Urinary Metabolic Disorders Associated with Urolithiasis in Cuban Pediatric Patients

INTRODUCTION Pediatric urinary lithiasis (urolithiasis) is an important health issue linked to urinary metabolic disorders. In the United States alone, annual costs associated with urolithiasis are $229 million for hospital admissions and $146 million for emergency care. OBJECTIVE Identify urinary metabolic disorders in Cuban pediatric patients with urolithiasis and better understand the relationship of age, demographic and anthropometric variables to urinary metabolic disorders strongly associated with urolithiasis. METHODS We carried out a descriptive, cross-sectional study. The study universe was comprised of Cuban patients aged 2 to 19 years with urinary lithiasis who underwent renal metabolic studies at the Dr Abelardo Buch López Nephrology Institute in Havana, Cuba, from 2008 through 2019. All data were obtained from reports of the aforementioned metabolic studies. We collected the following variables: age, sex, nutritional status, urinary volume, plasma and urinary creatine concentrations; and calcium, uric acid, oxalate and citrate urinary excretions collected during a 24-hour period. We included results of urinary cystine tests and urine mini-cultures. We obtained frequency distributions for categorical and qualitative variables and calculated means and standard deviations for quantitative variables. We also evaluated homogeneity of metabolic disorders between children and adolescents. RESULTS We studied 1592 pediatric patients, of whom 67.7% (1078/1592) were adolescents. The main metabolic disorders included hypercalciuria (39.1%; 622/1592), decreased urinary fl ow (22.4%; 357/1592) and hypocitraturia (18.2%; 289/1592). Hypercalciuria, hypocitraturia and hyperoxaluria were more common in children, while decreased urinary fl ow and hyperuricosuria were more common in adolescents. Hyperuricosuria was more frequent in male patients (6.3%; 40/639 vs. 1.8%; 8/439) and had the greatest impact on lithogenesis. Hypercalciuria was more frequent in undernourished children (62.5%; 30/48) than in overweight children (21.7%; 10/46), or those with obesity (33.3%; 15/45). CONCLUSIONS The main metabolic disorders among Cuban pediatric patients with urinary lithiasis are: hypercalciuria, decreased urinary fl ow and hypocitraturia. Hypercalciuria, hypocitraturia and hyperoxaluria are more common in children, and decreased urinary fl ow and hyperuricosuria are more common in adolescents. Identifying urinary metabolic disorders facilitates formulation of treatment plans tailored to decreasing the likelihood of urolithiasis.


INTRODUCTION
Urinary lithiasis is an important health problem in the general population and, although its occurrence is comparatively rare in pediatric ages, evidence suggests that its incidence is increasing in this population. [1,2] Additionally, the disease is characterized by high recurrence rates that can require surgical intervention and is associated with high treatment costs and the risk of developing renal dysfunction requiring renal replacement therapy (dialysis or transplantation). [3] Added to these risks is the decrease in bone mineral density in patients who develop calcium stones, which increases the risk of fractures and can compromise patients' quality of life. [4] Although overall incidence of pediatric hospital admissions for urinary lithiasis in high-income countries is 1%-3%, [5] the USA has experienced an increase from 18.4 cases per 100,000 admissions in 1999 to 57 cases per 100,000 admissions in 2008, an adjusted annual increase of 10.6%. [1,5] Estimated recurrence rates in that country range from 16% to 67%, [6] and annual estimated costs for pediatric patients with urinary lithiasis are $229 million in hospital admissions and $146 million for emergency care. [7] Development of urinary stones in children and adolescents is associated primarily with urinary metabolic disorders and urinary tract malformations. [8] Consequently, patients presenting with urolithiasis must be rigorously evaluated in order to identify underlying urinary metabolic disorders promoting lithogenesis, and treatments must be tailored to prevent recurrence. [8,9] Data are scarce on metabolic disorders in the Cuban and Latin American pediatric populations, making it diffi cult to implement strategies for primary and secondary prevention of urolithiasis. [9][10][11] The objective of this study was to identify urinary metabolic disorders in the Cuban pediatric population with urolithiasis according to age, demographic and anthropometric variables, and the relation of these variables to disorders with the greatest lithogenic potential.

Design and participants
We conducted a descriptive, crosssectional observational study. The study universe consisted of all Cuban patients aged 2-19 years who presented with urinary lithiasis (demonstrated by imaging studies, or by spontaneous or instrumental elimination) who underwent renal metabolic studies at the Dr Abelardo Buch López Nephrology Institute in Havana, Cuba from January 2008 through December 2019.

IMPORTANCE
This study comprises the best evidence currently available in Cuba on urinary metabolic disorders predisposed to causing urolithiasis in children and adolescents, and offers insights for its primary and secondary prevention.

