Assessment of Cardiac Functions and Subclinical Cardiovascular Risk In Children with Urolithiasis: An Initial Study

Information on cardiovascular problems related to childhood urinary stone disease is limited. The aim of this study is to assess the ventricular functions and subclinical cardiovascular risk in children with urolithiasis using echocardiographic measurements. Children readily diagnosed with urolithiasis were enrolled in the study as well as children with no urinary stone disease conrmed via urinary ultrasonography. Body mass index (BMI) and blood pressures were noted as well as basic serum parameters. Carotid intima media thickness (cIMT), epicardial fat tissue (EFT) thickness and periaortic fat tissue (PFT) thickness were measured via transthoracic echocardiography in addition to pulsed and tissue Doppler imaging. Myocardial performance indexes were also calculated and correlation analyses were made. A total of 17 patients (10 boys) were enrolled in the study with a mean age of 8.57 ± 2.62 years. There were 17 children (12 boys) in the control group and their mean age was 9.53 ± 1.72 years. There was no statistically signicant difference between the two groups in terms of demographic and laboratory variables. Tissue Doppler echocardiography revealed that Tei indexes of left ventricle, right ventricle and septum were signicantly higher in the study group than in the controls (p < 0.001 for all). The cIMT (0.041 ± 0.012 vs. 0.025 ± 0.002), EFT (0.432 ± 0.083 vs. 0.325 ± 0.032) and PFT thicknesses (0.138 ± 0.029 vs. 0.113 ± 0.008) of the study group were statistically higher than the control group (p < 0.001, p < 0.001 and p = 0.002, respectively) indicating a higher CVD risk.


Introduction
Urolithiasis is a global disease with an increasing prevalence in recent years and it causes signi cant morbidity for all ages. In the USA, 11% of men and 7% of women are diagnosed with urinary stone disease throughout their lives [1]. Further, recurrences may be observed in 30 to 50% of the cases in 5-10 years after the rst stone incidence [2]. As a chronic disease, urolithiasis affects 3-5% of the population whereas exact incidence in children is not known [3]. Pediatric emergency room (ER) visits due to urinary stone disease comprised 8 of 100,000 in 1996 while this increased to 18 of 100,000 in 2007 [4]. It is verywell documented that pediatric urolithiasis is strictly related to metabolic abnormalities [5]. Thus, it has higher risk for recurrence and morbidity in children [6]. Moreover, chronic kidney disease prevalence is 1.5 times higher and end stage kidney disease may be seen 2.4 times in pediatric kidney stone formers [7].
Chronic in ammation is an independent factor for subclinical atherosclerosis and thus, cardiovascular complications. It was shown that in ammatory cytokines have a role in the progression of atherosclerosis [8]. Studies in adult population indicated that urolithiasis is related to chronic in ammation such as hypertension, diabetes and metabolic syndrome. Also, it has been shown that myocardial infarction, stroke and coronary artery disease are more commonly found in patients with urinary stone disease [9]. A recent study that looked into adolescents with urolithiasis reported increased levels of urinary cytokines that point out presence of chronic in ammation [10].
Furthermore, studies showed oxidative stress and in ammation induces Randall's plaque which is the initial step in stone formation [11]. In CARDIA study, researchers revealed a positive correlation between urolithiasis and subclinical atherosclerosis in young adults [12]. To date, there is a single study that investigated atherosclerosis and cardiovascular complications in children. Carotid intima media thickness (cIMT) which is a sign of subclinical atherosclerosis was found to be increased in children with nephrolithiasis this study [13].
In current practice, cIMT is used as a marker to identify subclinical atherosclerosis as well as epicardial fat tissue (EFT) thickness and periaortic fat tissue (PFT) thickness. Several researchers demonstrated the use of these markers of subclinical atherosclerosis in different chronic diseases in pediatric population [14; 15]. The aim of this initial prospective case-control study is to assess the risk of subclinical cardiovascular risk in children with urolithiasis using cIMT, EFT and PFT thickness in addition to tissue Doppler imaging (TDI) echocardiographic evaluation of cardiac functions.

