PEDIATRIC UROLOGY Utility of Urine Interleukines in Children with Vesicoureteral Reflux and Renal Parenchymal Damage Azar Nickavar1, Baranak Safaeian2*, Ehsan Valavi3, Homa Davoodi2 Purpose: Vesicoureteral reflux (VUR) is the most common risk factor of urinary tract infection in children. Cur- rently, diagnosis of VUR depends on invasive imaging studies, with a high radiologic burden. Therefore, different biomarkers have been introduced for the evaluation of these patients. The objective of this study was to identify alteration of urinary interleukins (ILs) excretion in children with primary VUR and renal parenchymal damage, for further clinical application. Materials and methods: Urinary concentrations of IL-1α, IL-1β, IL-6, and IL-8 were evaluated in 34 children with VUR (cases) and 36 without VUR (control), during 2018-2019. Urinary concentrations of IL-1, IL-1, IL-6 and IL-8 were measured, using polyclonal antibody ELISA kit, and standardized to urine creatinine (Cr). Patients with infectious or inflammatory disorders, urolithiasis, immune deficiency, acute or chronic kidney disease, and secondary VUR were excluded from the study. Results: Mean age of cases (36.00 ± 27.66) had no significant difference with the control (32.86±29.31) group (p=0.44). The majority of patients had moderate VUR (58.8%), followed by severe (35.3%) and mild (5.9%) grades. Urinary concentration of all ILs/Cr were significantly higher in patients with VUR, compared with those without VUR. There was no significant correlation between urine ILs/Cr with age, gender, serum electrolytes, urine specific gravity, renal ultrasound, laterality or severity of VUR, and DMSA renal scan. All urine ILs/Cr had acceptable sensitivity and accuracy for workup of children with primary VUR. Conclusion: Urine IL-1α, IL-1β, IL-6 and IL-8/Cr were sensitive and accurate additionary screening biomarkers in children with primary VUR. Keywords: vesicoureteral reflux; interleukin; cytokine; renal damage INTRODUCTION Vesicoureteral reflux (VUR) accounts for 30–50% of urinary tract infections (UTI) in children. About 8.5–18% of chronic kidney disease occurs secondary to VUR in pediatrics. Therefore, early diagnosis and ap- propriate management of VUR might prevent its long- term complications, such as hypertension, proteinuria, and decreased renal function(1). Nowadays, identification of VUR depends on invasive and expensive imaging modalities, with high radiolog- ic exposure. Meanwhile, recently introduced noninva- sive biomarkers such as urine interleukins (ILs) have been suggested as alternative diagnostic approach- es in patients with VUR and their high risk siblings (2,3). Cytokines are small soluble proteins, and regulate both humoral and cellular immunity(4). IL-1α, IL-6, and IL-8 are proinflammatory cytokines, which stimulate periph- eral neutrophilia, chemokine secretion, and scar forma- tion in different tissues(5-7). Lymphocyte and plasma cell infiltration is responsi- ble for increased urinary IL excretion in patients with VUR or reflux associated nephropathy(7). Although inflammatory processes and immune system dysfunc- tion have been suggested in the pathogenesis of renal parenchymal damage (RPD), however, a correlation between VUR and urinary cytokines excretion remains controversial(7,8). The purpose of this study was to iden- tify alteration of urinary ILs excretion in children with primary VUR and RPD. MATERIALS AND METHODS This is a cross sectional multicentric case-control study on children admitted to 3 pediatric nephrology clinics during 2018-2019. It was approved by the institutional ethics committee (ethical code; ir.goums. rec.1396.02), and informed consent was obtained from legal guardi- ans. Children with a history of recurrent UTIs, urosep- sis, UTI with abnormal ultrasound, atypical UTI, and asymmetrical kidneys who had a definite cystography and 99mTc-DMSA scintigraphy were included in this study. All of them were in healthy condition with nor- mal body mass index at the time of the study. Totally, 70 children (35 females, 35 males) were evalu- ated. Of them, 34 had VUR (case group) and 36 did not 