Urinary NGAL and Renalase as Non-Invasive Biomarkers for Detection of Deterioration of Kidney Function and Kidney Scarring in Children with Neurogenic Bladder

Urinary NGAL and Renalase as Non-Invasive Biomarkers for Detection of Deterioration of Kidney Function and Kidney Scarring in Children with Neurogenic Bladder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Urinary NGAL and Renalase as Non-Invasive Biomarkers for Detection of Deterioration of Kidney Function and Kidney Scarring in Children with Neurogenic Bladder Anjali Srivastava, Sachit Anand, Himalaya Kumar, Jitendra Kumar Meena, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5998098/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 May, 2025 Read the published version in Pediatric Nephrology → Version 1 posted 5 You are reading this latest preprint version Abstract Background Urinary biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) and Renalase hold promise for assessing kidney health, yet their role in the pediatric neurogenic bladder (NB) remains unclear. This study evaluates their clinical utility in detecting kidney dysfunction and their association with disease severity. Methods A cross-sectional study included 44 NB patients and 45 age- and gender-matched healthy children (reference group). Urinary NGAL and Renalase levels were measured using ELISA. NB patients were categorized based on glomerular filtration rate (GFR) and kidney scarring. Biomarker levels were compared using the Mann-Whitney U test, and their correlations with functional parameters (DTPA, DMSA) were assessed using Spearman’s correlation. Results Urinary NGAL and Renalase levels were significantly higher in NB patients than in the reference group (NGAL: 31.86 vs. 23.40 pg/mg creatinine, p = 0.0345; Renalase: 2.75 vs. 1.76 ng/mg creatinine, p = 0.0493). NB patients with GFR < 60 mL/min/1.73 m² or kidney scarring had elevated NGAL (46.90 vs. 26.76 pg/mg creatinine, p = 0.0406) and Renalase (3.76 vs. 1.82 ng/mg creatinine, p = 0.0050). Both biomarkers correlated inversely with GFR (NGAL: r = -0.3344, p = 0.0326; Renalase: r = -0.4054, p = 0.0085) and increased with kidney scarring, suggesting their potential role in assessing kidney injury severity. Conclusions Urinary NGAL and Renalase are elevated in pediatric NB patients, particularly in those with kidney dysfunction, and correlate with GFR and kidney scarring. These findings highlight their potential as non-invasive markers for early detection and monitoring of kidney impairment in NB. Future longitudinal studies are warranted to validate their diagnostic and prognostic utility. Neurogenic Bladder Urinary Biomarkers NGAL Renalase Kidney Dysfunction Chronic Kidney Disease Kidney Scar Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Neurogenic bladder (NB) is a chronic urological condition affecting patients of all ages. In the pediatric population, it is often secondary to congenital or acquired neurological abnormalities, such as spina bifida or spinal cord injuries [ 1 ]. Spina bifida affects approximately 1 in 1000 live births globally, making it a leading cause of NB [ 2 ]. NB is characterized by impaired bladder control and dysfunctional voiding, which can result in severe complications, including recurrent urinary tract infections (UTIs), kidney scarring, and progressive kidney damage if untreated [ 2 ]. Early identification and management of kidney dysfunction in NB patients are critical to reducing morbidity and preserving kidney function. Traditional diagnostic tools, such as serum creatinine, glomerular filtration rate (GFR) estimations, and imaging, have limited sensitivity, particularly for detecting early-stage kidney injury [3]. In addition, the available radiological and nuclear imaging modalities for the assessment of upper urinary tract function are associated with radiation exposure. Thus, there is a need for non-invasive biomarkers offering more sensitive and dynamic assessments of kidney health. Among these, urinary neutrophil gelatinase-associated lipocalin (NGAL) and Renalase have gained attention for their potential roles in detecting kidney injury and monitoring disease progression [ 4 , 5 ]. NGAL, a protein released by injured kidney tubular epithelial cells, is a well-established early marker of acute kidney injury (AKI) [ 6 ]. It has been linked to tubular damage in various kidney disorders, including AKI, chronic kidney disease (CKD), and urinary tract obstructions [ 4 ]. Previous studies have specifically explored the role of NGAL in the pediatric population, highlighting its potential in assessing tubular injury and disease progression [ 7 – 9 ]. A recent study examined NGAL and KIM-1 as tubular injury markers in pediatric NB, reinforcing the need for further investigation into these biomarkers in this subgroup [ 10 ]. However, limited studies have investigated its utility in pediatric NB patients, where chronic and recurrent kidney insults are prevalent. Similarly, Renalase, a flavoprotein involved in catecholamine metabolism and oxidative stress regulation, has shown promise as a biomarker for CKD in adult populations [ 5 ]. While it has shown promise in adult studies, its application in pediatric populations, particularly those with NB, remains underexplored [ 11 ]. This study aims to evaluate the utility of urinary NGAL and Renalase as biomarkers of deterioration of kidney function in pediatric NB by comparing the levels of these biomarkers between patients vs. reference group. Also, by comparing biomarker levels across distinct clinical subgroups within NB patients, classified based on GFR and kidney scarring, we sought to determine their association with the severity of kidney injury. Methods Study Design and Setting This single-center cross-sectional study was conducted between September-November 2024, in the Department of Pediatric Surgery at the All India Institute of Medical Sciences, New Delhi, India. Ethical approval was obtained from the institutional review board (Ref no: AIIMSA2015/06.09.2024) prior to study initiation, and all procedures adhered to institutional guidelines. Written informed consent was obtained from the parents or legal guardians of all children, and assent was provided by participants as age-appropriate. Study Population The study included a total of 44 consecutive children (< 18 years) with NB who were in regular follow-up in the outpatient department (OPD). The diagnosis of NB was based on clinical, radiological, and urodynamic criteria [ 12 ]. Inclusion criteria for the NB group were confirmed diagnosis of NB, regular follow-up at our institution, and availability of clinical and