Patients
After obtaining ethical board approval (Number: 419011325-050.99), patients were prospectively included for this study beginning from December 2019 -May 2020. Patients >5 years of age and had kidney stones (>3 mm) that were con rmed by two consecutive ultrasonographic images or a computed tomography and those had normal serum chemistries were included in the study. Those with congenital heart diseases, chronic kidney disease, in ammatory bowel disease, monogenic stone phenotypes, normal serum chemistries, urogenital malformations (vesicoureteral re ux, posterior urethral valves, neuropathic bladder etc.), obesity, hypertension, chronic diseases (i.e., diabetes mellitus) and patients who were passive smokers were excluded.
Blood pressures were measured from the left arm using age-appropriate manual sphygmomanometer cuffs after 5 minutes of resting and mean of 3 measurements were noted. All blood samples including urea, creatinine, glucose, uric acid, total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) were obtained a.m. after 8 hours of fasting. Glomerular Filtration Rate (GFR) was calculated by Schwartz formula [16].
Control group consisted of health children who have normal BMI and blood pressure (adjusted for age) with no known history of urolithiasis (con rmed by urinary ultrasonography). Pulsed Doppler echocardiographic measurements Ventricular functions were evaluated using the following pulse-wave Doppler (PWD) echocardiographic parameters: early (E) and late (A) mitral/tricuspid diastolic velocities, E/A ratio, and LV/right ventricle (RV) ejection times. Standard measurement techniques were used for evaluation [17]. We calculated the myocardial performance index (MPI) using the formula, the sum of isovolumic contraction and relaxation times divided by the ejection time [19].
Tissue Doppler echocardiographic measurements Tissue Doppler velocities; peak early diastolic myocardial (e'), peak atrial systolic (a'), and peak systolic (s') myocardial velocities were measured by standard technique [17]. Also, e'/a' and E/e' ratios were calculated. The Tei index (MPI) was calculated as de ned above.

Measurement of epicardial fat tissue thickness
The EFT thickness was identi ed as an echo-free space in the pericardial layers on 2-dimensional echocardiography, and its thickness was measured perpendicularly on the free wall of the RV at end diastole from the parasternal long-axis views [20]. The mean EFT thickness was calculated from 3 consecutive measurements.

Measurement of common carotid artery intima-media thickness
Longitudinal images of the common carotid artery were obtained by combined 2-dimensional mode and color Doppler examinations. On a longitudinal echocardiographic image of the posterior wall of the carotid artery was displayed as 2 bright white lines separated by a hypoechogenic space [21]. The mean cIMT was calculated from the 3 consecutive measurements of the maximum far wall thickness obtained from 10 mm below the carotid bulb.

Measurements of periaortic fat thickness
Measurement of PFT thickness was done with conventional methods from the adventitia layer of the abdominal aorta and the adventitial layer of the aorta adjacent to the form of the measurement of the linear echogenic line. Periaortic fat tissue cannot be directly distinguished with echocardiographic and ultrasonographic images in deep tissue. Therefore, it should be measured with adventitia. Measurements were taken in the axial plane in the supine position at the L1-2 level (just above the umbilicus), proximal to the iliac bifurcation. Evaluation was repeated three times and the mean value was calculated.

Statistical Analysis
The compatibility of numerical variables to normal distribution was examined using the Shapiro-Wilk test.
Descriptive ndings were presented as number, percentage mean and standard deviation. Comparisons between groups were made by Chi-Square test for categorical variables, and t test for independent groups if assumptions were met for numerical variables, otherwise by Mann-Whitney U test. Statistical signi cance level was accepted as p <0.05.