Original Research
Individuals who had suffered renal colic in the six weeks prior to recruitment or who had poorly collected samples were excluded from our research, as were pregnant individuals, foreign patients, vegetarians, amputees, those with chronic diarrhea or muscular dystrophies, or who were short for their age, according to Cuban height-for-age tables. [12] These patients were excluded because they presented with factors that could either interfere with the quality of the urine sample or could modify excretion of urinary solutes.
Variables Age (two groups: 2-9 years and 10-19 years); sex (female, male); nutritional status (undernourished, thin, normal weight, overweight, obese); urinary volume; plasma and urinary creatinine concentrations; urinary excretion of calcium, uric acid, oxalate and citrate over a 24-hour period; and the results of urinary cystine tests and urine mini-cultures.
Procedures We interviewed all patients (or patients' caregivers, if patients were pre-verbal) to ensure that they did not meet any exclusion criteria. All medications were suspended for fi ve days before the study. We collected 24-hour urine samples (in washed bottles with 5% thymol included as a preservative) and 5 mL fasting blood samples. Independent urine samples were used for the mini-cultures. We collected anthropometric data (height and weight), estimated the nutritional status of patients according to body mass index (BMI) in kg/m 2 , and classifi ed patients according to the Cuban BMI-for-age tables. [13] Body surface area (BS) was calculated using the Dubois-Dubois formula. [14] A urine sample was considered 'well-collected' when creatinine excretion was less than ±1 standard deviation (SD) from the reference value according to the patient's age, height and sex, as pathologies that may infl uence the generation and excretion of creatinine were exclusion criteria. [15] Analytical determinations were made with a Microlab 300 spectrophotometer (Elitech Clinical Systems, Germany) in the case of urinary citrate and oxalate. For all other analyses, we used a HumaStar 80 spectrophotometric autoanalyzer (Human Diagnostics, Magdeburg, Germany). We followed manufacturer's guides for both. Citrate and oxalate concentrations were determined via direct ferric cyanide/ citrate reaction or formic acid/chromotrophic reaction methods, respectively. Creatinine was measured using the Jaffé kinetic method (alkaline picrate); uric acid using the enzymatic uricase/peroxidase method; and calcium using the calciumarsenazo method. All reagents were provided by the Carlos J. Finlay Pharmaceutical Laboratory Company in Havana, Cuba. Urinary cystine tests were performed by reaction with sodium nitroprusside (Merck, Darmstadt, Germany). Urine mini-cultures were cultured in MacConkey's Agar and CLED (cysteine lactose electrolyte defi cient) Agar media (National Biopreparations Center, BIOCEN, Cuba).
Renal function was measured by creatinine clearance in mL/ min/1.73m 2 body surface (BS) and serum creatinine in mg/dL. Cystinuria and urinary tract infections were diagnosed by qualitative methods, the results used to classify patients as positive or negative.

Metabolic disorder diagnostic cut-off values
Hypercalciuria Urinary calcium >4.0 mg or 0.10 mmol/kg/day Hyperuricosuria Uric acid >815 mg or 4.85 mmol/1.73 m 2 / day Hyperoxaluria Urinary oxalate >45 mg or 0.5 mmol/1.73 m 2 /day Hypocitraturia Daily urinary citrate <365 mg or 1.90 mmol /1.73 m 2 in males and <310 mg or 1.61 mmol/1.73 m 2 in females Low urinary fl ow Urinary volume <20 mL/ kg /day [16] Data collection and analysis We obtained information for the above selected variables from the fi rst metabolic study conducted on each patient. All information was processed using the Statistical Package for Social Science (SPSS) version 22.0 for Windows (IBM, USA). We constructed frequency distributions for qualitative variables and calculated absolute frequencies and percentages. For quantitiative variables, we calculated means and standard deviations. The age distribution was divided into quartiles. In order to analyze associations between patient demographics and nutritional status, and metabolic disorders with the greatest lithogenic potential (hypercalciuria, hyperuicosuria, hyperoxaluria and hypocitraturia) we constructed double-and triple-entry contingency tables to provide a descriptive analysis of homogeneity.
Ethics This study was approved by the Scientifi c Council and the Research Ethics Committee of the Dr Abelardo Buch López Nephrology Institute. Confi dentiality of all data obtained during the investigation was guaranteed.