1Department of pediatric nephrology, Iran university medical sciences, Tehran, Iran. 2Neonatal and children’s health research center, Golestan university of medical sciences, Gorgan, Iran. 3Chronic renal failure research center, Ahvaz Jundishapur university of medical sciences, Ahvaz, Iran. *Correspondence: Baranak Safaeian and Homa Davoodi, Neonatal and children’s health research center, Golestan university of medical sciences, Gorgan, Iran. Tel: 0098-2122226127. Email: Baranak54@yahoo.com. Received January 2020 & Accepted July 2020 Urology Journal/Vol 18 No. 2/ March-April 2021/ pp. 199-202. [DOI: 10.22037/uj.v16i7.5957] have VUR (control group). Conventional cystography (VCUG) or radioisotopic cystography (direct RNC) was done under prophylactic antibiotic treatment in all patients. 99mTc-DMSA renal scan was performed in patients with documented VUR or other inclusion cri- teria 9. Patients with a history of UTI in the preceding 3 months, inflammatory disorders, active infections, ongoing an- tibiotic treatment, secondary VUR, neurogenic bladder, obstructive uropathy, urolithiasis, immune deficiency, malnutrition, obesity, hypertension, and chronic kidney disease were excluded from the study. Based on imaging studies, VUR was classified as mild (I, II), moderate (III), and severe (IV, V) grades. The highest grade was taken in to consideration in patients with bilateral VUR. Parenchymal damage was defined as decreased cortical uptake or renal outline defect in 99mTc-DMSA scintigraphy. A spot morning urine sample was obtained from all in- dividuals and frozen at _80°_ C within 3 hours of col- lection. Urinary level of cytokines was measured using polyclonal antibody ELISA kit. To avoid dilutional effects, urinary ILs were expressed as the ratio of cy- tokine-to-urine creatinine (Cr) excretion (pg/mg). Statistical analysis was performed using SPSS ver. 24.0 and 15.4 Med calc. Values are presented as mean±SD. Student’s t test, nonparametric Mann-Whitney test, and Chi2 were used for comparison of variables between two groups. Correlations between urine ILs with oth- er variables were determined, using the Spearman’s, Mann-Whitney, and Kruskal Walis tests. A receiver operating characteristic (ROC) curve was constructed to determine the cutoff values of each cytokine with the best sensitivity, specificity, and accuracy. P values < 0.05 considered to be statistically significant. RESULTS A total of 34 children with VUR (M/F=1), and 36 with- out VUR (M/F=1) were enrolled in this study. VUR was bilateral in 21 (61.8%) patients. Two patients (1 unilateral, 1 bilateral) had mild VUR, followed by 20 with moderate (7 unilateral, 13 bilateral) and 12 with severe (5 unilateral, 7 bilateral) grades. Laboratory findings including renal function, serum electrolytes, serum bicarbonate, and urine specific gravity had no significant difference between the two groups. Renal ultrasound and 99mTc-DMSA renal scan were normal in the majority of patients, with no signifi- cant difference between the two groups (Table 1). Mean urinary concentration of all ILs/Cr were signif- icantly higher in children with VUR, compared with those without VUR (Table 2). None of the urine ILs had a significant correlation with quantitative (age, sodium, potassium, bicarbonate, urine SG) and qualitative (gender, ultrasound, VUR lat- erality, VUR grade) variables, except for a direct cor- relation between urine IL-1β/Cr (p = 0.033) and IL-6/ Cr (p = 0.037) with DMSA renal parenchymal damage. However, Multivariate analysis showed no association between DMSA scan, unilateral or bilateral VUR, and severity of VUR with urine ILs/Cr excretion (Table 3). The optimal cutoff values of urine ILs/Cr with the highest sensitivity, specificity, and accuracy are shown in Table 4 and Figure 1. Accordingly, all ILs/Cr had acceptable sensitivity and accuracy for the workup of children with VUR. DISCUSSION Increased urinary cytokine excretion occurs secondary to tubular damage and interstitial fibrosis in patients with reflux associated nephropathy(3,7). Therefore, measurement of urine ILs has been suggested for early identification of VUR, prior