laboratory data. Exclusion criteria included the presence of an active UTI (defined as positive urine culture with > 10⁵ CFU/mL and associated symptoms), prior history of kidney transplantation, presence of other primary kidney diseases, and use of nephrotoxic drugs within three months prior to sampling. In addition, 45 age- and gender-matched healthy children (reference group) were also recruited during the same period. Inclusion criteria for the reference group were healthy children attending the general pediatric OPD with no history of neurological or urological disorders and the absence of any chronic illnesses. Exclusion criteria for this group was any known congenital or acquired kidney disease, prior history of UTI, and abnormal findings on routine urine analysis. Urine Collection and Biomarker Analysis Single spot urine samples were collected from each participant in the morning to minimize circadian variation. For those who were able to void (including reference group), a midstream clean-catch urine sample was obtained. For patients who were unable to void or who were on clean intermittent catheterization (CIC), samples were obtained aseptically via catheterization. Collected samples were centrifuged and the supernatants were stored at − 80°C until analysis. Urinary NGAL and renalase levels were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits following manufacturers' instructions. Absorbance was read at 450 nm using a microplate reader (Infinite 200 PRO, Tecan, Switzerland), and all values were normalized to urinary creatinine, measured via Jaffe’s method, to adjust for urinary concentration variability [ 13 ]. Upper Tract Functional Investigations As per the Institutional Protocol, upper tract functional assessment using nuclear scintigraphy studies, i.e., technetium-99m diethylene triamine pentaacetic acid (DTPA) scan for glomerular filtration rate (GFR) assessment and technetium-99m dimercaptosuccinic acid (DMSA) scan for evaluation of kidney scars (defined as focal or diffuse cortical defects with reduced tracer uptake), are performed at recruitment and yearly follow-up visits. Thus, recent GFR values and scarring status of all patients were recorded and they were divided into two groups. Group 1 comprised patients without a significant kidney function impairment, whereas Group 2 included patients with a significant kidney function impairment, defined by a GFR < 60 mL/min/1.73 m² or evidence of scarring in one or both kidneys as confirmed by DMSA scintigraphy [ 14 ]. Statistical Analysis Data entry was done using Microsoft Excel spreadsheets and the analysis was performed using GraphPad Prism 5 software. Data were presented as median (interquartile range, IQR) for continuous variables based on the Shapiro-Wilk test for normality. Urinary NGAL and renalase levels were compared between the patients and reference group using the Mann-Whitney U test. Spearman’s correlation coefficients were used to evaluate correlations between urinary biomarker levels and the different parameters of functional investigations (DTPA and DMSA). Group comparisons of urinary NGAL and renalase levels across different patient groups (based on CKD stages and scarring status) were performed using the Kruskal-Wallis test, followed by Dunn's multiple comparison tests for pairwise comparisons. A p-value of < 0.05 was considered statistically significant. Results Patient Characteristics The patients with NB (n = 44) consisted of 20 girls (45.46%) and 24 boys (54.54%). Among the reference group, the gender distribution was B: G = 24 (53.33%): 21 (46.67%). The median age of the NB group was 120 months (Range: 9–192 months), while the reference group had a median age of 96 months (Range: 12–180 months). The age difference between the two groups was not statistically significant (p = 0.1833). Results of the DTPA and DMSA scans were available for 41/44 and 42/44 patients respectively. The distribution of patients among Group 1 and Group 2 were 27 (64.29%) and 15 (35.71%) respectively. Further, stratification by GFR revealed that 9.75% of patients had GFR 90 mL/min/1.73 m². In terms of kidney scarring, as assessed by DMSA scintigraphy, 69.05% had no scarring while 30.95% of the patients had scars in their kidneys. Biomarker Concentrations in the Cohort The median concentrations of urinary NGAL in patients and reference group were 31.86 (IQR: 20.41–55.34) pg/mg creatinine and 23.40 (IQR: 15.14–34.24) pg/mg creatinine respectively (Fig. 1 A). Upon comparison, a statistically significant difference was observed among patients vs. reference group (p = 0.0345). Similarly, the levels of urinary renalase were significantly elevated in the NB group (median = 2.75 ng/mg creatinine, IQR: 1.42–4.66 ng/mg creatinine) compared to the reference group (median = 1.76 ng/mg creatinine, IQR: 1.10–2.99 ng/mg creatinine); p = 0.0493 (Fig. 1 B). Urinary Biomarker Levels and Deterioration of Kidney Function Based on the availability of GFR and DMSA reports, 42 patients were categorized into subgroups for further analysis. Urinary biomarker profiles revealed significant differences between NB patients with and without significant kidney function impairment. The median urinary NGAL levels for Group 1 and Group 2 were 26.76 (IQR: 15.55–49.25) and 46.90 (IQR: 23.82–83.61) pg/mg creatinine respectively, demonstrating a statistically significant difference (U = 124.0, p = 0.0406) (Fig. 2 A). Similarly, the urinary renalase levels for Group 1 and Group 2 were 1.82 (IQR: 1.03–3.28) and 3.75 (IQR: 2.33–7.85) ng/mg creatinine respectively, reflecting a significant difference between the two groups (U = 95.0, p = 0.0050) (Fig. 2 B). Relationship of Urinary NGAL and Renalase levels with falling GFR The analysis of urinary biomarker levels in relation to GFR categories indicated a potential association between biomarker levels and the severity of chronic kidney disease. Patients with GFR < 60 mL/min/1.73 m² exhibited the highest median NGAL levels (63.11 pg/mg creatinine), followed by those in the 60–89 mL/min/1.73 m² category (31.16 pg/mg creatinine), and the lowest levels were observed in patients with GFR > 90 mL/min/1.73 m² (26.75 pg/mg creatinine) (Fig. 3 A). A similar trend was observed for renalase levels, with median concentrations of 5.31 ng/mg creatinine in the GFR 90 mL/min/1.73 m² (Fig. 3 B). Correlation analysis further demonstrated a moderate negative and statistically significant correlation of NGAL levels with GFR (r = -0.3344, p = 0.0326), suggesting that as kidney function declines, NGAL levels tend to increase. Similarly, renalase levels showed a significant negative correlation with GFR (r = -0.4054, p = 0.0085), indicating a trend toward higher renalase levels with decreased kidney function. Relationship of Urinary NGAL and Renalase Levels with Kidney Scarring Interestingly, urinary biomarker levels revealed a clear association with kidney scarring. The participants were categorized into three groups: reference group, patients with no kidney scars, and patients with scarring. NGAL levels for these groups were 23.91 pg/mg creatinine, 27.89 pg/mg creatinine, and 46.90 pg/mg creatinine respectively (Fig. 4 A). Renalase levels also showed a similar trend, with median concentrations of 1.76 ng/mg creatinine in the reference group, 2.14 ng/mg creatinine in patients with no kidney scars, and 2.92 ng/mg creatinine among the patients with scarring (Fig. 4 B). Discussion Our study underscores the clinical relevance of urinary NGAL and Renalase as potential biomarkers for assessing kidney dysfunction and scarring in pediatric NB patients. Our findings demonstrate a significant association between elevated biomarker levels, declining GFR, and kidney scarring, underscoring their utility in identifying early kidney injury and stratifying patients based on the severity of kidney impairment [ 15 – 17 ]. Multiple studies have reported that NGAL levels rise rapidly in response to ischemic kidney injury, correlating with histological tubular damage [ 18 ]. Similarly, Liu et al. demonstrated that NGAL is a strong predictor of chronic kidney disease progression [ 19 ]. In our study, elevated urinary NGAL levels in NB patients with scarring and reduced GFR align with these findings, emphasizing its relevance as an early marker of tubular damage in this specific population. Renalase, a flavin adenine dinucleotide (FAD)-dependent oxidase, is secreted into circulation by the kidneys and acts on catecholamines such as adrenaline and dopamine, reducing their levels and mitigating oxidative stress [ 5 ]. This activity plays a protective role by preventing sustained hypertension and vascular damage. However, during kidney injury, the compromised secretion of Renalase paradoxically leads to local catecholamine accumulation, which exacerbates oxidative stress and triggers pro-inflammatory pathways. This cascade promotes endothelial dysfunction, tubular damage, and fibrosis [ 5 ]. Wisniewska et al. have reported increased urinary Renalase levels in CKD patients, suggesting a compensatory response to oxidative stress and catecholamine dysregulation [ 20 ]. Its role as a biomarker of kidney injury has been reported in conditions such as diabetic nephropathy and hypertensive kidney disease [ 5 , 21 ]. Also, previously published studies have demonstrated increased urinary Renalase levels in adult patients with CKD and requiring hemodialysis, correlating inversely with kidney function [ 20 ]. The current study extends these observations to pediatric NB, with significantly higher urinary Renalase levels in patients with scarring or low GFR, reflecting ongoing oxidative stress and potential maladaptive kidney repair mechanisms. The inverse correlations between GFR and both biomarkers indicate a progressive increase in biomarker levels with worsening kidney function. These results corroborate with the findings of prior studies, such as those, reporting a similar relationship between NGAL levels and GFR in CKD patients,[ 22 ] and those showing an association of Renalase with kidney functional decline [ 23 ]. The observed increase in NGAL and Renalase levels with confirmed kidney scarring further supports their role not only as functional markers of kidney injury but also as indicators of structural damage. This connection may be explained by the interplay of chronic inflammation, oxidative stress, and impaired tubular repair mechanisms in NB-related kidney dysfunction. Kidney scarring may further amplify these processes, thus, creating a vicious cycle of injury and maladaptive repair. While our study provides valuable insights, some limitations should be acknowledged. First, the cross-sectional design limits our ability to establish temporal or causal relationships between biomarker levels and disease progression. A longitudinal study would provide more definitive evidence of the predictive value of NGAL and Renalase in NB patients. Second, our cohort size, though carefully selected, remains relatively small, which may restrict the generalizability of our findings. Expanding the study to include larger, multicenter cohorts could address this limitation. Third, while we normalized biomarker levels to urinary creatinine, individual variations in hydration status and muscle mass could introduce variability. Pairing urinary biomarkers with serum levels or other normalization strategies may enhance accuracy in future studies. Looking ahead, our findings open several pathways for future research. Investigating the dynamic changes in NGAL and Renalase levels in response to therapeutic interventions, such as clean intermittent catheterization (CIC) or anticholinergic therapy, could validate their role as markers of treatment response. Additionally, integrating these biomarkers into risk prediction models alongside clinical parameters, imaging, and other biomarkers (e.g., KIM-1, MCP-1, etc.) could enhance their utility in routine clinical practice. The incorporation of these biomarkers into standardized monitoring protocols could improve early detection and guide personalized treatment decisions for pediatric NB patients. Their ability to detect kidney dysfunction at an early stage could aid in risk stratification, enabling timely interventions to prevent further kidney deterioration. Additionally, these biomarkers could play a crucial role in differentiating between varying degrees of kidney injury, helping clinicians tailor the treatment strategies based on disease severity. Future research should focus on evaluating their clinical utility in distinguishing between different NB phenotypes and disease progression patterns. The potential role of NGAL and Renalase in predicting long-term outcomes, such as progression to CKD stage 5 or kidney failure, also warrants exploration. Future longitudinal studies are necessary to validate these findings and explore their utility in disease management. Conclusion Urinary NGAL and Renalase levels were significantly elevated in pediatric NB patients compared to the reference group, as well as in NB patients with deteriorated kidney function compared to those without a functional deterioration. A significant negative correlation of these biomarkers with GFR underscores their relevance in evaluating kidney function. In addition, their association with kidney