Original Research
in the relative frequencies of hypercalciuria, hypocitraturia and hyperoxaluria, which were higher in children; and also for decreased urinary fl ow and hyperuricosuria, which were more common in adolescents. Urinary tract infection was low in overall frequency and similar between the two groups ( Table 2).
In terms of frequency of urinary metabolic disorders, it should be noted that we found minimal differences between the sexes in either children or adolescents. The greatest differences were found in hypocitraturia in children: 45.5% (101/222) in girls versus 37.3% (109/292) in boys, and in hyperuricosuria in adolescents; 1.8% (8/439) in female patients versus 6.3% (40/639) in male (Table 3).
There were notable differences in the frequencies of hypercalciuria and hypocitraturia in children and in the frequencies of hyperuricosuria and hypocitraturia in adolescents (Table 4).
There were also appreciable differences for hypercalciuria and hypocitraturia in children, and for hypercalciuria, hyperuricosuria and hypocitraturia in adolescents when comparing disorder frequencies with groups according to patient nutritional status (      Peer Reviewed

Original Research
The higher frequency of urolithiasis we found in the male sex coincides with results from both national and international epidemiological studies in adults. [18,19] However, the results in the pediatric population are less consistent. [20][21][22] Prevalence of lithiasis has also been described in adolescents, [23] and it is believed that the recent increase of the disorder in pediatric patients has occurred mainly within this age group. However, we can make no such assertions in the context of this study, since it was not conducted in an open population but only in urolithiastic patients who underwent urinary composition studies. It should also be noted that while urinary composition studies may have been conducted during adolescence, a fi rst lithiastic episode could have happened during childhood. But to conduct such tests in children is diffi cult, as problems with 24-hour urine collection are more common in younger individuals. [24] Worth noting is that approximately one fi fth of the children in our study were in either the thin or undernourished categories. This may be due to the fact that several causes of lithiasis during childhood have an impact on nutritional status. One of the most important is renal tubular acidosis, whose incidence peaks during childhood and is a known cause of malnutrition in children. We were not able to explore this possible link since our research did not include urinary acidifi cation studies. [25] It is also noteworthy that nearly a quarter of adolescents were either overweight or obese, both well-known risk factors for lithogenesis in adults and whose rates have increased in adolescent populations worldwide. [26] Renal function was preserved in the vast majority of patients, to be expected given their young age, as the damage to renal function caused by stones in the urinary tract is cumulative, and often due to obstructive nephropathy. Another consideration is that urinary tract infections are often associated with lithiasis, which can contribute to impaired kidney function. [27] However, in this study, the frequency of urinary tract infections was low and similar between all groups.
In the absence of 24-hour urinary composition studies in healthy Cuban children, for this study we used internationally-accepted reference values in defi ning each disorder. [16] One problem with these defi nitions is that the risk of lithiasis increases with an increase in excretion or in concentration of the metabolite in question, which does not necessarily occur within a defi ned threshold, even when healthy individuals have been used to determine these thresholds. [20] Frequencies of hypocitraturia and low urinary flow are higher in the pediatric populations with lithiasis than in the Cuban adult population with lithiasis. However, frequency of hyperuricosuria and urinary infections is higher in the adult population, consistent with findings in other studies. [8,28,29] Relative frequencies of urinary metabolic disorders in pediatric patients are similar to those found by Celiksoy in Turkey [30] and Amancio in Brazil. [10] Other studies, including Yang in China, [31] and Akhavan in Iran, [32] found hypocitraturia to be the most common disorder. These differences may be due to dietary habits or genetics.
Unfortunately, relative frequencies of disorders in these kinds of studies are not usually stratifi ed in children and adolescents. Our fi ndings are similar to those of the Cambareri study, which was carried out in four US centers and identifi ed higher frequencies of hypercalciuria and hyperoxaluria in children ≤10 years of age and decreased urinary fl ow in those over the age of 10. [33] Our fi nding of low urinary fl ow in adolescents stands out, and its cause should be investigated. This could be due to engaging in demanding physical activity without adequate hydration, which is common in school, sports and military settings, or to the trend in these age groups, as identifi ed in the general pediatric population in the United States, to fail to comply with dietary recommendations. [34] We consider promoting increased water intake a fundamental therapeutic recommendation for these age groups, and vital for both primary and secondary urolithiasis prevention.
