Serum α-Klotho as a Potential Biomarker in Pediatric Urolithiasis

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The α-Klotho protein, known for its role in mineral homeostasis and oxidative stress modulation, has been implicated in adult urolithiasis, but its relevance in children remains unexplored. Aim of the study was to assess serum α-Klotho levels in children with urinary stone disease and evaluate its potential as a biomarker associated with clinical features and risk of recurrence. Methods: A prospective study was conducted in 40 pediatric patients with urolithiasis and 40 healthy controls. Serum α-Klotho levels were measured using ELISA. Additional parameters included calcium-phosphate metabolism, 24-hour urine collections, treatment type, recurrence, and modifiable risk factors. Results: Serum α-Klotho levels were significantly lower in children with urolithiasis compared to controls (1622 vs. 2184 pg/ml, p = 0.017). Lower α-Klotho levels were associated with positive family history ( p = 0.049), recurrence of renal colic ( p = 0.001), and need for surgical intervention during recurrence ( p = 0.029). α-Klotho levels positively correlated with serum phosphate ( p = 0.045) but showed no significant relationship with other laboratory results. Increased physical activity was associated with higher Klotho levels ( p = 0.023), while no association was found with diet or fluid intake. Conclusions: This is the first study to investigate α-Klotho in pediatric urolithiasis. Our findings suggest that reduced serum α-Klotho levels may precede metabolic abnormalities and correlate with recurrence risk, supporting its potential role as an early biomarker in children with urinary stone disease. Klotho pediatric urolithiasis urinary stone disease biomarker recurrence calcium-phosphate metabolism Figures Figure 1 Figure 2 Figure 3 Introduction Urolithiasis, affecting approximately 2% of children, is an emerging health concern in the pediatric population [ 1 – 2 ]. This condition, classified among lifestyle-related diseases, is characterized by a complex and multifactorial etiology involving both genetic and environmental components [ 2 ]. The formation of urinary stones is a multistep process influenced by imbalances between promoters and inhibitors of crystallization, oxidative stress, injury to renal tubular cells, and enhanced generation of reactive oxygen species (ROS) [ 3 , 4 ]. The majority of stones contain calcium compounds, primarily in the form of oxalates and phosphates, underscoring the critical role of calcium-phosphate homeostasis in the pathogenesis of urolithiasis [ 1 – 4 ]. The discovery of the KLOTHO gene in 1997 marked a turning point in understanding the interplay between aging, calcium-phosphate axis and metabolic disorders [ 5 ]. Experimental suppression of KLOTHO expression in mice resulted in a dramatic reduction in lifespan—from two years to approximately two months—and the development of a premature aging phenotype, along with biochemical alterations such as hyperphosphatemia and hypercalcemia [ 5 – 8 ]. These findings identified α-Klotho protein as a key regulator of calcium-phosphate metabolism and an endogenous modulator of oxidative stress [ 6 – 8 ]. Its deficiency has been implicated in several pathophysiological processes, including chronic kidney disease, cardiovascular conditions, and more recently, urolithiasis [ 6 , 9 – 14 ]. The aim of the present study is to evaluate serum α-Klotho concentrations in children with urolithiasis and to analyze their association with clinical and laboratory parameters of the disease, as well as to investigate the potential utility of α-Klotho as a diagnostic and monitoring biomarker in this context. To the best of our knowledge, this is the first study to assess α-Klotho protein levels in pediatric patients with urolithiasis. Existing evidence on this topic is extremely limited and based solely on adult populations [ 6 , 15 – 21 ], highlighting a significant knowledge gap in pediatric nephrology. Materials and Methods 2.1. Study Design and Patient Assessment This study included 40 children aged 0–18 years hospitalized due to urolithiasis, either symptomatic or incidentally detected during imaging studies. The control group comprised 40 children with no history of urolithiasis. Family and medical histories were collected for all participants. Children with comorbidities or chronic disorders—particularly nephrological, endocrinological, autoimmune, or gastrointestinal conditions that could influence serum α-Klotho levels—were excluded from the study. Body weight and height were measured, and body mass index (BMI) was calculated and interpreted using age- and sex-specific percentile charts. Based on these, children were classified as underweight ( 97 percentile). In the urolithiasis group, modifiable risk factors such as fluid intake, dietary habits, and physical activity were assessed using a structured questionnaire. Stone characteristics, including size and location, were determined by abdominal ultrasound. The type of treatment—conservative or surgical—was recorded. Serum α-Klotho concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) with the Human Soluble α-Klotho Assay Kit (Immuno-Biological Laboratories, Japan), according to the manufacturer’s instructions. Additional serum parameters included ionized and total calcium, phosphorus, sodium, potassium, magnesium, creatinine, urea, 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), and alkaline phosphatase. Twenty-four-hour urine collections were analyzed to assess the daily excretion of calcium, phosphorus, sodium, magnesium, uric acid, and creatinine. A follow-up was conducted six months after enrollment, consisting of a clinical interview to determine the frequency of renal colic episodes, hospitalizations, and any interventions performed within that period. 2.2. Ethical Considerations The study was approved by the Local Bioethics Committee (PCN/CBN/0022/KB1/25/I/20/21). Written informed consent was obtained from parents or legal guardians, and assent was acquired from children capable of understanding the study objectives. The research was conducted in accordance with the principles outlined in the Declaration of Helsinki. 2.3. Statistical Analysis Statistical analysis was performed using MedCalc software (version 18.1, MedCalc Software Ltd., Ostend, Belgium). Qualitative variables were presented as counts and percentages; continuous variables were expressed as medians (Me) with interquartile ranges (IQR). The Shapiro-Wilk test was used to assess the distribution of continuous variables. The chi-square test was used for categorical variables, while the Mann-Whitney U and Kruskal-Wallis tests were applied to compare continuous variables between groups. Correlations were evaluated using Spearman’s rank correlation coefficient (rho), with a p -value < 0.05 considered statistically significant. Results 4.1. Characteristics of the Study Group The study included 40 pediatric patients diagnosed with urinary tract stones. Detailed characteristics of the study population are presented in Table 1 . Table 1 Characteristics of the study group. Categorical variables are presented as counts and percentages, while continuous variables are expressed as medians with interquartile ranges (IQR); BMI – body mass index; URSL – ureteroscopy with laser lithotripsy; DJ – double-J; ESWL – extracorporeal shock wave lithotripsy. Variable Study Group sex (female) 25 (62.5%) age [years] 15 (11–16) weight [kg] 55 (43.75–67.5) height [cm] 161.5 (157–171) BMI underweight 4 (10%) lean 3 (7.5%) normal weight 25 (62.5%) overweight 4 (10%) obese 4 (10%) family history 30 (75%) Localization of calculi Ureter 20 (50%) Kidney 11 (27.5%) ureter + kidney 8 (20%) bladder 1 (2.5%) Type of stone