scarring also highlights their potential role in assessing kidney impairment. These findings suggest that urinary NGAL and Renalase could serve as non-invasive biomarkers for the early detection and monitoring of kidney dysfunction in pediatric NB patients. Declarations The abstract of this work has been submitted for consideration for presentation at the 35th European Society for Paediatric Urology (ESPU) Congress. Author Contributions: Conceptualization, S.A.; experimentation, A.S., and S.A.; sample collection, H.K.; formal analysis, writing, and original draft preparation, A.S., and S.A.; review and editing, J.M, A.V., and P.H.; access to lab facility and essential reagents, K.L.; S.A. will act as the guarantor of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: None Conflicts of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability Statement: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. References Brownrigg, N., Lorenzo, A. J., Rickard, M., & Dos Santos, J. (2024). The urological evaluation and management of neurogenic bladder in children and adolescents-what every pediatric nephrologist needs to know. 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Biomedicines, 12 (8). doi:10.3390/biomedicines12081715 Cite Share Download PDF Status: Published Journal Publication published 14 May, 2025 Read the published version in Pediatric Nephrology → Version 1 posted Editorial decision: Accept as is 29 Apr, 2025 Reviewers agreed at journal 22 Mar, 2025 Reviewers invited by journal 22 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 19 Mar, 2025 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. 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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-5998098","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432497432,"identity":"acca98f7-e884-48f0-823a-4a3fb595e3c4","order_by":0,"name":"Anjali Srivastava","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-7246-9586","institution":"All India Institute of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Anjali","middleName":"","lastName":"Srivastava","suffix":""},{"id":432497433,"identity":"592bd5b2-e370-40ec-883c-cdaf3edb9f9e","order_by":1,"name":"Sachit Anand","email":"","orcid":"https://orcid.org/0000-0002-0447-2350","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sachit","middleName":"","lastName":"Anand","suffix":""},{"id":432497434,"identity":"8420d1b3-1c48-4fc9-a5d4-2393553c968d","order_by":2,"name":"Himalaya Kumar","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Himalaya","middleName":"","lastName":"Kumar","suffix":""},{"id":432497435,"identity":"f0bac57c-3a28-4f4c-bc12-3d66be77ee9b","order_by":3,"name":"Jitendra Kumar Meena","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jitendra","middleName":"Kumar","lastName":"Meena","suffix":""},{"id":432497436,"identity":"6330c67a-862f-4437-bd6c-185adfcd5906","order_by":4,"name":"Ajay Verma","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Verma","suffix":""},{"id":432497437,"identity":"1572bc78-e2f0-4628-8330-e5c08674308f","order_by":5,"name":"Kalpana Luthra","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kalpana","middleName":"","lastName":"Luthra","suffix":""},{"id":432497438,"identity":"475f3149-7992-4057-b390-8b55088d3a23","order_by":6,"name":"Pankaj Hari","email":"","orcid":"","institution":"All India Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Hari","suffix":""}],"badges":[],"createdAt":"2025-02-10 10:12:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5998098/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5998098/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00467-025-06808-z","type":"published","date":"2025-05-14T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79322351,"identity":"e6896018-1924-45d2-aa53-3fecc0f37e80","added_by":"auto","created_at":"2025-03-27 04:38:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of NGAL and Renalase Levels Between Reference group and NB Patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoxplots showing the distribution of (A) NGAL and (B) Renalase concentrations (normalized to creatinine) in the reference group and NB patients. Mann-Whitney U test was used to analyze data and presented as median (IQR). Statistical significance is denoted by * p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5998098/v1/f41da6fc05023bd149da59a4.jpg"},{"id":79322353,"identity":"5e76a0b0-984f-4a42-b063-75086039afd9","added_by":"auto","created_at":"2025-03-27 04:38:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of Urinary Biomarker Levels in Patients with and without Kidney Function Impairment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoxplots showing the distribution of (A) NGAL and (B) Renalase concentrations (normalized to creatinine) in Group 1 (no significant kidney function impairment) and Group 2 (GFR \u0026lt;60 mL/min/1.73 m² or scarring on DMSA). Mann-Whitney U test was used to analyze data and presented as median (IQR). Statistical significance is denoted by * p \u0026lt; 0.05, ** p \u0026lt; 0.01\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5998098/v1/c958569d925546fe4ce1233d.jpg"},{"id":79322364,"identity":"5df14a6b-b544-4cc5-9d3c-64f8df20fe86","added_by":"auto","created_at":"2025-03-27 04:38:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of Urinary NGAL and Renalase Levels with Declining GFR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLine graphs demonstrating the distribution of urinary (A) NGAL and (B) Renalase levels across declining GFR (mL/min/1.73 m²). Data are presented as median values.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5998098/v1/d30685e2abdeaf831f82ef89.jpg"},{"id":79322366,"identity":"d8b22ff2-3178-46b9-a8d9-93f8d2a6138c","added_by":"auto","created_at":"2025-03-27 04:38:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation of Urinary NGAL and Renalase Levels with Kidney Scarring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLine graphs demonstrating the distribution of urinary (A) NGAL and (B) Renalase levels in the reference group, patients with no kidney scars, and patients with scarring. Data are presented as median values.