representative group of patients with obesity. In addition, uric acid is considered a marker connecting obesity with metabolic syndrome and is also indicative of the development of cardiovascular diseases, including arterial hypertension, atherosclerosis, and renal impairment [53]. The above reports are confirmed in the present study by a positive correlation between the uric acid level and markers of the metabolic syndrome: concentration of triglycerides, HDL, and LVMI indicating left ventricular hypertrophy and as a risk factor of cardiovascular diseases, including arterial hypertension.

In the present study, the BMI, describing simple obesity in children and adolescents, correlated the most with two oral status markers: PCR and BOP. Additionally, the project revealed a high correlation between BMI Z-score and gingivitis, estimated by the BOP index. The presented results may initiate the discussion and our future studies of the role of gingivitis in the progression of obesity. Moreover, the study evidenced a significant correlation between BOP index and concentration of kidney markers: uric acid, cystatin C, and indicators of metabolic syndrome: concentration of triglycerides, HDL, and glucose. Based on the above information and other reports [54], we suggest that gingivitis may represent an early marker of metabolic syndrome and kidney injury such as obesity-related glomerulopathy. In particular, the strongest correlation was found between the BOP index and uric acid. It seems to be the link between local inflammation in the oral cavity and systemic inflammatory response, which occurs in patients with obesity and morphological or functional changes in kidneys, described as obesity-related glomerulopathy.

Showing the usefulness of dental and biochemical markers, which are routinely assessed during diagnostics of obesity complications, may contribute to improved identification of the group of children with a risk of ORG and, therefore, chronic kidney disease in adulthood. In this way, invasive diagnostics of kidney damage, i.e., kidney biopsy, could be avoided. Early introduction of nephroprotective treatment and intensification of body weight reduction in this group of patients may significantly contribute to a decrease in the number of patients with obesity who may develop end-stage renal failure later in adulthood.

#### *4.4. Limitations of the Study*

The study has other limitations that have to be underlined in our interpretation of the results. No renal biopsies were performed in the group of patients with obesityrelated glomerulopathy and healthy children and adolescents to confirm the clinical results received from the study. ORG is defined by glomerular hypertrophy (glomerulomegaly), adaptive focal segmental glomerulosclerosis, and tubulointerstitial fibrosis with tubular atrophy [55,56]. Renal biopsy is too invasive a procedure in children and adolescents (in Poland, it is usually used in case of acute renal failure without the onset point). At the time of the survey, the hormonal profiles of the subjects were not investigated, and oral status was examined without any pre-diagnostic dental history (baseline). Further, family members were not studied at all, and even they presented with overweight body mass. Finally, fewer than 100 participants may not be a representative study group for distinct conclusions. On the other hand, a homogenous group of children affected by obesity was collected, which was examined in one hospital and tested by the same diagnostic laboratory. Further, the assessment of salivary levels of such markers as cystatin C and uric acid should be considered in search of an alternative, still non-invasive method of ORG diagnosis in children [57]. The circumstances mentioned above would add valuable data to general analysis. Oral health indicators may motivate to begin obesity treatment and support diminishing the risk of developing or progressing obesity-related glomerulopathy and metabolic syndrome in pediatric patients. Obtained results highlight the differences in oral status between obese and non-obese young patients, as well as the need for prophylactic programs, especially in times of pandemic, with unpredictable access to public spaces and enforced lockdowns [58].

#### **5. Conclusions**

No specific relation to markers of kidney disease was found; however, more frequent gingivitis/bacterial colonization and significant differences in oral status between obese and non-obese patients were revealed. Susceptibility to inflammation may be conducive to developing metabolic syndrome and kidney damage in the form of obesity-related glomerulopathy and contribution to dental caries progression in the future.

Increased serum cystatin C and uric acid might aspire to be early markers of kidney damage leading to obesity-related glomerulopathy. GFR based on serum concentration of cystatin C represents a more precise index of hyperfiltration, which is characteristic of ORG. Uric acid seems to indicate metabolic syndrome and cardiovascular complications (LVMI > 95 percentiles).