management conservative 20 (50%) URSL 15 (37.5%) DJ stent 4 (10%) ESWL 1 (2.5%) Follow-up Recurrence of renal colic 13 (32,5%) Hospitalization 12 (30%) Urological intervention 3 (7.5%) 4.2. Klotho Protein in Pediatric Patients with Urolithiasis Significant differences in serum α-Klotho concentrations were observed between the urolithiasis group and the control group, as shown in Table 2 . Additional significant differences were found among patients based on family history, the presence of recurrence, and the type of treatment during subsequent episodes. Table 2 Differences in α-Klotho serum levels between the study and control groups, as well as among subgroups within the study group. Variables are presented as medians (Me) with interquartile ranges (IQR). Comparison Group Klotho [pg/ml] p-value Study vs. Control group 1622 (1065–2351) vs. 2184 (1375–3442) 0.017 Family history (Positive vs. Negative) 1531 (488–5866) vs. 2118 (577–9129) 0.049 Initial treatment (Surgical vs. Conservative) 1822 (488–9129) vs. 1577 (558–4684) 0.570 Recurrence (Yes vs. No) 1054 (488–2323) vs. 1843 (577–9129) 0.001 Recurrence treatment (Surgical vs. Conservative) 753 (558–1115) vs. 1742 (488–9129) 0.029 The median α-Klotho serum level in patients with urolithiasis was 1622 pg/ml (IQR: 1065–2351), which was significantly lower than in the control group (2184 pg/ml, IQR: 1375–3442; p = 0.017). Patients without a family history of stone disease had significantly higher Klotho levels (2118 pg/ml, IQR: 577–9129) than those with a positive family history (1531 pg/ml, IQR: 488–5866; p = 0.049). No significant difference in Klotho levels was found between patients treated conservatively and those who underwent endoscopic urological procedures ( p = 0.570). However, patients who experienced a recurrence of renal colic within six months of the initial hospitalization showed significantly lower Klotho levels (1054 pg/ml, IQR: 488–2323) compared to those without recurrence (1843 pg/ml, IQR: 577–9129; p = 0.001). Similarly, children who required a urological intervention during recurrence had lower Klotho concentrations (753 pg/ml, IQR: 558–1115) than those managed conservatively (1742 pg/ml, IQR: 488–9129; p = 0.029). A moderate inverse correlation was identified between serum Klotho levels and age among patients with urolithiasis (rho = -0.476, p = 0.002) (Fig. 1 ), whereas no significant correlation was observed in the control group (rho = -0.247, p = 0.125). 4.3. Klotho Protein and Laboratory Parameters in Pediatric Urolithiasis The majority of patients demonstrated normal calcium-phosphate metabolism, with the exception of a high prevalence of vitamin D deficiency ( n = 34/40; 85%). Hyperuricemia was observed in nearly half of the patients. All children maintained normal renal function, as reflected by normal glomerular filtration rates. Most had no significant abnormalities in 24-hour urinary excretion of electrolytes, apart from relatively frequent hypomagnesuria ( n = 19/40; 47.5%) and occasional hypercalciuria ( n = 7/40; 17.5%). A statistically significant positive correlation was found between Klotho levels and serum phosphorus (rho = 0.318, p = 0.045), suggesting a link between Klotho and phosphate metabolism (Fig. 2 ). No significant correlations were observed between Klotho and other metabolic parameters, including total calcium (rho = 0.276, p = 0.085), PTH (rho = -0.104, p = 0.523), or vitamin D (rho = 0.097, p = 0.552). Furthermore, Klotho levels did not correlate with urinary excretion of calcium, phosphorus, or magnesium. 4.4. Klotho Protein and Modifiable Risk Factors for Urolithiasis in Children A statistically significant trend was observed indicating that higher physical activity was associated with elevated Klotho levels ( p = 0.023), with a notable difference emerging only in patients engaging in 3–7 physical activity sessions per week (H = 9.558, df = 3) (Fig. 3 ). No significant associations were found between Klotho levels and other modifiable risk factors, including adequate fluid intake ( U = 134, p = 0.316), frequent consumption of salty snacks, fast food ( U = 128, p = 0.065), or oxalate-rich foods ( U = 158, p = 0.348). Discussion 5.1. Serum Klotho level as a potential biomarker in children at risk for urinary stone disease The assessment of serum α-Klotho protein levels as a potential marker for urinary stone risk aligns with a growing body of research focused on identifying sensitive, reproducible, and clinically relevant biomarkers of pathophysiological processes underlying chronic and lifestyle-related diseases. Over the past decade, significant advances in the understanding of urolithiasis pathogenesis have highlighted the roles of inflammation, oxidative stress, and calcium-phosphate dysregulation in the initiation and recurrence of stone formation [ 3 , 22 ]. In animal models, renal crystal deposition has been shown to trigger the production of reactive oxygen species (ROS), activation of inflammasomes, and a proinflammatory cascade, leading to tissue damage and the formation of Randall's plaques [ 3 , 22 ]. In this context, the α-Klotho protein has emerged as a nephroprotective factor, with its antioxidant properties reducing mitochondrial oxidative stress and enhancing the activity of cellular antioxidant enzymes [ 4 , 15 – 17 ]. Α-Klotho has been extensively studied in chronic kidney disease (CKD), where its deficiency has gained recognition as an early biomarker of renal injury—even before classical laboratory abnormalities emerge [ 23 ]. The first clinical reports linking α-Klotho to urolithiasis appeared in the last decade, initially focusing on gene polymorphisms. Telci et al., Gürel et al., and Lanka et al. demonstrated associations between specific KLOTHO gene variants and an increased risk of urinary tract stones, particularly through mechanisms involving hypercalciuria and phosphate handling [ 18 – 20 ]. Xu et al. further suggested that certain KLOTHO polymorphisms may increase α-Klotho expression, providing a protective effect by preventing calcium oxalate crystal formation in renal epithelial cells [ 21 ]. Subsequent studies have evaluated circulating α-Klotho levels in adult patients with urolithiasis, showing that lower serum α-Klotho concentrations correlate with increased oxidative stress and stone recurrence [ 4 , 15 , 17 ]. Gurtan et al. reported the lowest α-Klotho levels and the highest oxidative stress index (OSI) in patients with recurrent stones, findings consistent with our observations in the pediatric population [ 4 ]. In our study, children with urolithiasis had significantly lower α-Klotho levels than controls. Furthermore, children with a positive family history or recurrent renal colic episodes demonstrated even lower α-Klotho levels, supporting its potential role as a prognostic marker for recurrence. This aligns with findings in adults and expands current understanding into the pediatric domain—to our knowledge, this is the first study to assess α-Klotho levels in children with urinary tract stones. It is well known that α-Klotho protein levels decrease with age; however, this relationship has not been thoroughly studied in children, and the available research provides inconclusive results [ 11 , 24 , 25 ]. In our study, we observed a slight decline in α-Klotho levels with age, but only in the group of children with urolithiasis. No such correlation was found among healthy children. This moderate yet significant association is consistent with the literature, which indicates that the physiological decline in α-Klotho levels during childhood and adolescence is not as pronounced as in adults [ 11 , 24 , 25 ]. A marked decrease in its concentration in children is primarily observed in the context of specific disease entities [ 11 ]. Among these, particularly