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5998098/v1/4e752772c279a0a16633d30a.jpg"},{"id":83067713,"identity":"c1eea75c-b5a3-4c16-a757-7ecd5a714bde","added_by":"auto","created_at":"2025-05-19 16:04:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":816036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5998098/v1/6f31a577-09a0-4c94-9862-dc06aff70f2f.pdf"}],"financialInterests":"","formattedTitle":"Urinary NGAL and Renalase as Non-Invasive Biomarkers for Detection of Deterioration of Kidney Function and Kidney Scarring in Children with Neurogenic Bladder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurogenic bladder (NB) is a chronic urological condition affecting patients of all ages. In the pediatric population, it is often secondary to congenital or acquired neurological abnormalities, such as spina bifida or spinal cord injuries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Spina bifida affects approximately 1 in 1000 live births globally, making it a leading cause of NB [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NB is characterized by impaired bladder control and dysfunctional voiding, which can result in severe complications, including recurrent urinary tract infections (UTIs), kidney scarring, and progressive kidney damage if untreated [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Early identification and management of kidney dysfunction in NB patients are critical to reducing morbidity and preserving kidney function.\u003c/p\u003e \u003cp\u003eTraditional diagnostic tools, such as serum creatinine, glomerular filtration rate (GFR) estimations, and imaging, have limited sensitivity, particularly for detecting early-stage kidney injury [3]. In addition, the available radiological and nuclear imaging modalities for the assessment of upper urinary tract function are associated with radiation exposure. Thus, there is a need for non-invasive biomarkers offering more sensitive and dynamic assessments of kidney health. Among these, urinary neutrophil gelatinase-associated lipocalin (NGAL) and Renalase have gained attention for their potential roles in detecting kidney injury and monitoring disease progression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNGAL, a protein released by injured kidney tubular epithelial cells, is a well-established early marker of acute kidney injury (AKI) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has been linked to tubular damage in various kidney disorders, including AKI, chronic kidney disease (CKD), and urinary tract obstructions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies have specifically explored the role of NGAL in the pediatric population, highlighting its potential in assessing tubular injury and disease progression [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A recent study examined NGAL and KIM-1 as tubular injury markers in pediatric NB, reinforcing the need for further investigation into these biomarkers in this subgroup [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, limited studies have investigated its utility in pediatric NB patients, where chronic and recurrent kidney insults are prevalent. Similarly, Renalase, a flavoprotein involved in catecholamine metabolism and oxidative stress regulation, has shown promise as a biomarker for CKD in adult populations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While it has shown promise in adult studies, its application in pediatric populations, particularly those with NB, remains underexplored [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to evaluate the utility of urinary NGAL and Renalase as biomarkers of deterioration of kidney function in pediatric NB by comparing the levels of these biomarkers between patients vs. reference group. Also, by comparing biomarker levels across distinct clinical subgroups within NB patients, classified based on GFR and kidney scarring, we sought to determine their association with the severity of kidney injury.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003eThis single-center cross-sectional study was conducted between September-November 2024, in the Department of Pediatric Surgery at the All India Institute of Medical Sciences, New Delhi, India. Ethical approval was obtained from the institutional review board (Ref no: AIIMSA2015/06.09.2024) prior to study initiation, and all procedures adhered to institutional guidelines. Written informed consent was obtained from the parents or legal guardians of all children, and assent was provided by participants as age-appropriate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Population\u003c/h3\u003e\n\u003cp\u003eThe study included a total of 44 consecutive children (\u0026lt;\u0026thinsp;18 years) with NB who were in regular follow-up in the outpatient department (OPD). The diagnosis of NB was based on clinical, radiological, and urodynamic criteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Inclusion criteria for the NB group were confirmed diagnosis of NB, regular follow-up at our institution, and availability of clinical and laboratory data. Exclusion criteria included the presence of an active UTI (defined as positive urine culture with \u0026gt;\u0026thinsp;10⁵ CFU/mL and associated symptoms), prior history of kidney transplantation, presence of other primary kidney diseases, and use of nephrotoxic drugs within three months prior to sampling. In addition, 45 age- and gender-matched healthy children (reference group) were also recruited during the same period. Inclusion criteria for the reference group were healthy children attending the general pediatric OPD with no history of neurological or urological disorders and the absence of any chronic illnesses. Exclusion criteria for this group was any known congenital or acquired kidney disease, prior history of UTI, and abnormal findings on routine urine analysis.\u003c/p\u003e\n\u003ch3\u003eUrine Collection and Biomarker Analysis\u003c/h3\u003e\n\u003cp\u003eSingle spot urine samples were collected from each participant in the morning to minimize circadian variation. For those who were able to void (including reference group), a midstream clean-catch urine sample was obtained. For patients who were unable to void or who were on clean intermittent catheterization (CIC), samples were obtained aseptically via catheterization. Collected samples were centrifuged and the supernatants were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eUrinary NGAL and renalase levels were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits following manufacturers' instructions. Absorbance was read at 450 nm using a microplate reader (Infinite 200 PRO, Tecan, Switzerland), and all values were normalized to urinary creatinine, measured via Jaffe\u0026rsquo;s method, to adjust for urinary concentration variability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eUpper Tract Functional Investigations\u003c/h3\u003e\n\u003cp\u003eAs per the Institutional Protocol, upper tract functional assessment using nuclear scintigraphy studies, i.e., technetium-99m diethylene triamine pentaacetic acid (DTPA) scan for glomerular filtration rate (GFR) assessment and technetium-99m dimercaptosuccinic acid (DMSA) scan for evaluation of kidney scars (defined as focal or diffuse cortical defects with reduced tracer uptake), are performed at recruitment and yearly follow-up visits. Thus, recent GFR values and scarring status of all patients were recorded and they were divided into two groups. Group 1 comprised patients without a significant kidney function impairment, whereas Group 2 included patients with a significant kidney function impairment, defined by a GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73 m\u0026sup2; or evidence of scarring in one or both kidneys as confirmed by DMSA scintigraphy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData entry was done using Microsoft Excel spreadsheets and the analysis was performed using GraphPad Prism 5 software. Data were presented as median (interquartile range, IQR) for continuous variables based on the Shapiro-Wilk test for normality. Urinary NGAL and renalase levels were compared between the patients and reference group using the Mann-Whitney U test. Spearman\u0026rsquo;s correlation coefficients were used to evaluate correlations between urinary biomarker levels and the different parameters of functional investigations (DTPA and DMSA). Group comparisons of urinary NGAL and renalase levels across different patient groups (based on CKD stages and scarring status) were performed using the Kruskal-Wallis test, followed by Dunn's multiple comparison tests for pairwise comparisons. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePatient Characteristics\u003c/h2\u003e \u003cp\u003eThe patients with NB (n\u0026thinsp;=\u0026thinsp;44) consisted of 20 girls (45.46%) and 24 boys (54.54%). Among the reference group, the gender distribution was B: G\u0026thinsp;=\u0026thinsp;24 (53.33%): 21 (46.67%). The median age of the NB group was 120 months (Range: 9\u0026ndash;192 months), while the reference group had a median age of 96 months (Range: 12\u0026ndash;180 months). The age difference between the two groups was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.1833).\u003c/p\u003e \u003cp\u003eResults of the DTPA and DMSA scans were available for 41/44 and 42/44 patients respectively. The distribution of patients among Group 1 and Group 2 were 27 (64.29%) and 15 (35.71%) respectively. Further, stratification by GFR revealed that 9.75% of patients had GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73 m\u0026sup2;, 29.27% had GFR between 60\u0026ndash;89 mL/min/1.73 m\u0026sup2;, and 60.98% had GFR\u0026thinsp;\u0026gt;\u0026thinsp;90 mL/min/1.73 m\u0026sup2;. In terms of kidney scarring, as assessed by DMSA scintigraphy, 69.05% had no scarring while 30.95% of the patients had scars in their kidneys.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiomarker Concentrations in the Cohort\u003c/h3\u003e\n\u003cp\u003eThe median concentrations of urinary NGAL in patients and reference group were 31.86 (IQR: 20.41\u0026ndash;55.34) pg/mg creatinine and 23.40 (IQR: 15.14\u0026ndash;34.24) pg/mg creatinine respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Upon comparison, a statistically significant difference was observed among patients vs. reference group (p\u0026thinsp;=\u0026thinsp;0.0345). Similarly, the levels of urinary renalase were significantly elevated in the NB group (median\u0026thinsp;=\u0026thinsp;2.75 ng/mg creatinine, IQR: 1.42\u0026ndash;4.66 ng/mg creatinine) compared to the reference group (median\u0026thinsp;=\u0026thinsp;1.76 ng/mg creatinine, IQR: 1.10\u0026ndash;2.99 ng/mg creatinine); p\u0026thinsp;=\u0026thinsp;0.0493 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eUrinary Biomarker Levels and Deterioration of Kidney Function\u003c/h2\u003e \u003cp\u003eBased on the availability of GFR and DMSA reports, 42 patients were categorized into subgroups for further analysis. Urinary biomarker profiles revealed significant differences between NB patients with and without significant kidney function impairment. The median urinary NGAL levels for Group 1 and Group 2 were 26.76 (IQR: 15.55\u0026ndash;49.25) and 46.90 (IQR: 23.82\u0026ndash;83.61) pg/mg creatinine respectively, demonstrating a statistically significant difference (U\u0026thinsp;=\u0026thinsp;124.0, p\u0026thinsp;=\u0026thinsp;0.0406) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Similarly, the urinary renalase levels for Group 1 and Group 2 were 1.82 (IQR: 1.03\u0026ndash;3.28) and 3.75 (IQR: 2.33\u0026ndash;7.85) ng/mg creatinine respectively, reflecting a significant difference between the two groups (U\u0026thinsp;=\u0026thinsp;95.0, p\u0026thinsp;=\u0026thinsp;0.0050) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRelationship of Urinary NGAL and Renalase levels with falling GFR\u003c/h2\u003e \u003cp\u003eThe analysis of urinary biomarker levels in relation to GFR categories indicated a potential association between biomarker levels and the severity of chronic kidney disease. Patients with GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73 m\u0026sup2; exhibited the highest median NGAL levels (63.11 pg/mg creatinine), followed by those in the 60\u0026ndash;89 mL/min/1.73 m\u0026sup2; category (31.16 pg/mg creatinine), and the lowest levels were observed in patients with GFR\u0026thinsp;\u0026gt;\u0026thinsp;90 mL/min/1.73 m\u0026sup2; (26.75 pg/mg creatinine) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A similar trend was observed for renalase levels, with median concentrations of 5.31 ng/mg creatinine in the GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73 m\u0026sup2; group, 2.76 ng/mg creatinine in the 60\u0026ndash;89 mL/min/1.73 m\u0026sup2; group, and 1.82 ng/mg creatinine in those with GFR\u0026thinsp;\u0026gt;\u0026thinsp;90 mL/min/1.73 m\u0026sup2; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrelation analysis further demonstrated a moderate negative and statistically significant correlation of NGAL levels with GFR (r = -0.3344, p\u0026thinsp;=\u0026thinsp;0.0326), suggesting that as kidney function declines, NGAL levels tend to increase. Similarly, renalase levels showed a significant negative correlation with GFR (r = -0.4054, p\u0026thinsp;=\u0026thinsp;0.0085), indicating a trend toward higher renalase levels with decreased kidney function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRelationship of Urinary NGAL and Renalase Levels with Kidney Scarring\u003c/h2\u003e \u003cp\u003eInterestingly, urinary biomarker levels revealed a clear association with kidney scarring. The participants were categorized into three groups: reference