Since the group with suspicion of ORG is small and comparisons with non-ORG patients are inconclusive, an analysis of the relationship between renal, metabolic, and oral health indicators among pediatric patients affected by simple obesity needs to be continued in future clinical trials.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10163723/s1, Figure S1: Flow chart of the study. Figure S2: STROBE Protocol Checklist.

**Author Contributions:** K.M.-L.: data curation, investigation, and software; D.O.-N.: conceptualization, funding acquisition, supervision, and revising the manuscript; J.Z.: supervision; E.P.: conceptualization, investigation, writing review, and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research and the APC were funded only by the Poznan University of Medical Sciences.

**Institutional Review Board Statement:** Before the experiment was started, approval was obtained from the Bioethics Committee at the Poznan University of Medical Sciences (protocol code No. 679/17 and date of approval 22 June 2017). The study followed the rules of the Declaration of Helsinki and complied with Good Clinical Practice guidelines. Before the subject evaluation, the whole course of the study was explained to all patients, and they were asked to express their opinion.

**Informed Consent Statement:** Written informed consent was obtained from all patients and their parents or legal guardians, or both for study participation and publication of this paper. Additionally, a parent or legal guardian approval was needed for inclusion in the study. A lack of acceptance from patients and their parents/legal guardians resulted in exclusion from the study.

**Data Availability Statement:** Data associated with the paper are not publicly available but are available from the corresponding author at reasonable request.

**Acknowledgments:** We appreciate any technical support in editing performed by Tomasz Maksymiuk.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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## *Article* **Serum Sclerostin Is Associated with Peripheral and Central Systolic Blood Pressure in Pediatric Patients with Primary Hypertension**

**Piotr Skrzypczyk 1,\*, Anna Ofiara 1, Michał Szyszka 2, Anna Stelmaszczyk-Emmel 3, Elzbieta G ˙ órska 3 and Małgorzata Pa ´nczyk-Tomaszewska 1**

**Abstract:** Recent studies showed the significance of the canonical Wnt/beta-catenin pathway and its inhibitor—sclerostin, in the formation of arterial damage, cardiovascular morbidity, and mortality. The study aimed to assess serum sclerostin concentration and its relationship with blood pressure, arterial damage, and calcium-phosphate metabolism in children and adolescents with primary hypertension (PH). Serum sclerostin concentration (pmol/L) was evaluated in 60 pediatric patients with PH and 20 healthy children. In the study group, we also assessed calcium-phosphate metabolism, office peripheral and central blood pressure, 24 h ambulatory blood pressure, and parameters of arterial damage. Serum sclerostin did not differ significantly between patients with PH and the control group (36.6 ± 10.6 vs. 41.0 ± 11.9 (pmol/L), *p* = 0.119). In the whole study group, sclerostin concentration correlated positively with height Z-score, phosphate, and alkaline phosphatase, and negatively with age, peripheral systolic and mean blood pressure, and central systolic and mean blood pressure. In multivariate analysis, systolic blood pressure (SBP) and height expressed as Z-scores were the significant determinants of serum sclerostin in the studied children: height Z-score (β = 0.224, (95%CI, 0.017–0.430)), SBP Z-score (β = −0.216, (95%CI, −0.417 to −0.016)). In conclusion, our results suggest a significant association between sclerostin and blood pressure in the pediatric population.

**Keywords:** sclerostin; primary hypertension; children; adolescents; arterial damage; blood pressure

#### **1. Introduction**

It is estimated that arterial hypertension (AH) affects 3–5% of the pediatric population, and its frequency increases with the age of the children studied [1]. Today, in Western countries, with adverse lifestyle changes, the incidence of primary hypertension (PH) is increasing and is slowly becoming the dominant form of AH also in the developmental age [2]. PH is now believed to be not only cardiovascular disease but a multifaceted syndrome involving abnormal body fat distribution, overactive renin-angiotensin-aldosterone, sympathetic and immune systems, and premature aging (including early vascular aging) [3].