in chronic kidney disease (CKD), α-Klotho has been much more extensively studied in pediatric populations [ 23 , 26 – 32 ]. Therefore, it may be reasonable to relate these findings to urolithiasis, although research on this condition in children remains limited. Although the classical approach to metabolic evaluation in stone disease involves serum calcium-phosphate balance and 24-hour urinary excretion of electrolytes [ 2 ], our study focused on α-Klotho as a novel biomarker. Consistent with adult studies [ 4 ], we found that hypercalciuria was present in only a minority (17.5%) of patients, while hypomagnesuria was more common (47.5%). We did not observe significant correlations between α-Klotho levels and 24-hour urinary excretion. The only statistically significant association was a positive correlation between serum phosphorus and α-Klotho, in line with α-Klotho’s known role in phosphate regulation [ 8 ]. This may suggest that reduced α-Klotho levels precede abnormalities in other classical parameters, making α-Klotho a potential early marker of urolithiasis. The idea that α-Klotho could serve as an early biomarker of stone risk and recurrence is inspired by its diagnostic and monitoring potential in CKD. Wan et al. demonstrated that in pediatric CKD patients, serum α-Klotho levels decline progressively with disease severity and are responsive to interventions such as kidney transplantation [ 23 ]. It is plausible that similar mechanisms operate in urolithiasis, especially in recurrent cases. In our study, children with stone recurrence had significantly lower α-Klotho levels, suggesting that low baseline α-Klotho may reflect greater vulnerability to recurrent renal colic and hospitalization. Although no differences were observed in α-Klotho levels based on initial treatment method (surgical vs. conservative), recurrence appeared more informative for predicting long-term risk. This further supports the use of α-Klotho as a monitoring tool rather than a marker of stone burden per se. We also explored associations between α-Klotho and modifiable lifestyle-related risk factors. Children with obesity had notably lower α-Klotho levels, consistent with adult data linking reduced α-Klotho to obesity and metabolic syndrome [ 33 – 35 ]. Our findings diverge from the only pediatric study by Socha-Banasiak et al., who reported elevated α-Klotho levels in obese children—though the authors themselves note this contradiction with adult data and call for further research [ 36 ]. Our results additionally confirmed that regular physical activity (3–7 sessions/week) was associated with higher α-Klotho levels. This is in line with a Brazilian meta-analysis identifying α-Klotho as an “exerkine”—a protein upregulated in response to physical exercise [ 37 ]. While the mechanisms remain unclear, skeletal muscle-derived cytokines may play a role in inducing renal or systemic α-Klotho expression [ 37 ]. Other dietary and lifestyle factors, including fluid intake and consumption of salty or oxalate-rich foods, showed no clear association with α-Klotho levels in our cohort. Nonetheless, given their well-established role in stone pathogenesis, early health education targeting these behaviors remains crucial [ 2 ]. 5.2. Study Limitations This study has several limitations. First, the sample size was relatively small, reflecting its design as a pilot study. Nonetheless, the observed trends are promising and justify further research in larger, multicenter pediatric populations. Second, lifestyle variables such as fluid intake, diet, and physical activity were assessed using questionnaires completed by patients and their parents, introducing a degree of subjectivity. While these tools provided valuable insight into modifiable risk factors, the data should be interpreted cautiously, and future studies should aim for more objective measurement methods where feasible. Summary Our study demonstrated that children with urinary stone disease exhibit significantly lower serum levels of α-Klotho protein compared to their healthy peers, underscoring the potential role of this protein in the pathogenesis of urolithiasis. These findings suggest that α-Klotho may serve as an early biomarker of stone formation, potentially preceding detectable changes in traditional laboratory parameters. This hypothesis is further supported by analogous findings in studies on chronic kidney disease in pediatric populations. Although no significant differences in α-Klotho levels were observed between children treated conservatively and those undergoing surgical interventions, lower α-Klotho concentrations were consistently associated with stone recurrence. This indicates a possible prognostic role for α-Klotho in identifying patients at risk for recurrent renal colic and hospitalization. This observation holds particular relevance in pediatric practice, where early diagnosis and close monitoring are essential for long-term care and prevention of kidney damage. A comprehensive approach—including assessment of metabolic risk factors, timely treatment of stones, and early implementation of preventive strategies—is vital to safeguard renal health across the lifespan. The identification and validation of novel biomarkers such as α-Klotho may significantly enhance these efforts, contributing to more effective disease monitoring and improved outcomes in children with urolithiasis. Declarations Competing Interests: There is no conflict of interests. 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Nutrition. 2013 Sep;29(9):1106-9. doi: 10.1016/j.nut.2013.02.005. Epub 2013 Jun 19. PMID: 23790542. Liu Y, Chen M. Emerging role of α-Klotho in energy metabolism and cardiometabolic diseases. Diabetes Metab Syndr. 2023 Oct;17(10):102854. doi: 10.1016/j.dsx.2023.102854. Epub 2023 Sep 14. PMID: 37722166. Orces CH. The Association of Obesity and the Antiaging Humoral Factor Klotho in Middle-Aged and Older Adults. ScientificWorldJournal. 2022 Aug 24;2022:7274858. doi: 10.1155/2022/7274858. PMID: 36061981; PMCID: PMC9433301. Socha-Banasiak A, Michalak A, Pacześ K et al. Klotho and fibroblast growth factors 19 and 21 serum concentrations in children and adolescents with normal body weight and obesity and their associations with metabolic parameters. BMC Pediatr. 2020 Jun 16;20(1):294. doi: 10.1186/s12887-020-02199-2. PMID: 32546231; PMCID: PMC7296965. Corrêa HL, Raab ATO, Araújo TM et al. A systematic review and meta-analysis demonstrating Klotho as an emerging exerkine. Sci Rep. 2022 Oct 20;12(1):17587. doi: 10.1038/s41598-022-22123-1. PMID: 36266389; PMCID: PMC9585050. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7294779","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499604226,"identity":"b33db650-00d5-4288-b2f3-1e51f4ab66ab","order_by":0,"name":"Agnieszka Wiernik","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4405-0822","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":true,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Wiernik","suffix":""},{"id":499604227,"identity":"be6d6c78-97df-42f3-bbc7-519e18974eee","order_by":1,"name":"Grzegorz Kudela","email":"","orcid":"","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":false,"prefix":"","firstName":"Grzegorz","middleName":"","lastName":"Kudela","suffix":""},{"id":499604228,"identity":"050f4acd-e3e1-4f68-8b1f-710388bb13e0","order_by":2,"name":"Anna Rokowska-Oleksa","email":"","orcid":"","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Rokowska-Oleksa","suffix":""},{"id":499604229,"identity":"cec5541b-679d-4c46-a1f8-263fe490838c","order_by":3,"name":"Agnieszka Jędzura","email":"","orcid":"","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Jędzura","suffix":""},{"id":499604230,"identity":"393bb180-a3a2-4c87-84be-174175e90f21","order_by":4,"name":"Lidia Hyla-Klekot","email":"","orcid":"","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":false,"prefix":"","firstName":"Lidia","middleName":"","lastName":"Hyla-Klekot","suffix":""},{"id":499604231,"identity":"228bf99f-0886-40c8-8a5d-82305aa43554","order_by":5,"name":"Tomasz Koszutski","email":"","orcid":"","institution":"Medical University of Silesia: Slaski Uniwersytet Medyczny w Katowicach","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Koszutski","suffix":""}],"badges":[],"createdAt":"2025-08-04 23:04:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7294779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7294779/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89560753,"identity":"5d657bb4-0579-48c2-a402-d1e752dc7567","added_by":"auto","created_at":"2025-08-21 10:20:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326978,"visible":true,"origin":"","legend":"\u003cp\u003eGraph showing the correlation between α-Klotho serum levels and age in the group of children with urolithiasis.