antenatal hydronephrosis ( ah ) is a relatively common finding on gestational ultrasound : it is observed in about 0.5% to 1% of pregnant women , especially after the 28th gestational week . ureteropelvic junction obstruction ( upjo ) is the most common finding in fetuses with ah and in neonates with unilateral hydronephrosis . it has been suggested that most babies do not need any treatment other than observation and close follow - up . in infants whose renal function is severely reduced owing to obstructive uropathy , prompt surgical treatment is recommended . the current tools for assessment of renal differential function and obstruction are renal scintigraphy modalities , such as tc - mag3 , tc - dtpa , and tc - ec scans . this technique has some limitations , including cost , exposure to ionizing radiation , invasiveness ( because of the need for injections in all cases and catheterization in many cases ) , and finally inaccessibility in some countries . therefore , development of a diagnostic modality that is less invasive and has no risk of exposure to toxic chemicals is necessary . urinary biomarkers are candidates for assessment of kidney injury and have been studied widely for this purpose . theoretically , these markers could be used for the early diagnosis of kidney damage in cases with obstruction . in this study , we assessed the urinary levels of 3 markers in neonates : endothelin-1 ( et-1 ) , monocyte chemotactic peptide-1 ( mcp-1 ) , and n - acetyl - beta - d - glucosaminidase ( nag ) . these biomarkers are potentially related to the renal cell injury that occurs before the clinical appearance of the insult . et-1 is a strong vasoconstrictor that has a role in the development of stenosis in the preglomerular arterioles . it is a mediator of vascular and cellular damage in the course of urinary system obstruction . mcp-1 is a peptide with 76 amino acids that belongs to the family of chemokines and exerts strong chemoattractant activities on monocytes , t cells , and natural killer cells . nag is a lysosomal enzyme that is abundantly present in the cells of the proximal tubule . it is considered to be a very sensitive marker of renal tubular impairment in various disease states . low nag levels are found in normal urine as a result of the exocytosis / endocytosis transport system of the proximal tubules . any perturbation of this system results in an elevated excretion of different nag isoenzymes , including intermediate forms , which then provides an excellent early indicator of any kind of tubular damage . the prospective study was approved by the research committee of mazandaran university of medical sciences ( approval number # 4/p/674 ) and was performed from march 2012 to january 2014 . all parents provided signed , written informed consent before enrollment of their neonates into this study . ah was defined as an anteroposterior ( ap ) pelvic diameter of > 7 mm after the 36th gestational week and > 5 mm before the 36th gestational week on an ultrasound exam . postnatal ultrasonography was performed on days 3 - 7 and weeks 6 - 8 on the basis of our hospital policy . the severity of hydronephrosis was assessed according to the society of fetal urology criteria and is presented as grade 1 to grade 4 . cystography was performed at week 1 for neonates with severe bilateral hydronephrosis and at weeks 6 - 8 in other infants with significant persistent postnatal hydronephrosis . neonates without vur who had persistent significant hydronephrosis ( grade 3 or 4 , or pelvic diameter > 15 mm ) were studied by dynamic renal scan by use of tc - mag3 , tc - dtpa , and tc - ec radiopharmaceuticals for assessment of the presence of any obstruction and the severity of renal function impairment . the obstruction was defined as a time to reduction of tracer uptake to half value ( t1/2 ) of more than 15 minutes . a differential function of less than 40% was considered as functional impairment of the renal unit . the indications for surgery were differential renal function < 40% , bilateral obstruction , unilateral obstruction in a solitary kidney , and severely symptomatic patients . in this study , patients were categorized into two groups : group 1 included neonates who had severe hydronephrosis and were candidates for surgical intervention on the basis of functional impairment of the involved kidney ( differential renal function , < 40% ) ; group 2 included neonates with less severe hydronephrosis with preserved renal function who did not need any surgical treatment . those with vur , posterior urethral valve , and other postbladder obstructions were excluded from the study . all neonates were assessed for blood urea nitrogen , plasma creatinine ( cr ) , and urinary levels of et-1 , mcp-1 , nag , and cr . fresh first - morning voided urine samples were obtained in the first week before contrast studies . one - milliliter aliquots were centrifuged at 4000g for 10 minutes and the supernatant fraction was stored at -80 until analysis . the urine was tested for the presence of blood or leucocytes by use of urine analysis strips ; samples containing blood or leucocytes were excluded . urinary levels of et-1 and mcp-1 were determined by use of commercially available immunoassays ( abcam , cambridge , uk ) according to the manufacturer 's protocol . in addition , urinary nag concentrations were measured by a colorimetric assay according to the manufacturer 's recommendation ( boehringer - mannheim gmbh , ingelheim am rhein , germany ) . in brief , samples were placed into wells coated with a mouse monoclonal antibody as a catcher . unbound antibodies washed away , and a horseradish peroxidase - conjugated streptavidin was used for detection of the presence of 3,3',5,5'-tetramethylbenzidine substrate . absorption was determined with an enzyme - linked immunosorbent assay ( elisa ) reader ( biotek elx800 , bio - tek instruments inc . , winooski , vt , usa ) at 450 nm . the assay sensitivities were 1.1 and 0.41 ng / l for mcp-1 and et-1 and 5 u / l for nag , respectively . the sensitivities of the assays were 1.1 ng / l for mcp-1 , 0.41 ng / l for et-1 , and 5 u / l for nag , respectively . all samples were analyzed in duplicate with the appropriate standards on 96-well microplates . for practical purposes , we collected random urinary measures and then normalized the results to urinary cr values to modulate the daily changes in urinary excretion of markers . categorical variables were presented as percentages , whereas continuous variables were expressed as meansstandard deviation or median ( 25 - 75th quartiles ) . statistical analysis of the difference between groups with normal distributions was determined by using the t - test or fisher exact test to compare two groups . nonparametric tests such as the mann - whitney test were used for variables that were not distributed normally . a receiver operating characteristic ( roc ) curve was drawn and the sensitivity and specificity of the different cutoff points for et-1 , mcp-1 , nag , et-1/cr , mcp-1/cr , and nag / cr were determined . the results were expressed with a 95% confidence interval ( ci ) . a p - value of < 0.05 was considered statistically significant . the prospective study was approved by the research committee of mazandaran university of medical sciences ( approval number # 4/p/674 ) and was performed from march 2012 to january 2014 . all parents provided signed , written informed consent before enrollment of their neonates into this study . ah was defined as an anteroposterior ( ap ) pelvic diameter of > 7 mm after the 36th gestational week and > 5 mm before the 36th gestational week on an ultrasound exam . postnatal ultrasonography was performed on days 3 - 7 and weeks 6 - 8 on the basis of our hospital policy . the severity of hydronephrosis was assessed according to the society of fetal urology criteria and is presented as grade 1 to grade 4 . cystography was performed at week 1 for neonates with severe bilateral hydronephrosis and at weeks 6 - 8 in other infants with significant persistent postnatal hydronephrosis . neonates without vur who had persistent significant hydronephrosis ( grade 3 or 4 , or pelvic diameter > 15 mm ) were studied by dynamic renal scan by use of tc - mag3 , tc - dtpa , and tc - ec radiopharmaceuticals for assessment of the presence of any obstruction and the severity of renal function impairment . the obstruction was defined as a time to reduction of tracer uptake to half value ( t1/2 ) of more than 15 minutes . a differential function of less than 40% was considered as functional impairment of the renal unit . the indications for surgery were differential renal function < 40% , bilateral obstruction , unilateral obstruction in a solitary kidney , and severely symptomatic patients . in this study , patients were categorized into two groups : group 1 included neonates who had severe hydronephrosis and were candidates for surgical intervention on the basis of functional impairment of the involved kidney ( differential renal function , < 40% ) ; group 2 included neonates with less severe hydronephrosis with preserved renal function who did not need any surgical treatment . those with vur , posterior urethral valve , and other postbladder obstructions were excluded from the study . all neonates were assessed for blood urea nitrogen , plasma creatinine ( cr ) , and urinary levels of et-1 , mcp-1 , nag , and cr . fresh first - morning voided urine samples were obtained in the first week before contrast studies . one - milliliter aliquots were centrifuged at 4000g for 10 minutes and the supernatant fraction was stored at -80 until analysis . the urine was tested for the presence of blood or leucocytes by use of urine analysis strips ; samples containing blood or leucocytes were excluded . urinary levels of et-1 and mcp-1 were determined by use of commercially available immunoassays ( abcam , cambridge , uk ) according to the manufacturer 's protocol . in addition , urinary nag concentrations were measured by a colorimetric assay according to the manufacturer 's recommendation ( boehringer - mannheim gmbh , ingelheim am rhein , germany ) . in brief , samples were placed into wells coated with a mouse monoclonal antibody as a catcher . unbound antibodies washed away , and a horseradish peroxidase - conjugated streptavidin was used for detection of the presence of 3,3',5,5'-tetramethylbenzidine substrate . absorption was determined with an enzyme - linked immunosorbent assay ( elisa ) reader ( biotek elx800 , bio - tek instruments inc . , winooski , vt , usa ) at 450 nm . the assay sensitivities were 1.1 and 0.41 ng / l for mcp-1 and et-1 and 5 u / l for nag , respectively . the sensitivities of the assays were 1.1 ng / l for mcp-1 , 0.41 ng / l for et-1 , and 5 u / l for nag , respectively . all samples were analyzed in duplicate with the appropriate standards on 96-well microplates . for practical purposes , we collected random urinary measures and then normalized the results to urinary cr values to modulate the daily changes in urinary excretion of markers . categorical variables were presented as percentages , whereas continuous variables were expressed as meansstandard deviation or median ( 25 - 75th quartiles ) . statistical analysis of the difference between groups with normal distributions was determined by using the t - test or fisher exact test to compare two groups . nonparametric tests such as the mann - whitney test were used for variables that were not distributed normally . a receiver operating characteristic ( roc ) curve was drawn and the sensitivity and specificity of the different cutoff points for et-1 , mcp-1 , nag , et-1/cr , mcp-1/cr , and nag / cr were determined . the results were expressed with a 95% confidence interval ( ci ) . a p - value of < 0.05 was considered statistically significant . forty - two neonates were enrolled in this study and classified into two groups : group 1 included neonates with ah who were candidates for surgical intervention ( n=24 , 21 males and 3 females ) ; group 2 included neonates with ah with milder degrees of obstruction who did not need any surgical intervention ( n=18 , 16 males and 2 females ) . there were no significant differences in sex , gestational maturity , or type of delivery between the groups ( p=0.89 , p=0.83 , and p=0.66 respectively ) . surgery was performed in all group 1 patients at a mean age of 4.24 months for functional impairment . in this study , 27 patients ( 64% ) had unilateral hydronephrosis ; however , there was no significant difference in laterality in the two groups of patients ( p=0.09 ) . as shown in table 2 , most neonates in group 1 had severe degrees of hydronephrosis in the pre- and postnatal period . the median urinary levels and 25th and 75th quartiles of the biomarkers ( et-1 , mcp-1 , and nag ) are presented in table 3 . for standardization purposes , we calculated values as a ratio to urinary cr ( et-1/cr , mcp-1/cr , and nag / cr ) . the kolmogorov - smirnov test was used for assessment of normality of distribution ; none of the distributions met the criteria for normality , so nonparametric tests were performed . in comparison between the two groups , there were no significant differences for absolute measures of et-1 , mcp-1 , and nag . when we standardized the values to urinary cr , however , some differences appeared . the nag / cr ratio was higher in group 1 than group 2 , but the difference was not statistically significant . the mcp-1/cr ratio was significantly higher in group 1 than in group 2 ( 1.47 ng / mg vs. 0.46 ng / mg , respectively ; p=0.012 ) . the sensitivity and specificity of the et-1/cr , mcp-1/cr , and nag / cr ratios were calculated from the roc curves ( fig . 