The importance of calcium-phosphate disorders, including vitamin D, in the development of AH and target-organ damage has become the subject of intensive research in recent years [4]. Although the recently published VITAL study (The Vitamin D and OmegA-3 Trial) did not demonstrate an impact of vitamin D supplementation on cardiovascular morbidity in adults [5], research is ongoing on the potential interplay between cardiovascular and bone health.

**Citation:** Skrzypczyk, P.; Ofiara, A.; Szyszka, M.; Stelmaszczyk-Emmel, A.; Górska, E.; Pa ´nczyk-Tomaszewska, M. Serum Sclerostin Is Associated with Peripheral and Central Systolic Blood Pressure in Pediatric Patients with Primary Hypertension. *J. Clin. Med.* **2021**, *10*, 3574. https://doi.org/ 10.3390/jcm10163574

Academic Editors: Katarzyna Taranta-Janusz, Kinga Musiał and Giuseppe Regolisti

Received: 15 June 2021 Accepted: 12 August 2021 Published: 13 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Sclerostin is a protein made up of 190 amino acids encoded by the *SOST* gene located on 17q12-21 chromosome, produced mainly by osteocytes, but also by chondrocytes, cementocytes, kidneys, liver, and vascular smooth muscle cells. Initially, it was thought that sclerostin belongs to the bone morphogenic protein (BMP) family [6]. However, it is now known that sclerostin, alongside the Dickkopf-1 protein (Dkk-1) is the most important inhibitor of the Wnt/beta-catenin pathway. Wnt ligands connect to the frizzled receptor and LRP5/6 (low-density lipoprotein receptor-related protein) coreceptor, stabilizing the beta-catenin structure, inducing its transfer to the nucleus, and affecting the transcription of genes encoding the proteins responsible for bone formation. Sclerostin combines with LRP5/6 coreceptors and inhibits the Wnt/beta-catenin pathway, which, in effect, reduces bone formation by inhibiting the formation of new osteoblasts and has a negative impact on the survival of already existing osteoblasts. Recent data indicate that sclerostin also stimulates osteoclast differentiation and increases bone resorption [7–9]. In 2019, the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) issued a positive opinion on the authorization of a monoclonal antibody against sclerostin (romosozumab) in postmenopausal women with osteoporosis and a high risk of fractures.

The Wnt/beta-catenin system might be the link between the bone and vascular system. Increased Wnt/beta-catenin activity was found in the experimental model of vascular calcification [10] and sclerostin expression was demonstrated in atherosclerotic plaques and calcifications within the human aorta [11]. The role of the Wnt/beta-catenin system and its endogenous inhibitors in circulatory system is unclear. Studies have shown both a positive [12,13] and negative [14,15] association, as well as no significant relationship [16,17] between sclerostin levels and arterial damage and cardiovascular risk in adults.

Considering the short duration of the disease and a small number of comorbidities, the population of children and adolescents with primary hypertension seems to be an ideal group to study the relationship between sclerostin, blood pressure, and subclinical arterial damage. Our study aims to assess serum sclerostin concentration in pediatric patients with PH and analyze its relationship with peripheral and central blood pressure, parameters of arterial damage, and parameters of calcium-phosphate metabolism.

#### **2. Materials and Methods**

#### *2.1. Study Group*

This single-center cross-sectional study included patients with PH treated in 2018– 2020 in one tertiary center of pediatric nephrology. The criterion for inclusion in the study was confirmed AH following ESH recommendations [1]. In all patients referred to the center, we confirmed hypertension using ambulatory blood pressure monitoring. Only patients with elevated both office and 24 h ambulatory blood pressure were included in the final analysis. The exclusion criteria were: secondary forms of hypertension, the coexistence of known bone pathology, abnormalities in biochemical parameters of calcium-phosphate metabolism, diabetes mellitus, chronic inflammatory disease, or acute infectious disease. Based on available literature on sclerostin, assuming a delta of 4.5 (effect size: 0.50), a 0.05 alpha level, and 80% power, we estimated the sample size at 60 patients [13,14,16,18–20]; 20 age- and sex-matched healthy subjects were included in the control group. In the statistical analysis, the control group was smaller than the study group due to the comparable variance of key parameters in both groups.