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7294779/v1/917f77e4859cd6e8d8c32e9c.png"},{"id":89560754,"identity":"97e5cf89-91f2-4f14-a5fb-a8b2900a31da","added_by":"auto","created_at":"2025-08-21 10:20:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337795,"visible":true,"origin":"","legend":"\u003cp\u003eGraph showing the correlation between serum Klotho protein levels and serum phosphate concentration in the group of children with urolithiasis.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7294779/v1/6c1f6f5bc7b556a90f865090.png"},{"id":89560756,"identity":"24ffe59b-0130-4670-aa4f-5fc7a5d918bb","added_by":"auto","created_at":"2025-08-21 10:20:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287611,"visible":true,"origin":"","legend":"\u003cp\u003eGraph showing the correlation between Klotho serum levels and physical activity in the group of children with urolithiasis.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7294779/v1/1144c08a58941ed9c0240a95.png"},{"id":94988499,"identity":"56e63054-5be2-49db-8bbb-98334eb38e71","added_by":"auto","created_at":"2025-11-03 07:09:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1593665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7294779/v1/18604bbe-dd2b-4760-b603-3e6923714542.pdf"}],"financialInterests":"","formattedTitle":"Serum α-Klotho as a Potential Biomarker in Pediatric Urolithiasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrolithiasis, affecting approximately 2% of children, is an emerging health concern in the pediatric population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This condition, classified among lifestyle-related diseases, is characterized by a complex and multifactorial etiology involving both genetic and environmental components [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The formation of urinary stones is a multistep process influenced by imbalances between promoters and inhibitors of crystallization, oxidative stress, injury to renal tubular cells, and enhanced generation of reactive oxygen species (ROS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The majority of stones contain calcium compounds, primarily in the form of oxalates and phosphates, underscoring the critical role of calcium-phosphate homeostasis in the pathogenesis of urolithiasis [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe discovery of the \u003cem\u003eKLOTHO\u003c/em\u003e gene in 1997 marked a turning point in understanding the interplay between aging, calcium-phosphate axis and metabolic disorders [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Experimental suppression of \u003cem\u003eKLOTHO\u003c/em\u003e expression in mice resulted in a dramatic reduction in lifespan\u0026mdash;from two years to approximately two months\u0026mdash;and the development of a premature aging phenotype, along with biochemical alterations such as hyperphosphatemia and hypercalcemia [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings identified α-Klotho protein as a key regulator of calcium-phosphate metabolism and an endogenous modulator of oxidative stress [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its deficiency has been implicated in several pathophysiological processes, including chronic kidney disease, cardiovascular conditions, and more recently, urolithiasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aim of the present study is to evaluate serum α-Klotho concentrations in children with urolithiasis and to analyze their association with clinical and laboratory parameters of the disease, as well as to investigate the potential utility of α-Klotho as a diagnostic and monitoring biomarker in this context. To the best of our knowledge, this is the first study to assess α-Klotho protein levels in pediatric patients with urolithiasis. Existing evidence on this topic is extremely limited and based solely on adult populations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], highlighting a significant knowledge gap in pediatric nephrology.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study Design and Patient Assessment\u003c/h2\u003e\u003cp\u003eThis study included 40 children aged 0\u0026ndash;18 years hospitalized due to urolithiasis, either symptomatic or incidentally detected during imaging studies. The control group comprised 40 children with no history of urolithiasis. Family and medical histories were collected for all participants. Children with comorbidities or chronic disorders\u0026mdash;particularly nephrological, endocrinological, autoimmune, or gastrointestinal conditions that could influence serum α-Klotho levels\u0026mdash;were excluded from the study.\u003c/p\u003e\u003cp\u003eBody weight and height were measured, and body mass index (BMI) was calculated and interpreted using age- and sex-specific percentile charts. Based on these, children were classified as underweight (\u0026lt;\u0026thinsp;10 percentile), lean (10\u0026ndash;25 percentile), normal weight (25\u0026ndash;90 percentile), overweight (90\u0026ndash;97 percentile) or obese (\u0026gt;\u0026thinsp;97 percentile). In the urolithiasis group, modifiable risk factors such as fluid intake, dietary habits, and physical activity were assessed using a structured questionnaire. Stone characteristics, including size and location, were determined by abdominal ultrasound. The type of treatment\u0026mdash;conservative or surgical\u0026mdash;was recorded.\u003c/p\u003e\u003cp\u003eSerum α-Klotho concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) with the Human Soluble α-Klotho Assay Kit (Immuno-Biological Laboratories, Japan), according to the manufacturer\u0026rsquo;s instructions. Additional serum parameters included ionized and total calcium, phosphorus, sodium, potassium, magnesium, creatinine, urea, 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), and alkaline phosphatase. Twenty-four-hour urine collections were analyzed to assess the daily excretion of calcium, phosphorus, sodium, magnesium, uric acid, and creatinine.