Results
A total of 17 patients, 10 boys and 7 girls (41% and 59%), were enrolled in the study. Mean age of the study group was 8.57 ± 2.62 years (range years). Eleven (64.7%) of the patients in the study group had positive family history for urolithiasis, 10 (58.8%) had multiple stones, 6 (35.3%) had bilateral stones. Mean stone size was 8.6 ± 4.3 mm and mean follow-up period was 16.2 ± 9.3 months. In terms of metabolic abnormality, 7 (41.2%) had hypocitraturia, 4 (23.5%) had hypercalciuria, 2 (11.8%) had hyperoxaluria while no abnormalities were detected in 4 (23.5%). At the time of enrollment, 8 (47.1%) children were on medical treatment (in the form of oral potassium citrate), 4 (23.5%) and 5 (29.4%) have underwent Extracorporeal Shock Wave Lithotripsy and surgical treatment (3 ureterorenoscopic intervention,2 percutaneous nephrolithotomy), respectively. There were 12 boys and 5 girls (71% and 29%) in the control group with mean age of 9.53 ± 1.72 years. There was no signi cant difference between the two groups in terms of age, gender, blood pressure, BMI, serum lipids, hemoglobin and GFR (Table 1).
There was no statistical difference between the M-mode echocardiography, LVM and LVMI results of study and control groups (  (Table 3).

