Pediatr Nephrol (2012) 27:2099–2106 DOI 10.1007/s00467-012-2217-6

ORIGINAL ARTICLE

Urinary NGAL, cystatin C, β2-microglobulin, and osteopontin significance in hydronephrotic children Mia Gebauer Madsen & Rikke Nørregaard & Johan Palmfeldt & Lars Henning Olsen & Jørgen Frøkiær & Troels Munch Jørgensen Received: 1 March 2012 / Revised: 25 April 2012 / Accepted: 25 April 2012 / Published online: 19 June 2012 # IPNA 2012

Abstract Background Ureteropelvic junction obstruction (UPJO) accounts for 35 % of all congenital hydronephrosis cases. The challenge in managing childhood hydronephrosis is to distinguish obstructive from nonobstructive cases and, thereby, identify patients requiring surgical intervention. This study aimed to examine four urinary proteins as potential biomarkers of obstruction in hydronephrosis. Methods Urine samples from 24 children with UPJO were collected pre-, peri-, and postoperatively, together with urine samples from healthy children. Neutrophil gelatinaseassociated lipocalin (NGAL), cystatin C (CyC), β2microglobulin (β2-M), and osteopontin (OPN) in the samples were measured simultaneously using multiplex sandwich immunoassay technology. Results Compared with controls, NGAL and β2-M were significantly increased in urine from patients with obstructed kidneys at the time of surgery. This increase was followed by a decrease and stabilization to the same level as that of the controls. Furthermore, age was negatively correlated with preoperative urinary concentrations of CyC, β2-M, and OPN. M. G. Madsen : L. H. Olsen : T. M. Jørgensen Department of Urology, Pediatric Section, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark M. G. Madsen (*) : R. Nørregaard : J. Frøkiær The Water and Salt Research Center, Institute of Clinical Medicine, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark e-mail: [email protected] J. Palmfeldt Research Unit for Molecular Medicine, Aarhus University Hospital, Brendstrupgaardsvej 21, 8200 Aarhus N, Denmark

Conclusions This study confirms increased concentrations of NGAL and β2-M in urine from obstructed kidneys and should be tested in larger studies to ascertain their ability to identify obstruction and to determine the importance of ageadjusted reference values. Keywords Hydronephrosis . Ureteropelvic junction obstruction . NGAL . Cystatin C . β2-microglobulin . Osteopontin

Introduction Hydronephrosis is diagnosed in 0.5–1 % of all newborns, and ureteropelvic junction obstruction (UPJO) is the most frequently observed cause [1]. Hydronephrosis requires close patient monitoring, as it may progress and if left untreated result in reduced kidney function. In patient management, the challenge is to preserve renal function by early identification of patients who require surgical treatment. Available tests of obstruction include repetitive ultrasonographies, invasive diuretic renograms, and monitoring of serum creatinine (Cr), all of which may provide information about disease progression but have no value as long-term prognostic indicators. This justifies the ongoing search for noninvasive urinary biomarkers with the potential to aid early identification of kidney injury, thus providing the clinician with a fast, specific, and reliable prognostic tool. Several potential urinary biomarkers have been suggested in the past decade, but none of these has yet been implemented in daily clinical practice due to their limitations and the need for further validation in clinical studies [2]. It is unlikely that a single protein will meet the required criteria for a urinary biomarker in UPJO, and given the multifactorial nature of UPJO, including the unpredictable course of the condition, we believe that a panel of urinary biomarkers

2100

will be necessary. In this study we evaluated, neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), β2-microglobulin (β2-M), and osteopontin (OPN) in urine samples from children who underwent surgery for UPJO and compared values to those of healthy children. NGAL is a protein synthesized in the distal nephrons of the kidney and secreted into the urine by the thick ascending limb of the loop of Henle and the collecting ducts of the kidney [3, 4]. In addition, a possible synthesis in the proximal tubules has been suggested [5–7]. An increased urinary NGAL (uNGAL) level is a well-established biomarker of kidney injury, and its role has been examined in various kidney diseases [3–5, 8–10]. In animal studies of obstructive nephropathy, NGAL was reported to accumulate in urine collected from a dilated pelvis [4], and recently, a study performed in children with severe UPJO suggests that increasing uNGAL concentrations are associated with worsening obstruction [11]. CyC is a cysteine protease inhibitor that is stably secreted from all nucleated cells, freely filtered through the glomerulus, and completely reabsorbed by the proximal tubule [12]. Urinary CyC (uCyC) was tested as an early detector of acute kidney injury following cardiac surgery and was found to be a potentially valuable tool for diagnosing tubular damage and dysfunction [13]. β2-M is a small protein filtered through the glomerulus and almost completely reabsorbed by proximal tubular cells [14]. It is present in small amounts in urine from healthy humans and to a much greater degree in urine from patients with tubular proteinemia, renal failure, or kidney transplants [15]. Bartoli et al. demonstrated a significantly higher concentration of urinary β2-M (uβ2-M) in children with UPJO compared with that of healthy controls [16]. OPN is a glycosylated phosphoprotein produced by renal tubular epithelial cells [17]. Several animal studies of unilateral ureteral obstruction show a dual action of OPN in the kidney, indicating its role in mediating early interstitial macrophage influx and fibrosis but also its functions as a survival factor for renal tubulointerstitial cells, where it suppresses apoptosis [18–20]. The four urinary proteins we examined are all sensitive biomarkers of early kidney injury. This clinical follow-up study was designed to assess whether they are of value for predicting renal function and thus may be useful as urinary biomarkers in hydronephrosis.

