Neonates with a prenatal history of hydronephrosis were enrolled in this study. 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. Those with vesicoureteral reflux (VUR) were excluded from the study.

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 ^99mTc-MAG3, ^99mTc-DTPA, and ^99mTc-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 (T_1/2) 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. The samples were collected in sterile polypropylene containers. One-milliliter aliquots were centrifuged at 4000×g 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. All samples were analyzed in duplicate and blinded to clinical status.

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. After washing, biotinylated antihuman MCP1, ET-1, or NAG antibody was added. Unbound antibodies washed away, and a horseradish peroxidase-conjugated streptavidin was used for detection of the presence of 3,3',5,5'-Tetramethylbenzidine substrate. Reference concentrations of ET-1, MCP-1, and NAG were used for assay calibration. 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. Inter- and intra-assay coefficients of variation were below 10%. For each assay, the results were calculated with reference to a standard curve. 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. The values were expressed in ng/mg or U/mg Cr.

Categorical variables were presented as percentages, whereas continuous variables were expressed as means±standard deviation or median (25-75th quartiles). The normality of distribution was assessed by using the Kolmogorov-Smirnov test. 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. The chi-square test was used for the qualitative data. 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 best cutoff point was chosen according to the ROC curve. In addition, the area under the curve was calculated. The results were expressed with a 95% confidence interval (CI). A p-value of <0.05 was considered statistically significant. All statistical analysis was performed by using SPSS ver. 16 (SPSS Inc., Chicago, IL, USA).

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). The demographic and other characteristics of the study population are summarized in Table 1. 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 ET-1/Cr ratio was not significantly different between the two obstructive groups. 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).