Participants were included in the study consecutively from among the patients after considering inclusion and exclusion criteria to exclude selection bias. In the analyzed period (2018–2020), there were also 31 patients with white coat hypertension and seven patients with masked hypertension who were excluded from further analysis.

#### *2.2. Ethical Issues*

The authors obtained approval of the local Bioethical Committee before initiating the research (approval no. KB/58/2016, 15 March 2016). All procedures involving human participants were in accordance with the highest ethical standards of the institutional research committee and were performed according to the Declaration of Helsinki on the treatment of human subjects and its later amendments. Informed consent was obtained from all participants' representatives and participants (≥16 years) before enrolling in the study.

#### *2.3. Clinical Parameters*

In all participants, the following clinical parameters were assessed: sex, age (years), body height (cm), body weight (kg), body mass index (kg/m2), antihypertensive medications taken, and duration of hypertension (months). Anthropometric parameters were compared with the standards for the local population and presented in the form of Zscore [21]. Overweight and obesity were defined according to WHO recommendations as a BMI Z-score above 1 and 2, respectively.

#### *2.4. Serum Sclerostin*

Serum sclerostin (ELISA enzyme-linked immunosorbent assay) was evaluated in all children studied using a kit from Biomedica Medizinprodukte GmbH, Vienna, Austria (BI-20492 Sclerostin ELISA). Blood samples were drawn on fasting from all the participants and thawed immediately after centrifugation at −84 ◦C.

#### *2.5. Biochemical Parameters*

All the participants had their calcium-phosphate metabolism evaluated on the basis of: serum concentrations of calcium (mg/dL), phosphate (mg/dL), 25-hydroxyvitamin D–25OHD (ng/mL), parathyroid hormone (pg/mL), and alkaline phosphatase activity (IU/L). In addition, the following biochemical parameters were evaluated: serum concentrations of creatinine (mg/dL), uric acid (mg/dL), total HDL- and LDL-cholesterol (mg/dL), and triglyceride (mg/dL). The estimated glomerular filtration rate (eGFR) was calculated in all patients (mL/min/1.73 m2) according to the revised 2009 Schwartz formula [22]. In line with local recommendations, 25OHD concentration was classified as severe deficiency (0–10 ng/mL), deficiency (>10–20 ng/mL), suboptimal (>20–30), optimal (>30–50 ng/mL), high (>50–100 ng/mL), and toxic (>100 ng/mL) levels [23]. Normal calcium (8.8–10.7 mg/dL), phosphate (2.8–5.6 mg/dL), parathyroid hormone (12–95 pg/mL), and alkaline phosphatase activity (45–515 IU/L) were derived from the manufacturer's reference values.

#### *2.6. Blood Pressure and Parameters of Arterial Damage*

Each participant had their blood pressure measured three times, and the mean of the second and third measurements was taken for further analysis. Blood pressure measurements were performed oscillometrically (Welch Allyn Patient Monitor, Welch Allyn, Skaneateles Falls, NY, USA) according to ESH recommendations and were analyzed using pediatric normative values [24]. All the patients studied also had 24 h ambulatory blood pressure measurement performed (Oscar 2 Suntech, SunTech Medical Inc., Morrisville, NC, USA) according to the American Heart Association guidelines [25]. The device was programmed to perform measurement every 15 min between 7 a.m. and 10 p.m. and every 30 min between 10 p.m. and 7 a.m., giving 78 measurements per 24 h. We analyzed only reports with at least 50 readings per 24 h. The mean number of successful measures was 69.3 ± 4.5 readings (from 54 to 78 readings). Activity and resting periods were assessed according to the patient's individual diaries. Systolic, diastolic, and mean pressures (SBP, DBP, MAP, respectively), blood pressure loads, and nighttime blood pressure dipping (DIP) were analyzed. DIP below 10% was considered as disturbed circadian rhythm [25]. For both office and ambulatory monitoring, the blood pressure cuff was chosen following ESH recommendations and the devices' instructions. In all individuals, the middle upper-arm circumference was measured, and an appropriate cuff was selected from the cuffs available (Welch Allyn VSM: 20–26 cm, 25–34 cm, 32–43 cm, Oscar 2 Suntech: 17–25 cm, 23–33 cm, and 31–40 cm).