\u003c/p\u003e\u003cp\u003eA follow-up was conducted six months after enrollment, consisting of a clinical interview to determine the frequency of renal colic episodes, hospitalizations, and any interventions performed within that period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Ethical Considerations\u003c/h2\u003e\u003cp\u003e The study was approved by the Local Bioethics Committee (PCN/CBN/0022/KB1/25/I/20/21). Written informed consent was obtained from parents or legal guardians, and assent was acquired from children capable of understanding the study objectives. The research was conducted in accordance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using MedCalc software (version 18.1, MedCalc Software Ltd., Ostend, Belgium). Qualitative variables were presented as counts and percentages; continuous variables were expressed as medians (Me) with interquartile ranges (IQR). The Shapiro-Wilk test was used to assess the distribution of continuous variables. The chi-square test was used for categorical variables, while the Mann-Whitney U and Kruskal-Wallis tests were applied to compare continuous variables between groups. Correlations were evaluated using Spearman\u0026rsquo;s rank correlation coefficient (rho), with a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Characteristics of the Study Group\u003c/h2\u003e\u003cp\u003eThe study included 40 pediatric patients diagnosed with urinary tract stones. Detailed characteristics of the study population are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of the study group. Categorical variables are presented as counts and percentages, while continuous variables are expressed as medians with interquartile ranges (IQR); BMI \u0026ndash; body mass index; URSL \u0026ndash; ureteroscopy with laser lithotripsy; DJ \u0026ndash; double-J; ESWL \u0026ndash; extracorporeal shock wave lithotripsy.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStudy Group\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003esex (female)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25 (62.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eage [years]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 (11\u0026ndash;16)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eweight [kg]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55 (43.75\u0026ndash;67.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eheight [cm]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e161.5 (157\u0026ndash;171)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eBMI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eunderweight\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (10%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003elean\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (7.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003enormal weight\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25 (62.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eoverweight\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (10%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eobese\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (10%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003efamily history\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 (75%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eLocalization of calculi\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eUreter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20 (50%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eKidney\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (27.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eureter\u0026thinsp;+\u0026thinsp;kidney\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (20%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ebladder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (2.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eType of stone management\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003econservative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20 (50%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eURSL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 (37.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eDJ stent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (10%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eESWL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (2.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eFollow-up\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eRecurrence of renal colic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13 (32,5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eHospitalization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (30%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eUrological intervention\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (7.5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Klotho Protein in Pediatric Patients with Urolithiasis\u003c/h2\u003e\u003cp\u003eSignificant differences in serum α-Klotho concentrations were observed between the urolithiasis group and the control group, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Additional significant differences were found among patients based on family history, the presence of recurrence, and the type of treatment during subsequent episodes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferences in α-Klotho serum levels between the study and control groups, as well as among subgroups within the study group. Variables are presented as medians (Me) with interquartile ranges (IQR).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComparison Group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eKlotho [pg/ml]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStudy vs. Control group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1622 (1065\u0026ndash;2351) vs. 2184 (1375\u0026ndash;3442)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.017\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily history (Positive vs. Negative)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1531 (488\u0026ndash;5866) vs. 2118 (577\u0026ndash;9129)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.049\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial treatment\u003c/p\u003e\u003cp\u003e(Surgical vs. Conservative)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1822 (488\u0026ndash;9129) vs. 1577 (558\u0026ndash;4684)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.570\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence (Yes vs. No)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1054 (488\u0026ndash;2323) vs. 1843 (577\u0026ndash;9129)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecurrence treatment\u003c/p\u003e\u003cp\u003e(Surgical vs. Conservative)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e753 (558\u0026ndash;1115) vs. 1742 (488\u0026ndash;9129)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.029\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe median α-Klotho serum level in patients with urolithiasis was 1622 pg/ml\u003c/p\u003e\u003cp\u003e(IQR: 1065\u0026ndash;2351), which was significantly lower than in the control group (2184 pg/ml, IQR: 1375\u0026ndash;3442; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017).\u003c/p\u003e\u003cp\u003ePatients without a family history of stone disease had significantly higher Klotho levels (2118 pg/ml, IQR: 577\u0026ndash;9129) than those with a positive family history (1531 pg/ml, IQR: 488\u0026ndash;5866; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049). No significant difference in Klotho levels was found between patients treated conservatively and those who underwent endoscopic urological procedures (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.570).\u003c/p\u003e\u003cp\u003eHowever, patients who experienced a recurrence of renal colic within six months of the initial hospitalization showed significantly lower Klotho levels (1054 pg/ml, IQR: 488\u0026ndash;2323) compared to those without recurrence (1843 pg/ml, IQR: 577\u0026ndash;9129; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Similarly, children who required a urological intervention during recurrence had lower Klotho concentrations (753 pg/ml, IQR: 558\u0026ndash;1115) than those managed conservatively (1742 pg/ml, IQR: 488\u0026ndash;9129; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029).