1 ) . groups 1 and 2 were compared to find the cutoff value for distinguishing neonates with severe obstructive hydronephrosis from those with milder forms without any indication for surgical intervention . for comparison of groups 1 and 2 , the cutoff values were measured as follows : 0.5709 ng / mg ( sensitivity , 75% ; specificity , 67% ; positive predictive value [ ppv ] , 71% ; negative predictive value [ npv ] , 71% ) for et-1/cr , 0.927 ng / mg ( sensitivity , 77% ; specificity , 72% ; ppv , 77% ; npv , 72% ) for mcp-1/cr , and 1.1913 iu / mg ( sensitivity , 62% ; specificity , 67% ; ppv , 68% ; npv , 60% ) for nag / cr ratios ( table 4 ) . forty - two neonates were enrolled in this study and classified into two groups : group 1 included neonates with ah who were candidates for surgical intervention ( n=24 , 21 males and 3 females ) ; group 2 included neonates with ah with milder degrees of obstruction who did not need any surgical intervention ( n=18 , 16 males and 2 females ) . there were no significant differences in sex , gestational maturity , or type of delivery between the groups ( p=0.89 , p=0.83 , and p=0.66 respectively ) . surgery was performed in all group 1 patients at a mean age of 4.24 months for functional impairment . in this study , 27 patients ( 64% ) had unilateral hydronephrosis ; however , there was no significant difference in laterality in the two groups of patients ( p=0.09 ) . as shown in table 2 , most neonates in group 1 had severe degrees of hydronephrosis in the pre- and postnatal period . the median urinary levels and 25th and 75th quartiles of the biomarkers ( et-1 , mcp-1 , and nag ) are presented in table 3 . for standardization purposes , we calculated values as a ratio to urinary cr ( et-1/cr , mcp-1/cr , and nag / cr ) . the kolmogorov - smirnov test was used for assessment of normality of distribution ; none of the distributions met the criteria for normality , so nonparametric tests were performed . in comparison between the two groups , there were no significant differences for absolute measures of et-1 , mcp-1 , and nag . the nag / cr ratio was higher in group 1 than group 2 , but the difference was not statistically significant . the mcp-1/cr ratio was significantly higher in group 1 than in group 2 ( 1.47 ng / mg vs. 0.46 ng / mg , respectively ; p=0.012 ) . the sensitivity and specificity of the et-1/cr , mcp-1/cr , and nag / cr ratios were calculated from the roc curves ( fig . 1 ) . groups 1 and 2 were compared to find the cutoff value for distinguishing neonates with severe obstructive hydronephrosis from those with milder forms without any indication for surgical intervention . for comparison of groups 1 and 2 , the cutoff values were measured as follows : 0.5709 ng / mg ( sensitivity , 75% ; specificity , 67% ; positive predictive value [ ppv ] , 71% ; negative predictive value [ npv ] , 71% ) for et-1/cr , 0.927 ng / mg ( sensitivity , 77% ; specificity , 72% ; ppv , 77% ; npv , 72% ) for mcp-1/cr , and 1.1913 iu / mg ( sensitivity , 62% ; specificity , 67% ; ppv , 68% ; npv , 60% ) for nag / cr ratios ( table 4 ) . the risk of permanent renal damage warrants surgical intervention in the first year of life . assessment of differential renal function by scintigraphic studies is at the center of decision - making strategies . the cellular effect of urinary obstruction comprises three basic mechanisms : ( 1 ) leukocyte ( especially macrophage ) migration and interstitial infiltration , ( 2 ) vasogenic effects and ischemia , and ( 3 ) cellular death and apoptosis . the markers for each of the 3 pathways are mcp-1 for the first stage , et-1 for the vascular pathway , and nag for the cellular stage . despite the common characteristics of the 3 markers , each biomarker has a unique mechanism of action . in our study , only the mcp-1/cr ratio was significantly higher in the severe obstruction group than in the mild obstruction group . the nag / cr ratio was not significantly higher in the severe obstruction group , and the et-1/cr ratio was similar in the 2 groups . the immunologic and tubulointerstitial changes in the obstructive process begin to develop in the first weeks of life , but other processes such as vascular or enzymatic processes take more time to develop . the participants in our study were neonates aged less than 2 weeks , and thus our results support this timing of events . they measured urinary mcp-1 levels by elisa and evaluated mcp-1 gene expression by in situ hybridization . they found that urinary mcp-1 concentrations were significantly positively correlated with the mean transit time of tc - mag3 through the cortex of the obstructed kidney . stephan et al .   performed an animal study on 48 surgically obstructed male juvenile wistar rats ( 28 days old ) . the renal pelvic urinary concentration and renal mrna expression of mcp-1 were correlated with the grade of obstruction and indicated the degree of subsequent renal damage in hydronephrosis . urinary mcp-1 increased significantly in the upjo group compared with the control group and significantly improved in the operated group . the results supported the role of urinary mcp-1 as a marker of tubulointerstitial damage in human obstructive nephropathy . taranta - janusz et al .   