Discussion
Besides urinary tract obstruction and related renal damage, urolithiasis may also cause the release of in ammatory cytokines due to crystal storage and this has been found to be associated with cardiovascular complications [22; 23]. Clinical and experimental studies have shown that there is a strong relationship between crystal adhesion and crystal formation in renal tubular cells and in ammation and oxidative stress [11; 24]. Taguchi et al. demonstrated genes related to oxidative stress and stated that proin ammatory conditions were highly expressed in calcium oxalate stone formers than normal renal papillary tissue [25]. Same group also demonstrated that M1 macrophages (in ammatory) stimulated renal calcium oxalate crystal deposition and M2 macrophages (anti-in ammatory) limited such crystal formation in a murine model of hyperoxaluria [23].
The persistent elevated levels of in ammatory cytokines result in a chronic condition, de ned as subclinical or low-grade in ammation, which have a fundamental role in the development of cardiovascular disease. It can be assumed that systemic in ammation cause both the onset and progression of cardiovascular disease [8]. Cytokines like IL-6 and IL-8 play an important role in the development of atherosclerosis by promoting in ammatory response, progression, angiogenesis and plaque formation. IL-6 levels are a predictor for the development of cardiovascular disease in healthy individuals and thus, it may be a potential biomarker of early-stage atherosclerosis [26 ; 27].
Clinically, cIMT is used as a reliable marker in the evaluation of atherosclerotic change in the early period.
In some studies that evaluate the progression of atherosclerosis, a strong relationship been reported between cIMT and IL-6 [26]. cIMT was reported to show subclinical atherosclerosis in different chronic diseases of childhood [14]. The only study in the literature in which cIMT was evaluated in children with urolithiasis, Kusumi et al. found cIMT was signi cantly higher in children aged 12-17 years and reported that urine osteopontin and bronectin-1could predict elevated cIMT [10]. Similarly, in our study, cIMT was signi cantly higher in children with urolithiasis than in the control group, and this shows the presence of subclinical atherosclerosis in patients with pediatric urolithiasis. On the other hand, mean age of our study group was younger than those included in their study. Thus, it can be suggested that subclinical atherosclerosis begins at an even earlier age in children with urolithiasis.
Body fat distribution is an important cardiovascular risk factor, and fat depositions are associated with all-cause deaths. One component of the abnormal body fat depot, called ectopic fat, is the accumulation of adipose tissue around organs and vessels. Ectopic adipose tissue, unlike subcutaneous adipose tissue, is not an ordinary place for lipid storage [28]. Epicardial and periaortic adipose tissue, like other adipose tissues, has endocrine functions that can produce in ammatory cytokines and hormone secretions. Moreover, they have been recently identi ed as strong risk factors for cardiovascular disease due to their role in the in ammatory process in atherosclerosis [20; 28]. It was reported that EFT is a reliable parameter for cardiovascular risk in adult chronic kidney disease and EFT thickness can predict coronary artery disease [29]. In obese children, an increase in EFT thickness was found to be associated with coronary artery disease, magni ed cIMT and arterial stiffness [30]. Studies also showed that in nonobese children with neurological disabilities, EFT thickness was signi cantly higher and correlated with clinical and metabolic risk factors [31]. Akyürek et al. evaluated the relationship between PFT thickness and cardiovascular risk in 135 children with type-1 DM and they showed a positive correlation between PFT thickness and cIMT and metabolic risk factors [15]. In our study, EFT and PFT thickness were signi cantly higher in children with urolithiasis.
It is known that E/e' ratio shows the strongest correlation with LV/RV diastolic lling pressure and LV/RV compliance [32], whereas E/A and e'/a' ratio correlates with relaxation type dysfunction [33]. Limited data from the children with chronic kidney diseases revealed that left ventricular E/A and e'/a' ratios decreases and E/e' ratio increases along with the worsening of renal functions from mild-moderate to severe renal failure [34]. On the other hand, Çelik et al. reported decreased E/A and increased e'/a' ratios in non-obesetreated hypertensive patients [35]. These studies showed dysfunction of LV relaxation and diastolic lling pressures, however, right ventricular functions were not studied and possibly RV dysfunction was underestimated. In this context, our study revealed that E/A ratio measured from RV and e'/a' ratios measured from LV were signi cantly lower in children with urolithiasis when compared with healthy controls (p = 0.026 and p = 0.018, respectively). Additionally, E/e' ratio measured from LV was detected to be increased in the patient group. This nding suggests that LV function and diastolic lling pressures are worsened in children with urolithiasis.
On the other hand, MPI or Tei index is a good predictor of ventricular systolic functions in children and adults [19]. MPI measured by PWD, M-mode and TDI methods is a valuable parameter indicating systolic and diastolic functions and also early deterioration in these parameters. The results from the study by Çelik et al. showed that left ventricular MPI was higher in non-obese-treated hypertensive children, but no signi cance was achieved [35]. However, in our study MPI values of LV was signi cantly higher in patients (p = 0.013) while no statistical signi cance was shown in right ventricular MPI. Besides, both Tei index values of LV, RV and septum were found to be signi cantly increased in the study group than in the controls (in all p < 0.001). By this way, we demonstrated a signi cant reduction of systolic and diastolic functions of LV and RV in children with urolithiasis compared to healthy children.

Limitations
Main limitation of our study is the limited number of patients. Also, in ammatory cytokines have not been studied in patients which is unfortunately, beyond the scope of our facility. However, extensive echocardiographic investigation for both the study and the control group were performed in this prospective study.

Conclusions
LV and RV early systolic and diastolic dysfunction, together with subclinical atherosclerosis, were detected in children with urolithiasis in early ages. Cardiovascular complications should be considered in the follow-up and treatment of these patients, and the pediatric urolithiasis patients deserve further studies in terms of cardiovascular risks. Longitudinal studies with long-term follow-up will enlighten the adulthood consequences of these ndings.  Tables   Table 1. Demographic and laboratory data of study population.
Data are expressed mean±SD. BMI: body mass index, SBP: systolic blood pressure, DBP: diastolic blood pressure, eGFR: estimated glomerular ltration rate, LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol, CRP: C-reactive protein. Table 2. M-mode echocardiographic measurements in patients with urolithiasis and control groups.  Early (E) and late (A) mitral/tricuspid diastolic velocities, LV: left ventricle, RV: right ventricle, MPI: myocardial performance index. Table 4. Tissue Doppler echocardiographic measurements in patients with urolithiasis and control groups.