\u003c/p\u003e\u003cp\u003eA moderate inverse correlation was identified between serum Klotho levels and age among patients with urolithiasis (rho = -0.476, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), whereas no significant correlation was observed in the control group (rho = -0.247, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.125).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Klotho Protein and Laboratory Parameters in Pediatric Urolithiasis\u003c/h2\u003e\u003cp\u003eThe majority of patients demonstrated normal calcium-phosphate metabolism, with the exception of a high prevalence of vitamin D deficiency (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;34/40; 85%). Hyperuricemia was observed in nearly half of the patients. All children maintained normal renal function, as reflected by normal glomerular filtration rates. Most had no significant abnormalities in 24-hour urinary excretion of electrolytes, apart from relatively frequent hypomagnesuria (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19/40; 47.5%) and occasional hypercalciuria (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7/40; 17.5%).\u003c/p\u003e\u003cp\u003eA statistically significant positive correlation was found between Klotho levels and serum phosphorus (rho\u0026thinsp;=\u0026thinsp;0.318, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045), suggesting a link between Klotho and phosphate metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNo significant correlations were observed between Klotho and other metabolic parameters, including total calcium (rho\u0026thinsp;=\u0026thinsp;0.276, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085), PTH (rho = -0.104, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.523), or vitamin D (rho\u0026thinsp;=\u0026thinsp;0.097, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.552). Furthermore, Klotho levels did not correlate with urinary excretion of calcium, phosphorus, or magnesium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Klotho Protein and Modifiable Risk Factors for Urolithiasis in Children\u003c/h2\u003e\u003cp\u003eA statistically significant trend was observed indicating that higher physical activity was associated with elevated Klotho levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023), with a notable difference emerging only in patients engaging in 3\u0026ndash;7 physical activity sessions per week (H\u0026thinsp;=\u0026thinsp;9.558, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNo significant associations were found between Klotho levels and other modifiable risk factors, including adequate fluid intake (\u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;134, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.316), frequent consumption of salty snacks, fast food (\u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;128, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.065), or oxalate-rich foods (\u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;158, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.348).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e5.1. Serum Klotho level as a potential biomarker in children at risk for urinary stone disease\u003c/h2\u003e\u003cp\u003eThe assessment of serum α-Klotho protein levels as a potential marker for urinary stone risk aligns with a growing body of research focused on identifying sensitive, reproducible, and clinically relevant biomarkers of pathophysiological processes underlying chronic and lifestyle-related diseases. Over the past decade, significant advances in the understanding of urolithiasis pathogenesis have highlighted the roles of inflammation, oxidative stress, and calcium-phosphate dysregulation in the initiation and recurrence of stone formation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn animal models, renal crystal deposition has been shown to trigger the production of reactive oxygen species (ROS), activation of inflammasomes, and a proinflammatory cascade, leading to tissue damage and the formation of Randall's plaques [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this context, the α-Klotho protein has emerged as a nephroprotective factor, with its antioxidant properties reducing mitochondrial oxidative stress and enhancing the activity of cellular antioxidant enzymes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eΑ-Klotho has been extensively studied in chronic kidney disease (CKD), where its deficiency has gained recognition as an early biomarker of renal injury\u0026mdash;even before classical laboratory abnormalities emerge [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The first clinical reports linking α-Klotho to urolithiasis appeared in the last decade, initially focusing on gene polymorphisms. Telci et al., G\u0026uuml;rel et al., and Lanka et al. demonstrated associations between specific \u003cem\u003eKLOTHO\u003c/em\u003e gene variants and an increased risk of urinary tract stones, particularly through mechanisms involving hypercalciuria and phosphate handling [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Xu et al. further suggested that certain \u003cem\u003eKLOTHO\u003c/em\u003e polymorphisms may increase α-Klotho expression, providing a protective effect by preventing calcium oxalate crystal formation in renal epithelial cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSubsequent studies have evaluated circulating α-Klotho levels in adult patients with urolithiasis, showing that lower serum α-Klotho concentrations correlate with increased oxidative stress and stone recurrence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Gurtan et al. reported the lowest α-Klotho levels and the highest oxidative stress index (OSI) in patients with recurrent stones, findings consistent with our observations in the pediatric population [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, children with urolithiasis had significantly lower α-Klotho levels than controls. Furthermore, children with a positive family history or recurrent renal colic episodes demonstrated even lower α-Klotho levels, supporting its potential role as a prognostic marker for recurrence. This aligns with findings in adults and expands current understanding into the pediatric domain\u0026mdash;to our knowledge, this is the first study to assess α-Klotho levels in children with urinary tract stones.\u003c/p\u003e\u003cp\u003eIt is well known that α-Klotho protein levels decrease with age; however, this relationship has not been thoroughly studied in children, and the available research provides inconclusive results [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our study, we observed a slight decline in α-Klotho levels with age, but only in the group of children with urolithiasis. No such correlation was found among healthy children. This moderate yet significant association is consistent with the literature, which indicates that the physiological decline in α-Klotho levels during childhood and adolescence is not as pronounced as in adults [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A marked decrease in its concentration in children is primarily observed in the context of specific disease entities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these, particularly in chronic kidney disease (CKD), α-Klotho has been much more extensively studied in pediatric populations [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, it may be reasonable to relate these findings to urolithiasis, although research on this condition in children remains limited.