group, patients with no kidney scars, and patients with scarring. NGAL levels for these groups were 23.91 pg/mg creatinine, 27.89 pg/mg creatinine, and 46.90 pg/mg creatinine respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Renalase levels also showed a similar trend, with median concentrations of 1.76 ng/mg creatinine in the reference group, 2.14 ng/mg creatinine in patients with no kidney scars, and 2.92 ng/mg creatinine among the patients with scarring (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study underscores the clinical relevance of urinary NGAL and Renalase as potential biomarkers for assessing kidney dysfunction and scarring in pediatric NB patients. Our findings demonstrate a significant association between elevated biomarker levels, declining GFR, and kidney scarring, underscoring their utility in identifying early kidney injury and stratifying patients based on the severity of kidney impairment [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Multiple studies have reported that NGAL levels rise rapidly in response to ischemic kidney injury, correlating with histological tubular damage [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, Liu et al. demonstrated that NGAL is a strong predictor of chronic kidney disease progression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In our study, elevated urinary NGAL levels in NB patients with scarring and reduced GFR align with these findings, emphasizing its relevance as an early marker of tubular damage in this specific population.\u003c/p\u003e \u003cp\u003eRenalase, a flavin adenine dinucleotide (FAD)-dependent oxidase, is secreted into circulation by the kidneys and acts on catecholamines such as adrenaline and dopamine, reducing their levels and mitigating oxidative stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This activity plays a protective role by preventing sustained hypertension and vascular damage. However, during kidney injury, the compromised secretion of Renalase paradoxically leads to local catecholamine accumulation, which exacerbates oxidative stress and triggers pro-inflammatory pathways. This cascade promotes endothelial dysfunction, tubular damage, and fibrosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Wisniewska et al. have reported increased urinary Renalase levels in CKD patients, suggesting a compensatory response to oxidative stress and catecholamine dysregulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Its role as a biomarker of kidney injury has been reported in conditions such as diabetic nephropathy and hypertensive kidney disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Also, previously published studies have demonstrated increased urinary Renalase levels in adult patients with CKD and requiring hemodialysis, correlating inversely with kidney function [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The current study extends these observations to pediatric NB, with significantly higher urinary Renalase levels in patients with scarring or low GFR, reflecting ongoing oxidative stress and potential maladaptive kidney repair mechanisms.\u003c/p\u003e \u003cp\u003eThe inverse correlations between GFR and both biomarkers indicate a progressive increase in biomarker levels with worsening kidney function. These results corroborate with the findings of prior studies, such as those, reporting a similar relationship between NGAL levels and GFR in CKD patients,[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and those showing an association of Renalase with kidney functional decline [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The observed increase in NGAL and Renalase levels with confirmed kidney scarring further supports their role not only as functional markers of kidney injury but also as indicators of structural damage. This connection may be explained by the interplay of chronic inflammation, oxidative stress, and impaired tubular repair mechanisms in NB-related kidney dysfunction. Kidney scarring may further amplify these processes, thus, creating a vicious cycle of injury and maladaptive repair.\u003c/p\u003e \u003cp\u003eWhile our study provides valuable insights, some limitations should be acknowledged. First, the cross-sectional design limits our ability to establish temporal or causal relationships between biomarker levels and disease progression. A longitudinal study would provide more definitive evidence of the predictive value of NGAL and Renalase in NB patients. Second, our cohort size, though carefully selected, remains relatively small, which may restrict the generalizability of our findings. Expanding the study to include larger, multicenter cohorts could address this limitation. Third, while we normalized biomarker levels to urinary creatinine, individual variations in hydration status and muscle mass could introduce variability. Pairing urinary biomarkers with serum levels or other normalization strategies may enhance accuracy in future studies.\u003c/p\u003e \u003cp\u003eLooking ahead, our findings open several pathways for future research. Investigating the dynamic changes in NGAL and Renalase levels in response to therapeutic interventions, such as clean intermittent catheterization (CIC) or anticholinergic therapy, could validate their role as markers of treatment response. Additionally, integrating these biomarkers into risk prediction models alongside clinical parameters, imaging, and other biomarkers (e.g., KIM-1, MCP-1, etc.) could enhance their utility in routine clinical practice. The incorporation of these biomarkers into standardized monitoring protocols could improve early detection and guide personalized treatment decisions for pediatric NB patients. Their ability to detect kidney dysfunction at an early stage could aid in risk stratification, enabling timely interventions to prevent further kidney deterioration. Additionally, these biomarkers could play a crucial role in differentiating between varying degrees of kidney injury, helping clinicians tailor the treatment strategies based on disease severity. Future research should focus on evaluating their clinical utility in distinguishing between different NB phenotypes and disease progression patterns. The potential role of NGAL and Renalase in predicting long-term outcomes, such as progression to CKD stage 5 or kidney failure, also warrants exploration. Future longitudinal studies are necessary to validate these findings and explore their utility in disease management.