The low frequency of cystinuria found in this study is consistent with fi ndings in Cuban adults with lithiasis, [28] and contrasts with other studies in China and Turkey, revealing considerably higher frequency. This may be due to genetics underlying the frequency of cystinuria, which exhibits great variability between populations with different ethnic origins. [8,30,31] The higher frequency of hyperuricosuria in male adolescents seems to be linked to increased concentration of male sex hormones that occurs in the second decade of life. These hormones do not have a direct effect on the frequency of hyperuricosuria, but act through other metabolic mediators. [21,35] Uric acid is produced in the liver as a byproduct of digestion, and, as it cannot be metabolized by humans, constitutes a fi nal metabolic product. This product is then eliminated in the gastrointestinal tract by bacteria that break down uric acid (intestinal uricholysis), a process that is responsible for one third of excretion (the remaining two thirds are excreted in urine). [35] Endogenous production of uric acid is the element that seems to be the most heavily infl uenced by sex hormones and thus may be responsible for an increase in urinary excretion (hyperuricosuria), which occurs in higher frequencies among overweight and obese adolescents, both of which are nutritional states that are linked to higher incidence rates of hyperuricemia and hyperuricosuria. [36] Both children and adolescents experience development through notable changes in relatively short periods of time, so it is important to understand how frequencies of disorders most prone to lithogenesis vary by age subgroups. However, there are no other studies that include comparisons between age groups, probably due to the relatively large number of patients that would be required to perform this stratifi cation.
The higher frequency of hypercalciuria in children aged <7 years is consistent with Bergland's fi ndings in male children aged <8 years, [37] and other evidence pointing to decreased urinary calcium excretion with aging. [38] No dietary modifi cations were made before this study was carried out, so hypercalciuria could not be classifi ed into renal, intestinal or bone (resorptive hypercalciuria). [20] The increased rate of hypocitraturia in earlier ages, both in children and adolescent subgroups, is likely due to a progressive increase in urinary citrate excretion with age, as identifi ed by Kirejczyk in a study of 2334 Polish children and adolescents. [39] This is of particular interest, considering the trend pointing to increased frequency of hypocitraturia in several studies of lithiasis patients. This increased rate also points to the importance of urinary citrate, which is the most abundant organic anion in urine and is a notable inhibitor of urinary calculi, as it bonds to ionic calcium and reduces urinary calcium saturation, which in turn prevents the growth of calcium crystals in addition to promoting an increase in urinary pH that decreases uric acid lithogenesis. [9,16,30,32,40] In light of the results of this study, young children and younger adolescents would benefi t the most from oral citrate salt supplements.
Although the infl uence of diet on urolithiasis is well known, surprisingly little work has been done exploring the relationship between urolithiasis and nutritional status. [41] This makes it diffi cult to draw larger comparisons using the results of this study.
Association between hypercalciuria and malnutrition has been studied in particular disorders, like renal tubular acidosis, but at least part of this association is due to a defi ned calcium cut-off value of 4 mg/kg/day for this disorder, as undernourished and lean individuals, who as a matter of defi nition have lower body weights than their peers, are diagnosed with hypercalciuria despite having levels of calcium excretion similar to that of other children their age. [16,25] Added to this is new evidence suggesting monogenic causes of urolithiasis, many of which are characterized by the presence of hypercalciuria, which in turn may affect patient nutritional status. [42] Association of hypocitraturia with thinness and undernutrition have been classically attributed to states of chronic metabolic acidosis that cause increased proximal citrate reabsorption and tissue catabolism. [20,25] Other causes include chronic diarrhea, use of drugs infl uencing urinary acidifi cation (which were not considered in our study, due to exclusion criteria), and distal renal tubular acidosis (including incomplete forms). [20,43] Although we did not evaluate urinary acidifi cation for the purpose of diagnosing renal tubular acidosis, and this precludes us from making categorical attributions, we are aware that idiopathic hypocitraturia must have made at least some contribution to our study results. [25] Association of hyperuricosuria with obesity in adolescents was expected, as the relationship between obesity and increased trunk fat with hyperuricemia and hyperuricosuria is known. This has been proposed as an explanation for the increased incidence of lithiasis in adolescents, although not all studies have drawn this conclusion. [36,[44][45][46] Hyperuricosuria not only causes uric acid lithiasis, but also urinary calculi. Obesity and states of insulin resistance create conditions of acidic urinary pH, which in turn favors the formation of urate crystals that can constitute nuclei leading to the formation of uric and calcium stones (a phenomenom known as epitaxis). [43,47,48] Prevention and treatment of obesity in adolescents should be included in guidelines for preventing pediatric lithiasis.
The main limitations of this study are the use of only one 24-hour urine sample per patient (more samples are ideal, but these are diffi cult to obtain in children), [49] the poor representation of children in the total study sample, the use of international cut-off values in defi ning metabolic disorders and the failure to study urinary acidifi cation or control patients' diets prior to sample collection, which would have allowed for greater diagnostic precision. However, this research included a considerable number of individuals from across Cuba, with a standardized study protocol, and constitutes the best evidence available for the country on urinary metabolic disorders prone to causing urolithiasis in the pediatric population. It can thus serve as a reference in designing strategies for primary and secondary urolithiasis prevention in Cuban pediatric patients.