\u003c/p\u003e\u003cp\u003eAlthough the classical approach to metabolic evaluation in stone disease involves serum calcium-phosphate balance and 24-hour urinary excretion of electrolytes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], our study focused on α-Klotho as a novel biomarker. Consistent with adult studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], we found that hypercalciuria was present in only a minority (17.5%) of patients, while hypomagnesuria was more common (47.5%). We did not observe significant correlations between α-Klotho levels and 24-hour urinary excretion. The only statistically significant association was a positive correlation between serum phosphorus and α-Klotho, in line with α-Klotho\u0026rsquo;s known role in phosphate regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This may suggest that reduced α-Klotho levels precede abnormalities in other classical parameters, making α-Klotho a potential early marker of urolithiasis.\u003c/p\u003e\u003cp\u003eThe idea that α-Klotho could serve as an early biomarker of stone risk and recurrence is inspired by its diagnostic and monitoring potential in CKD. Wan et al. demonstrated that in pediatric CKD patients, serum α-Klotho levels decline progressively with disease severity and are responsive to interventions such as kidney transplantation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is plausible that similar mechanisms operate in urolithiasis, especially in recurrent cases.\u003c/p\u003e\u003cp\u003eIn our study, children with stone recurrence had significantly lower α-Klotho levels, suggesting that low baseline α-Klotho may reflect greater vulnerability to recurrent renal colic and hospitalization. Although no differences were observed in α-Klotho levels based on initial treatment method (surgical vs. conservative), recurrence appeared more informative for predicting long-term risk. This further supports the use of α-Klotho as a monitoring tool rather than a marker of stone burden per se.\u003c/p\u003e\u003cp\u003eWe also explored associations between α-Klotho and modifiable lifestyle-related risk factors. Children with obesity had notably lower α-Klotho levels, consistent with adult data linking reduced α-Klotho to obesity and metabolic syndrome [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our findings diverge from the only pediatric study by Socha-Banasiak et al., who reported elevated α-Klotho levels in obese children\u0026mdash;though the authors themselves note this contradiction with adult data and call for further research [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our results additionally confirmed that regular physical activity (3\u0026ndash;7 sessions/week) was associated with higher α-Klotho levels. This is in line with a Brazilian meta-analysis identifying α-Klotho as an \u0026ldquo;exerkine\u0026rdquo;\u0026mdash;a protein upregulated in response to physical exercise [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. While the mechanisms remain unclear, skeletal muscle-derived cytokines may play a role in inducing renal or systemic α-Klotho expression [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther dietary and lifestyle factors, including fluid intake and consumption of salty or oxalate-rich foods, showed no clear association with α-Klotho levels in our cohort. Nonetheless, given their well-established role in stone pathogenesis, early health education targeting these behaviors remains crucial [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5.2. Study Limitations\u003c/h2\u003e\u003cp\u003eThis study has several limitations. First, the sample size was relatively small, reflecting its design as a pilot study. Nonetheless, the observed trends are promising and justify further research in larger, multicenter pediatric populations. Second, lifestyle variables such as fluid intake, diet, and physical activity were assessed using questionnaires completed by patients and their parents, introducing a degree of subjectivity. While these tools provided valuable insight into modifiable risk factors, the data should be interpreted cautiously, and future studies should aim for more objective measurement methods where feasible.\u003c/p\u003e\u003c/div\u003e"},{"header":"Summary","content":"\u003cp\u003eOur study demonstrated that children with urinary stone disease exhibit significantly lower serum levels of α-Klotho protein compared to their healthy peers, underscoring the potential role of this protein in the pathogenesis of urolithiasis. These findings suggest that α-Klotho may serve as an early biomarker of stone formation, potentially preceding detectable changes in traditional laboratory parameters. This hypothesis is further supported by analogous findings in studies on chronic kidney disease in pediatric populations.\u003c/p\u003e\u003cp\u003eAlthough no significant differences in α-Klotho levels were observed between children treated conservatively and those undergoing surgical interventions, lower α-Klotho concentrations were consistently associated with stone recurrence. This indicates a possible prognostic role for α-Klotho in identifying patients at risk for recurrent renal colic and hospitalization.\u003c/p\u003e\u003cp\u003eThis observation holds particular relevance in pediatric practice, where early diagnosis and close monitoring are essential for long-term care and prevention of kidney damage.\u003c/p\u003e\u003cp\u003eA comprehensive approach\u0026mdash;including assessment of metabolic risk factors, timely treatment of stones, and early implementation of preventive strategies\u0026mdash;is vital to safeguard renal health across the lifespan. The identification and validation of novel biomarkers such as α-Klotho may significantly enhance these efforts, contributing to more effective disease monitoring and improved outcomes in children with urolithiasis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e There is no conflict of interests. This work was funded by resources allocated for the implementation of a research project as part of maintaining the research potential of the University \u0026ndash; statutory work, contract for the year 2020 (contract number: PCN-1-212/N/0/k).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePanzarino V. Urolithiasis in Children. Adv Pediatr. 2020 Aug;67:105-112. doi: 10.1016/j.yapd.2020.03.004. Epub 2020 May 26. PMID: 32591054.\u003c/li\u003e\n \u003cli\u003eJobs K, Rakowska M, Paturej A. 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Klotho, Oxidative Stress, and Mitochondrial Damage in Kidney Disease. Antioxidants (Basel). 2023 Jan 20;12(2):239. doi: 10.3390/antiox12020239.\u003c/li\u003e\n \u003cli\u003eAihemaitijiang B, Ruotian L, Qi Y, Mahemuti M. Correlation between sKL and Nrf2 plasma levels and calcium oxalate urolithiasis. Int Urol Nephrol. 2023 Jul;55(7):1671-1676. doi: 10.1007/s11255-023-03615-z. Epub 2023 May 17. PMID: 37198517.\u003c/li\u003e\n \u003cli\u003eHu X, Li X, Ye N, Zhou Z, Li G, Jiang F. Association of serum soluble α‑klotho with risk of kidney stone disease: a population-based cross-sectional study. World J Urol. 2024 Apr 8;42(1):219. doi: 10.1007/s00345-024-04837-1. PMID: 38587631.\u003c/li\u003e\n \u003cli\u003eAhmatjan B, Ruotian L, Rahman A et al. Klotho inhibits the formation of calcium oxalate stones by regulating the Keap1-Nrf2-ARE signaling pathway. Int Urol Nephrol. 2023 Feb;55(2):263-276. doi: 10.1007/s11255-022-03398-9. Epub 2022 Nov 7. PMID: 36336747.\u003c/li\u003e\n \u003cli\u003eTelci D, Dogan AU, Ozbek E et al. KLOTHO gene polymorphism of G395A is associated with kidney stones. Am J Nephrol. 2011;33(4):337-43. doi: 10.1159/000325505.\u003c/li\u003e\n \u003cli\u003eGürel A, Üre İ, Temel HE et al. The impact of klotho gene polymorphisms on urinary tract stone disease. World J Urol. 2016 Jul;34(7):1045-50. doi: 10.1007/s00345-015-1732-z.\u003c/li\u003e\n \u003cli\u003eLanka P, Devana SK, Singh SK, Sapehia D, Kaur J. Klotho gene polymorphism in renal stone formers from Northwestern India. Urolithiasis. 