Patients and methods Study population Twenty-four children diagnosed with unilateral UPJO were included in a prospective study from 2007 to 2011. They

Pediatr Nephrol (2012) 27:2099–2106

were included at referral for a scheduled Anderson–Hynes pyeloplasty at the Department of Urology, Aarhus University Hospital, Denmark. The day before the planned surgery, patients underwent renal ultrasonography by an experienced pediatric radiologist to assess the anterior–posterior (AP) diameter. The degree of hydronephrosis was graded using the Society of Fetal Urology (SFU) system: grade 1 0 visualization of the renal pelvis; grade 2 0 a dilated renal pelvis and a few visualized calyces; grade 3 0 a dilated renal pelvis with many identified calyces; grade 4 0 a similar appearance as grade 3, but the involved kidney has parenchymal thinning when compared with the normal kidney [21]. The patients also underwent technetium-99mmercaptoacetyltriglycine (99mTc-MAG-3) renography to determine differential renal function (DRF): a DRF of the obstructed kidney < 40 % was considered abnormal. A pyeloplasty was advised for patients with ipsilateral flank pain and for those with declining function of the hydronephrotic kidney >5 % and a DRF < 40 %. Exclusion criteria were bilateral hydronephrosis; urinary tract infections within the past 3 months; previous surgery in the urinary system; deformations in the lower part of the ureter, bladder or urethra; urinary stones; vesicoureteral reflux; neurogenic bladder dysfunction; and noncompliance. All patients were operated on by a pediatric urologist with a robot-assisted retroperitoneoscopic pyeloplasty with insertion of a thin stent (Salle Pyeloplasty Stent 4.7 Cook Urological, Spencer, IN, USA) to reduce the load on the anastomosis between the pelvis and ureter. After the stent was inserted, the anastomosis was sutured and the stent guided through the skin and carefully attached with a bandage. The stent was closed on the first postoperative day and removed without anesthetic at the outpatient clinic after 3 weeks. Follow-ups, including renal ultrasonography and 99mTc-MAG-3 renography, were scheduled at the nearest regional hospital at 3 months and 1 year after the procedure. Study design Urine samples were collected six times: 1. Preoperative bladder urine (the day before surgery). 2. Perioperative urine from the affected pelvis and bladder urine (i.e., from the nonobstructed kidney collected after opening the affected pelvis). 3. Postoperative (1 day) urine from the stent (i.e., from the obstructed kidney) and the bladder (i.e., from the nonobstructed kidney, as the stent was open). 4. Postoperative (3 weeks) urine from the stent and the bladder (i.e., from both kidneys, as the stent was closed until sample collection). 5. Postoperative (3 months bladder urine).

Pediatr Nephrol (2012) 27:2099–2106

6. Postoperative (1-year) bladder urine. Preoperative, and postoperative (3 months and 1 year) bladder urine samples were collected as a voided midstream urine sample. After an announcement of inclusion of healthy children as controls to a clinical study, 13 healthy sex and agematched children were included. Controls were not exposed to any intervention, and therefore, they did not attend a 1-year follow-up exam. A voided midstream urine sample was collected from all controls. Morning urine was avoided to reduce the degradation of proteins in the bladder/urinary tract. Urine samples were rapidly frozen and stored at −80°C until assayed. All analyses were performed in 2011 to limit interassay variation by using assays with the same batch number. Multiple freeze/thaw cycles were avoided. Analysis Protein profiles were measured by a bead-based multiplex sandwich immunoassay that uses the xMAP® detection technology developed by Luminex (Luminex, Austin, TX, USA). The simultaneous detection and quantification of multiple analytes was made possible by measuring spectral properties of the multifluorescent-coded beads and the amount of associated fluorescence, followed by generation of the mean fluorescence intensity (MFI) by Luminex 100 IS Total System equipped with StarStation version 2.3 software (Applied Cytometry, Sheffield, UK). NGAL, CyC, β2M, and OPN were measured simultaneously in urine using a Bead Human Kidney Toxicity/Injury Panel 2 (Widescreen: EMD Chemicals Inc., Merck, Darmstadt, Germany). The assay was set up for duplicate measurements of standard analyte mixtures and urine samples according to the manufacturer’s instructions. A five-parameter logarithmic algorithm was used to generate a standard curve. This curve-fitting equation was used to calculate the concentration of unknown samples using MFI as the input value. The standard recovery was calculated by taking the ratio of the calculated concentration value divided by the expected amount of the standard and expressing that as a percentage. All data were in the recovery range between 70 % and 130 %, which was considered acceptable. The limit of detection and the lower and higher limits of quantification were determined. If the measured concentration in the samples was lower than the limit, the sample concentration was set to 0. The measured concentrations were normalized to urinary Cr concentration [measured by enzymatic methods (Vitros 950: Johnson & Johnson, Denmark)].