Arterial structure and function tests were performed using following methods: ultrasonographic examination of the common carotid artery (ALOKA Prosound Alpha 6, Hitachi Ltd., Tokyo, Japan)—common carotid artery intima media thickness (cIMT) (mm), Z-score [26], and common carotid artery local stiffness (E-tracking); applanation tonometry (Sphygmocor, ATCOR, Sydney, Australia)—central blood pressure, pulse wave analysis, and aortic (carotid-femoral) pulse wave velocity (aPWV) (m/s) Z-score [27]. The detailed description of the methods used was presented in the previous manuscripts of the research group [4,28]. In brief, arterial structure and function measurements were performed in a quiet room with ambient temperature (20 ± 5 ◦C) after 5 min of resting. Peripheral pressure waveforms were recorded from the radial artery at the right wrist (patient in sitting position, with back supported). Once 20 sequential waveforms had been acquired, the transfer function was used to generate the central aortic pressure waveform. aPWV was measured in a supine position and calculated as a difference in the carotid-to-femoral path length divided by the difference in R wave to the foot of the pressure wave taken from the superimposed ECG and pressure tracings. The distance was measured as the distance from the right carotid sampling site to the jugular notch, subtracted from the distance from the jugular notch to the right femoral sampling site in accordance with pediatric normative values by Reusz et al. [27]. Measurements of peripheral pressure waveform and aPWV were obtained three times in each subject, and the mean value of these measurements were taken for further analysis. cIMT was measured in a patient in a supine position using a manual method approximately 1 cm proximal to the carotid bulb on the distal carotid wall. Six determinations of cIMT—three on the left and three on the right side—were obtained and averaged. All arterial measurements were performed by a single experienced investigator (P.S.).

#### *2.7. Statistical Analysis*

The results were statistically analyzed using TIBCO Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA). The numerical data obtained were presented as mean, standard deviation (SD), and interquartile range (IQR, Q1, Q3). The normality of variables was studied using the Shapiro–Wilk test. Normally distributed data were compared with Student *t*-test for independent groups and non-normally distributed data using the Mann– Whitney U test. The relationship between the two groups of variables was analyzed using Pearson correlation or Spearman rank correlation (depending on the distribution). Percentages in both groups were compared using the chi-square test and Fisher's exact test. Multivariate analysis was performed using a general linear model. Parameters correlating with each other with r > 0.600 were excluded from the final model to avoid collinearity. The results of the multivariate analysis were presented with beta coefficients, confidence intervals (CI), and *p*-value. A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Clinical, Laboratory Parameters, Blood Pressure, and Arterial Damage*

Clinical and biochemical parameters in the PH and control groups are presented in Table 1, and data supporting the results can be found in Table S1. Patients with PH had significantly higher body weight, BMI, higher serum concentrations of uric acid and triglycerides, and lower concentration of HDL-cholesterol. In the PH group, 14 (23.3%) children were overweight, and 18 (30.0%) were obese. The duration of hypertension was 19.0 ± 25.4 (3–24) months. Twenty-five subjects were treated with antihypertensive medications and amlodipine was the most commonly used drug. The blood pressure and arterial parameters analysis revealed significantly higher office central and peripheral blood pressure and 24 h ambulatory blood pressure in the PH group (Table 2). Patients with PH were characterized by significantly higher cIMT, aPWV, larger diameter of common carotid artery, and slower time to maximal artery diameter.