to the development of RPD and its serious complications(4,10). This study was per- formed to identify alteration of urine ILs excretion in children with primary VUR and RPD. IL-6 is a proinflammatory cytokine which is produced by endothelial and mesangial cells, fibroblasts, activat- ed T cells and B cells, macrophages, and destructive renal tubular cells. Urinary concentration of IL-6 might reflect intrarenal production of this cytokine(7,10). It has a central role in T cell and B cell differentiation, mesangi- al cell proliferation, and promotion of tubulointerstitial damage. Urine IL-6 has been considered a noninvasive Urine interleukins and VUR-Nickavar et al. Table 1. Comparison of qualitative and quantitative variables in children with and without VUR. Variables VUR (mean±SD) No VUR (mean±SD) P-value Age (m) 36.00 ± 27.66 32.86 ± 29.31 0.444 Gender (M/F) 17(50%)/17(50%) 18(50%)18((50%) 1 Serum Na 138.36 ± 6.40 137.22 ± 5.16 0.412 Serum K 4.17 ± 0.55 4.28 ± 0.48 0.172 Serum Cr 0.51 ± 0.10 0.54 ± 0.09 0.278 Serum HCO3 21.37 ± 2.45 21.46 ± 2.47 0.752 Urine SG 1014.12 ± 3.85 1013.67 ± 4.36 0.762 Ultrasound (N/H) 22(64.7%)/12(35.3%) 23(63.9%)/13(36.11%) 0.943 DMSA renal scan (N/D/S) 17(50%)/10(29.4%)/7(20.6%) 27(75%)/6(16.7%)/3(8.3%) 0.09 Abbreviations: m: month, M:male, F: female, Na: sodium, K: potassium, Cr: creatinine, HCO3: bicarbonate, SG: specific gravity, N: normal, H: hydronephrosis, D: decreased cortical uptake, S: scar Variables VUR (mean±SD) No VUR (mean±SD) P-value IL1α/Cr 5.88 ± 7.62 3.52 ± 10.28 < 0.001 IL1β/Cr 343.44 ± 462.37 72.66 ± 193.24 < 0.001 IL6/Cr 8.19 ± 11.01 1.90 ± 7.32 < 0.001 IL8/Cr 14.94 ± 21.78 0.8 ± 1.51 < 0.001 Table 2. Comparison of urine ILs/Cr in children with and without VUR. Vol 18 No 2 March-April 2021 200 biomarker for monitoring the progression of RPD in pa- tients with reflux associated nephropathy(4,6,10). Urine IL-6/Cr was higher in our children with VUR, with acceptable sensitivity and accuracy. Similarly, Krzemier et al showed increased urine IL-6/Cr in 8/33 children, aged 1-24 months with first time febrile UTI and mild- moderate VUR(8). In addition, Gokce found increased urine IL-6/Cr in a study on 114 patients in 4 groups with or without VUR and RPD(7). However, urine IL-6 level was below the lower detection lim- it with no clinical importance in Haraoka et al. study on 17 renal units with VUR (2 mild, 12 moderate and 3 high grade)(11). Fernández et al. found no significant difference of urine IL-6/Cr excretion in a case control study on 40 children with documented VUR(12). Urine IL-6 had no significant correlation with RPD in multivariate analysis and seems to be an unreliable bi- omarker for the prediction of RPD in our study. Simi- larly, Renata et al. found no correlation between urine IL-6 concentration and renal scarring, and urine IL-6 was not higher in those who developed renal scar than those without scar(13). However, urine IL-6 was higher in children with severe renal damage than those without renal scar in Wang et al study on 66 patients aged 10-18 years with a history of antireflux surgery(10) IL-8 is a major proinflammatory chemokine, which is produced by mesangial and destructive renal tubular epithelial cells in patients with RPD,(7) and consider a useful biomarker for localization and determination of the severity of urinary tract inflammation. It has an important role in neutrophil chemoattraction and IL-6 secretion(13). Increased urinary IL-8 concentration has been reported in patients with urinary tract infection, VUR, and congenital kidney urinary tract abnormalities (CAKUT). It has been suggested a sensitive and non- specific screening test for diagnosis of VUR and RPD in the previous studies(3,7). Urine IL-8/Cr level was higher in our children with VUR, compared with the control group. Urine IL-8/ Cr>0.6 pg/ml was a sensitive, specific, and accurate bi- omarker for evaluation of VUR in our patients. There- fore, we suggested evaluation of urine IL-8 as a valu- able test for prediction of VUR. Similarly, urine IL-8 was a noninvasive diagnostic biomarker of isolated VUR in some of the previous studies, which