performed a prospective case - control study in children with hydronephrosis caused by upjo . children were categorized into two study groups : the first group comprised 15 surgical cases with severe hydronephrosis due to a unilateral , critical degree of ureteral stenosis and who underwent pyeloplasty ; the second group comprised 21 conservative cases with mild , nonobstructive hydronephrosis who did not require any surgery . those authors reported that urinary mcp-1/cr levels from voided urine before and after surgery and concentrations obtained from the affected pelvis were significantly greater than in the second group and the control group ( p<0.05 ) . the difference in the mcp-1/cr level from voided urine and the concentration in the affected pelvis was not statistically significant . .   reported on 28 children with unilateral upjo and 13 healthy sex- and age - matched children . there was a further increase in the cytokine in the peri- and postoperative ( 1 day and 3 weeks ) samples followed by a decline , but the concentrations in the postoperative ( 3 months and 1 year ) samples were not significantly different from the controls . one or 2 weeks of postnatal life are not long enough for the vascular or enzymatic pathways of cellular damage induced by obstruction to begin . sharifian et al .   determined a cutoff point of 50 pg / dl for the urinary et-1 level for diagnosis of hydronephrosis in children . the expression of et-1 in the obstructed ureteropelvic junction was significantly higher than in control tissue . ma et al .   analyzed the clinical urinary nag value in evaluating the degree of damage to hydronephrotic kidneys in children . urine nag levels in healthy and hydro nephrotic kidneys were 3.31(100.11)10 iu / l and 5.91(100.17)10 iu / l , respectively ( p=0.05 ) . there was a significant positive correlation between urinary nag levels from hydronephrotic kidneys and pathologic grades ( r=0.769 , p<0.05 ) . studied 31 children with hydronephrosis diagnosed by use of abdominal ultrasonography either prenatally or postnatally . the urinary nag / cr ratio was significantly higher in the patients with hydronephrosis in comparison with the reference data . this finding , of no relation between the urinary nag / cr level and the severity of obstruction , is similar to the results of our study . therefore , our hypothesis that the age of the children is the most important factor in the discrepancy in urinary levels of the three biomarkers may be only a simple assumption . the urinary mcp-1/cr ratio is significantly elevated in neonates with severe obstruction requiring surgical intervention . these results suggest that urinary mcp-1/cr may be useful in the identification of severe obstructive hydronephrosis in neonates .
purposeantenatal hydronephrosis ( ah ) is found in 0.5%-1% of neonates . 
 the aim of the study was to assess the urinary concentrations of 3 biomarkers , endothelin-1 ( et-1 ) , monocyte chemotactic peptide-1 ( mcp-1 ) , and n - acetyl - glucosaminidase ( nag ) in severely hydronephrotic neonates.materials and methodsneonates with a history of prenatal hydronephrosis were enrolled in the prospective study in 2 groups . 
 group 1 included neonates with severe forms of obstruction requiring surgical intervention and group 2 included neonates with milder forms of obstruction without any functional impairment . 
 fresh voided urinary levels of et-1 , mcp-1 , and nag were measured and their ratios to urinary cr were calculated.resultsfourty-two neonates were enrolled into the 2 groups : group 1 , 24 patients ( 21 male , 3 female ) ; group 2 , 18 neonates ( 16 male , 2 female ) . 
 there were no statistically significant differences between urinary et-1 , nag , mcp-1 values , and et-1/cr and nag / cr ratios in groups 1 and 2 . 
 the urinary mcp-1/cr ratio was significantly higher in group 1 than in group 2 . 
 for comparison of groups 1 and 2 , the cut - off values were measured as 0.5709 ng / mg ( sensitivity , 75% ; specificity , 67% ; positive predictive value [ ppv ] , 71% ; negative predictive value [ npv ] , 71% ) , 0.927 ng / mg ( sensitivity , 77% ; specificity , 72% ; ppv , 77% ; npv , 72% ) , and 1.1913 iu / mg ( sensitivity , 62% ; specificity , 67% ; ppv , 68% ; npv , 60% ) for et-1/cr , mcp-1/cr , and nag / cr ratios , respectively.conclusionsthe urinary mcp-1/cr ratio is significantly elevated in neonates with severe obstruction requiring surgical intervention . based upon these results , urinary mcp-1/cr may be useful in identification of severe obstructive hydronephrosis in neonates .