2021 Jun;49(3):195-199. doi: 10.1007/s00240-020-01226-2. Epub 2020 Nov 11. PMID: 33174123.\u003c/li\u003e\n \u003cli\u003eXu C, Song RJ, Yang J et al. Klotho gene polymorphism of rs3752472 is associated with the risk of urinary calculi in the population of Han nationality in Eastern China. Gene. 2013 Sep 10;526(2):494-7. doi: 10.1016/j.gene.2013.06.001. Epub 2013 Jun 10. PMID: 23756195.\u003c/li\u003e\n \u003cli\u003eKhan SR, Canales BK. Unified theory on the pathogenesis of Randall's plaques and plugs. Urolithiasis. 2015 Jan;43 Suppl 1(0 1):109-23. doi: 10.1007/s00240-014-0705-9.\u003c/li\u003e\n \u003cli\u003eWan M, Smith C, Shah V et al. Fibroblast growth factor 23 and soluble klotho in children with chronic kidney disease. Nephrol Dial Transplant. 2013 Jan;28(1):153-61. doi: 10.1093/ndt/gfs411. Epub 2012 Nov 23. PMID: 23180879.\u003c/li\u003e\n \u003cli\u003eYamazaki Y, Imura A, Urakawa I et al. Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects. Biochem Biophys Res Commun. 2010 Jul 30;398(3):513-8. doi: 10.1016/j.bbrc.2010.06.110. Epub 2010 Jul 1. PMID: 20599764; PMCID: PMC4130489.\u003c/li\u003e\n \u003cli\u003eGkentzi D, Efthymiadou A, Kritikou D, Chrysis D. Fibroblast growth factor 23 and Klotho serum levels in healthy children. Bone. 2014 Sep;66:8-14. doi: 10.1016/j.bone.2014.05.012. Epub 2014 May 29. PMID: 24880094.\u003c/li\u003e\n \u003cli\u003eCano FJ, Freundlich M, Ceballos ML et al. Longitudinal FGF23 and Klotho axis characterization in children treated with chronic peritoneal dialysis. Clin Kidney J. 2014 Oct;7(5):457-63. doi: 10.1093/ckj/sfu074. Epub 2014 Aug 8. PMID: 25878777; PMCID: PMC4379333.\u003c/li\u003e\n \u003cli\u003eGamrot Z, Adamczyk P, Świętochowska E, Roszkowska-Bjanid D, Gamrot J, Szczepanska M. Tumour necrosis factor alpha (TNFα) and alpha-Klotho (αKL) in children and adolescents with chronic kidney disease (CKD). Endokrynol Pol. 2021;72(6):625-633. doi: 10.5603/EP.a2021.0082. Epub 2021 Oct 14. PMID: 34647605.\u003c/li\u003e\n \u003cli\u003eSawires HK, Essam RM, Morgan MF, Mahmoud RA. Serum klotho: relation to fibroblast growth factor-23 and other regulators of phosphate metabolism in children with chronic kidney disease. Nephron. 2015;129(4):293-9. doi: 10.1159/000377633. Epub 2015 Mar 10. PMID: 25766835.\u003c/li\u003e\n \u003cli\u003eTranæus Lindblad Y, Olauson H, Vavilis G et al. The FGF23-Klotho axis and cardiac tissue Doppler imaging in pediatric chronic kidney disease-a prospective cohort study. Pediatr Nephrol. 2018 Jan;33(1):147-157. doi: 10.1007/s00467-017-3766-5. Epub 2017 Aug 9. PMID: 28795324; PMCID: PMC5700222.\u003c/li\u003e\n \u003cli\u003eOkarska-Napierała M, Skrzypczyk P, Pietrzak R et al. Serum Klotho is correlated to cardiovascular complications of chronic kidney disease in children. Clin Nephrol. 2020 Oct;94(4):163-172. doi: 10.5414/CN110049. PMID: 32729817.\u003c/li\u003e\n \u003cli\u003eKubota M, Hamasaki Y, Hashimoto J et al. Fibroblast growth factor 23-Klotho and mineral metabolism in the first year after pediatric kidney transplantation: A single-center prospective study. Pediatr Transplant. 2023 Mar;27(2):e14440. doi: 10.1111/petr.14440. Epub 2022 Dec 5. PMID: 36471536.\u003c/li\u003e\n \u003cli\u003eDonate-Correa J, Matos-Perdomo E, González-Luis A et al. The Value of Klotho in Kidney Transplantation. Transplantation. 2023 Mar 1;107(3):616-627. doi: 10.1097/TP.0000000000004331. Epub 2022 Oct 18. PMID: 36253904.\u003c/li\u003e\n \u003cli\u003eAmitani M, Asakawa A, Amitani H et al. Plasma klotho levels decrease in both anorexia nervosa and obesity. Nutrition. 2013 Sep;29(9):1106-9. doi: 10.1016/j.nut.2013.02.005. Epub 2013 Jun 19. PMID: 23790542.\u003c/li\u003e\n \u003cli\u003eLiu Y, Chen M. Emerging role of α-Klotho in energy metabolism and cardiometabolic diseases. Diabetes Metab Syndr. 2023 Oct;17(10):102854. doi: 10.1016/j.dsx.2023.102854. Epub 2023 Sep 14. PMID: 37722166.\u003c/li\u003e\n \u003cli\u003eOrces CH. The Association of Obesity and the Antiaging Humoral Factor Klotho in Middle-Aged and Older Adults. ScientificWorldJournal. 2022 Aug 24;2022:7274858. doi: 10.1155/2022/7274858. PMID: 36061981; PMCID: PMC9433301.\u003c/li\u003e\n \u003cli\u003eSocha-Banasiak A, Michalak A, Pacześ K et al. Klotho and fibroblast growth factors 19 and 21 serum concentrations in children and adolescents with normal body weight and obesity and their associations with metabolic parameters. BMC Pediatr. 2020 Jun 16;20(1):294. doi: 10.1186/s12887-020-02199-2. PMID: 32546231; PMCID: PMC7296965.\u003c/li\u003e\n \u003cli\u003eCorrêa HL, Raab ATO, Araújo TM et al. A systematic review and meta-analysis demonstrating Klotho as an emerging exerkine. Sci Rep. 2022 Oct 20;12(1):17587. doi: 10.1038/s41598-022-22123-1. PMID: 36266389; PMCID: PMC9585050.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Klotho, pediatric urolithiasis, urinary stone disease, biomarker, recurrence, calcium-phosphate metabolism","lastPublishedDoi":"10.21203/rs.3.rs-7294779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7294779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eUrolithiasis is an emerging problem in the pediatric population, with multifactorial etiology involving genetic, metabolic, and environmental factors. The α-Klotho protein, known for its role in mineral homeostasis and oxidative stress modulation, has been implicated in adult urolithiasis, but its relevance in children remains unexplored. Aim of the study was to assess serum α-Klotho levels in children with urinary stone disease and evaluate its potential as a biomarker associated with clinical features and risk of recurrence.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eA prospective study was conducted in 40 pediatric patients with urolithiasis and 40 healthy controls. Serum α-Klotho levels were measured using ELISA. Additional parameters included calcium-phosphate metabolism, 24-hour urine collections, treatment type, recurrence, and modifiable risk factors.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eSerum α-Klotho levels were significantly lower in children with urolithiasis compared to controls (1622 vs. 2184 pg/ml, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017). Lower α-Klotho levels were associated with positive family history (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049), recurrence of renal colic (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), and need for surgical intervention during recurrence (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029). α-Klotho levels positively correlated with serum phosphate (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045) but showed no significant relationship with other laboratory results. Increased physical activity was associated with higher Klotho levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023), while no association was found with diet or fluid intake.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e\u003cp\u003eThis is the first study to investigate α-Klotho in pediatric urolithiasis. Our findings suggest that reduced serum α-Klotho levels may precede metabolic abnormalities and correlate with recurrence risk, supporting its potential role as an early biomarker in children with urinary stone disease.\u003c/p\u003e","manuscriptTitle":"Serum α-Klotho as a Potential Biomarker in Pediatric Urolithiasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 10:20:10","doi":"10.21203/rs.3.rs-7294779/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46eaf644-e46f-4ee6-9add-d09a85ab8b5f","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-01T22:34:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 10:20:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7294779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7294779","identity":"rs-7294779","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}