suggested mild inflammatory process in these patients, and inde- pendent to the severity of VUR(3,11,12,14). Galanakis et al. performed a study on 59 infants in 3 groups (24 with VUR, 14 with a history of UTI and no VUR and 21 with a history of impaired renal function), and recommend- ed screening of VUR in patients with increased urine IL-8 excretion(2). Urine IL-8 was higher in patients with VUR and RPD or isolated renal scar in the other stud- ies, which suggested urine IL-8 as a predictive biomark- er of RPD with a direct correlation to the severity of renal damage(7,11,13). However, we showed no correlation between urine IL-8 excretion and DMSA uptake defect in our patients. IL-1 is the first line cytokine of antigen recognition and anti-inflammatory function, which has been considered for prediction of late renal scar in children with acute pyelonephritis(5). Both urine IL-1α/Cr and IL-1β/Cr were significantly higher in our children with VUR, irrespective to its se- verity, with acceptable sensitivity and accuracy for the prediction of VUR. Therefore, alteration of urine IL-1α and IL-1β were valuable biomarkers for the prediction of VUR in our patients. In addition, urine IL-1α/Cr or IL-1β/Cr had no significant correlation with renal dam- age in multivariate analysis. Sheu et al. in a study on 69 children, aged 1-121 months, found no alteration of urine IL-1β excretion in patients with VUR, but lower level in those with renal cortical scarring, and suggested protective effect of urine IL-1β against renal scarring during the active phase of acute pyelonephritis(5). We found no significant correlation between urine ILs with age, gender, serum electrolytes, renal function, lat- erality or severity of VUR, and imaging studies (ultra- sound, DMSA scan) in our patients. Similarly, age and gender had no significant effect on urine IL-1, IL-6, and IL-8 excretion in the previous studies(5,7). We concluded that urine IL-1α/Cr, IL-1β/Cr, IL-6/Cr, and IL-8/Cr were sensitive and accurate noninvasive additionary biomarkers in children with primary VUR. In addition, none of these ILs had significant value for the prediction of renal damage in these patients. The major limitation of this study was the low number of patients with RPD, which needs further studies for accurate diagnosis. Meanwhile, multiple collections of urine ILs over years might benefit the prediction of late renal scar in these patients. In addition, future studies with a larger patient popu- lation are recommended to confirm the potential ap- plication of these biomarkers in suspected patients to primary VUR, especially those with negative imaging studies, and siblings of an index case. Further studies DMSA scan Severity of VUR uunilateral or bilateral VUR Variables Regression coefficient Pv Regression coefficient Pv Regression coefficient P-value IL1α/Cr -0.430 0.793 -.229 0.923 1.219 0.654 IL1β/Cr -14.309 0.886 -52.854 0.717 -61.644 0.710 IL6/Cr -0.538 0.822 -.597 0.894 1.012 0.798 IL8/Cr -1.351 0.788 -1.414 0.839 1.859 0.815 Table 3. Multivariate analysis of urine ILs/Cr excretion (Regression model). Variables Cut point Sensitivity Specificity AUC CI (95%) SE P-value IL1α/Cr >0.83 78.79 69.44 0.744 0.625-0.842 0.0617 < 0.001 IL1β/Cr >12.38 97 63.90 0.854 0.749-0.928 0.0456 < 0.001 IL6/Cr >0.49 90.91 72.22 0.875 0.773-0.942 0.0428 < 0.001 IL8/Cr >0.6 97 77.80 0.929 0.841-0.977 0.0440 < 0.001 Table 4. Sensitivity, specificity and accuracy of different urine ILs/Cr in patients with VUR. Urine interleukins and VUR-Nickavar et al. Pediatric Urology 201 are recommended for differentiation of primary VUR from secondary suspected patients with controversial results. CONFLICT OF INTEREST None declared by the authors. REFERENCES 1. Nickavar A, Valavi E, Safaeian B, Moosavian M.Validity of urine neutrophile gelatinase- associated lipocalin in children with primary vesicoureteral reflux. Int Urol Nephrol. 2020;52:599-602. 2. Galanakis E, Bitsori M, Dimitriou H, Giannakopoulou C, Karkavitsas NS, Kalmanti M. Urine interleukin-8 as a marker of vesicoureteral reflux in infants. 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