**Table 1.** Clinical and biochemical data of the studied children.

BMI: body mass index; eGFR: estimated glomerular filtration rate; HDL: high-density lipoprotein; LDL: low-density lipoprotein.

**Table 2.** *Cont.*

SBP: systolic blood pressure; DBP: diastolic blood pressure; AoSBP: aortic (central) systolic blood pressure; AoDBP: aortic (central) diastolic blood pressure; SBPL: systolic blood pressure load; DBPL: diastolic blood pressure load; DIP: dipping; cIMT: common carotid artery intima-media thickness; aPWV: aortic pulse wave velocity; AIx75HR: augmentation index normalized to heart rate of 75 beats per minute; SEVR: subendocardial viability ratio; beta: stiffness index; Ep: pressure strain elasticity modulus; AC: arterial compliance; AIx: augmentation index; D\_max: maximal diameter of the right common carotid artery; D\_min: minimal diameter of the right common carotid artery; DAT\_max: acceleration time to the right common carotid artery maximal diameter.

#### *3.2. Sclerostin and Parameters of Calcium-Phosphate Metabolism*

The concentration of sclerostin and parameters of calcium-phosphate metabolism are shown in Table 3. The groups did not differ significantly in sclerostin concentration (Figure 1). All the patients studied had normal calcium, phosphate, parathyroid hormone, and alkaline phosphatase levels, but the serum calcium level was significantly higher in the PH group without any other differences between the groups. Severe deficiency and deficiency of vitamin D were found in more than half (56.3%) of the children studied. In children with PH, we showed no difference in serum sclerostin concentration between boys and girls (38.2 ± 10.0 vs. 34.1 ± 11.2 (pmol/L), *p* = 0.142) and between patients treated and untreated with antihypertensive agents (34.3 ± 10.6 vs. 38.3 ± 10.4 (pmol/L), *p* = 0.151).

**Table 3.** Sclerostin and parameters of calcium-phosphate metabolism in studied children.

25OHD—25-hydroxyvitamin D.

**Figure 1.** Serum sclerostin in children and adolescents with primary hypertension and in the control group (SE—standard error, SD—standard deviation).

#### *3.3. Correlations of Sclerostin with Clinical and Biochemical Parameters, Blood Pressure, and Parameters of Arterial Damage*

As there was no significant difference in serum sclerostin between PH and healthy children, both groups were analyzed together. Correlations of serum sclerostin concentration with analyzed clinical and biochemical parameters in 80 studied children are presented in Table 4. The serum sclerostin correlated positively with the height Z-score, serum phosphate, and alkaline phosphatase activity, and negatively with systolic and mean blood pressure. Negative correlations of serum sclerostin with peripheral and central systolic blood pressure are presented in Figure 2, Figure 3, respectively. There were no significant correlations of sclerostin and markers of arterial damage (aPWV (m/s): r = −0.065, *p* = 0.567, aPWV Z-score: r = −0.062, *p* = 0.584, AIx75HR (%): r = −0.053, *p* = 0.638, cIMT (mm): r = −0.084, *p* = 0.459, and cIMT Z-score: r = −0.092, *p* = 0.417).

**Table 4.** Significant correlations of sclerostin with clinical and biochemical parameters in the study group.

SBP: systolic blood pressure; MAP: mean arterial pressure; AoSBP: aortic (central) systolic blood pressure; AoMAP: aortic (central) mean blood pressure.

**Figure 2.** Correlation of serum sclerostin with peripheral systolic blood pressure Z-score in the studied children (SBP—peripheral systolic blood pressure).

**Figure 3.** Correlation of serum sclerostin with central (aortic) systolic blood pressure in the studied children (AoSBP—aortic systolic blood pressure).

The results of multivariate analysis (general linear model) are shown in Table 5. Systolic blood pressure and height expressed as Z-scores were the significant independent determinants of serum sclerostin in the studied children.

**Table 5.** Multivariate analysis of sclerostin determinants in the studied children.

SBP: systolic blood pressure.