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUrinary NGAL and Renalase levels were significantly elevated in pediatric NB patients compared to the reference group, as well as in NB patients with deteriorated kidney function compared to those without a functional deterioration. A significant negative correlation of these biomarkers with GFR underscores their relevance in evaluating kidney function. In addition, their association with kidney scarring also highlights their potential role in assessing kidney impairment. These findings suggest that urinary NGAL and Renalase could serve as non-invasive biomarkers for the early detection and monitoring of kidney dysfunction in pediatric NB patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe abstract of this work has been submitted for consideration for presentation at the 35th European Society for Paediatric Urology (ESPU) Congress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, S.A.; experimentation, A.S., and S.A.; sample collection, H.K.; formal analysis, writing, and original draft preparation, A.S., and S.A.; review and editing, J.M, A.V., and P.H.; access to lab facility and essential reagents, K.L.; S.A. will act as the guarantor of the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrownrigg, N., Lorenzo, A. J., Rickard, M., \u0026amp; Dos Santos, J. (2024). 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Circulating Renalase as Predictor of Renal and Cardiovascular Outcomes in Pre-Dialysis CKD Patients: A 5-Year Prospective Cohort Study. \u003cem\u003eLife (Basel), 11\u003c/em\u003e(3). doi:10.3390/life11030210\u003c/li\u003e\n\u003cli\u003eWoo, K. S., Choi, J. L., Kim, B. R., Kim, J. E., An, W. S., \u0026amp; Han, J. Y. (2012). Urinary neutrophil gelatinase-associated lipocalin levels in comparison with glomerular filtration rate for evaluation of renal function in patients with diabetic chronic kidney disease. \u003cem\u003eDiabetes Metab J, 36\u003c/em\u003e(4), 307-313. doi:10.4093/dmj.2012.36.4.307\u003c/li\u003e\n\u003cli\u003eSerban-Feier, L. F., Cuiban, E., Gogosoiu, E. B., Stepan, E., \u0026amp; Radulescu, D. (2024). Renalase Potential as a Marker and Therapeutic Target in Chronic Kidney Disease. \u003cem\u003eBiomedicines, 12\u003c/em\u003e(8). doi:10.3390/biomedicines12081715\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pediatric-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pnep","sideBox":"Learn more about [Pediatric Nephrology](http://link.springer.com/journal/467)","snPcode":"467","submissionUrl":"https://www.editorialmanager.com/pnep/default2.aspx","title":"Pediatric Nephrology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Neurogenic Bladder, Urinary Biomarkers, NGAL, Renalase, Kidney Dysfunction, Chronic Kidney Disease, Kidney Scar","lastPublishedDoi":"10.21203/rs.3.rs-5998098/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5998098/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUrinary biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) and Renalase hold promise for assessing kidney health, yet their role in the pediatric neurogenic bladder (NB) remains unclear. This study evaluates their clinical utility in detecting kidney dysfunction and their association with disease severity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA cross-sectional study included 44 NB patients and 45 age- and gender-matched healthy children (reference group). Urinary NGAL and Renalase levels were measured using ELISA. NB patients were categorized based on glomerular filtration rate (GFR) and kidney scarring. Biomarker levels were compared using the Mann-Whitney U test, and their correlations with functional parameters (DTPA, DMSA) were assessed using Spearman\u0026rsquo;s correlation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUrinary NGAL and Renalase levels were significantly higher in NB patients than in the reference group (NGAL: 31.86 vs. 23.40 pg/mg creatinine, p\u0026thinsp;=\u0026thinsp;0.0345; Renalase: 2.75 vs. 1.76 ng/mg creatinine, p\u0026thinsp;=\u0026thinsp;0.0493). NB patients with GFR\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73 m\u0026sup2; or kidney scarring had elevated NGAL (46.90 vs. 26.76 pg/mg creatinine, p\u0026thinsp;=\u0026thinsp;0.0406) and Renalase (3.76 vs. 1.82 ng/mg creatinine, p\u0026thinsp;=\u0026thinsp;0.0050). Both biomarkers correlated inversely with GFR (NGAL: r = -0.3344, p\u0026thinsp;=\u0026thinsp;0.0326; Renalase: r = -0.4054, p\u0026thinsp;=\u0026thinsp;0.0085) and increased with kidney scarring, suggesting their potential role in assessing kidney injury severity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eUrinary NGAL and Renalase are elevated in pediatric NB patients, particularly in those with kidney dysfunction, and correlate with GFR and kidney scarring. These findings highlight their potential as non-invasive markers for early detection and monitoring of kidney impairment in NB. Future longitudinal studies are warranted to validate their diagnostic and prognostic utility.\u003c/p\u003e","manuscriptTitle":"Urinary NGAL and Renalase as Non-Invasive Biomarkers for Detection of Deterioration of Kidney Function and Kidney Scarring in Children with Neurogenic Bladder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:38:23","doi":"10.21203/rs.3.rs-5998098/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept as is","date":"2025-04-29T08:42:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-22T15:59:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-22T14:57:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T09:49:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pediatric Nephrology","date":"2025-03-20T01:55:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pediatric-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pnep","sideBox":"Learn more about [Pediatric Nephrology](http://link.springer.com/journal/467)","snPcode":"467","submissionUrl":"https://www.editorialmanager.com/pnep/default2.aspx","title":"Pediatric Nephrology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6fee0263-5f33-4bbd-b2d1-7e19020c0edc","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T15:59:22+00:00","versionOfRecord":{"articleIdentity":"rs-5998098","link":"https://doi.org/10.1007/s00467-025-06808-z","journal":{"identity":"pediatric-nephrology","isVorOnly":false,"title":"Pediatric Nephrology"},"publishedOn":"2025-05-14 15:57:08","publishedOnDateReadable":"May 14th, 2025"},"versionCreatedAt":"2025-03-27 04:38:23","video":"","vorDoi":"10.1007/s00467-025-06808-z","vorDoiUrl":"https://doi.org/10.1007/s00467-025-06808-z","workflowStages":[]},"version":"v1","identity":"rs-5998098","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5998098","identity":"rs-5998098","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}