2101

Statistics The STATA/IC 11.0 (StataCorp LP, College Station, TX, USA) software package was used for statistical analyses. Due to the small sample size, nonparametric statistical analysis was used. A Mann–Whitney U test was used to compare controls and patients. Kruskal–Wallis and Wilcoxon signed rank tests were used to analyze the patient group. Correlations were calculated using Spearman’s test. The receiver operating characteristic (ROC) curve was used to determine cutoff values of uNGAL and uβ2-M that yielded the best sensitivity and specificity. A p value<0.05 was considered significant. Values are presented as medians with ranges in parenthesis. Ethics statement The study was approved by the Local Ethics Committee of the Central Denmark Region and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Parents gave informed consent prior to the inclusion of their child in the study.

Results Clinical parameters of the patient group are presented in Table 1. The control group was comparable with the patient group with respect to gender (eight boys and five girls) and age [median 8.3 (3.5–14.5) years]. Median patient age was 8.0 (3.5–14.5) years. Two patients were excluded after time point 3 due to pain and infection, respectively. One patient was excluded after time point 4 due to severe pain after removal of the stent, which was followed by insertion of a nephrostomy catheter. One patient did not attend the 3-month control evaluation, and three patients did not attend the 1-year control evaluation. Eighteen patients attended the 1-year follow-up exam. Preoperatively, the median DRF of the obstructed kidney was 44 % (20–54 %), and six patients had a DRF <40 %. There were no correlations between measured urinary proteins and DRF, AP diameter, SFU grading, gender, or operative findings. On the contrary, a negative correlation was found between age and preoperative uβ2M (r0 −0.430, p00.04), uCyC (r0 −0.689, p00.0003), and uOPN (r0−0.686, p00.0003), respectively. Median urinary concentrations of the four studied proteins in all samples are presented in Table 2, and several measurements in the patient samples were significantly higher than those of controls (*p<0.05). Figure 1 depicts the dynamics of urinary excretion of the four proteins after obstruction relief. Concentrations of NGAL and β2-M showed significantly increased concentrations in the

2102

Pediatr Nephrol (2012) 27:2099–2106

Table 1 Clinical parameters of the patient group Patient characteristics Gender (male/female) Laterality (left/right) Age at diagnosis (years) Age at operation (years) Age groups 3–6 years 6.1–9 years 9.1–12 years

14/10 11/13 6.5 (0–14) 8.0 (3.5–14.5) 7 8 7

12.1–15 years Differential renal function (< 40 %/>40 %) Operative finding (stenotic segment/accessory artery) SFU grading (grade 1/2/3/4)

2 6/18 8/16 0/3/15/6

Anterior–posterior diameter (mm) Preoperative (n024) Three months postoperative (n020) One year postoperative (n018) Differential renal function (%) Preoperative (n024) Three months postoperative (n020) One year postoperative (n018)

35.5 (13–65) 16.0 (5–40) 20.0 (7–30) 44 (20–54) 48 (20–53) 49 (33–53)

Values are presented as medians with ranges given in parenthesis SFU Society for Fetal Urology

perioperative urine [NGAL: 48.5 (5.1–896.2) ng/mg Cr; β2M: 191.7 (13.9–1431.8) ng/mg Cr] compared with controls [NGAL: 8.1 (3.4–19.6) ng/mg Cr; β2-M: 113.6 (1.6–219.2)

ng/mg Cr]. This was followed by a decline in values, which were not significantly different from those of controls when measured in both the 3-month and 1-year follow-up samples. Figure 2 illustrates urinary concentrations in individual patients at the perioperative (i.e., from obstructed kidney) and postoperative time points. Decreases in uNGAL and uβ2-M were significant (p00.0125, p00.0072, respectively), and these decreases were observed in 75 % and 81 % of patients, respectively. The CyC urinary excretion pattern in the patient group exhibited significant changes, with an increase in perioperative and postoperative (1-day) samples. However, there were no differences compared with controls. OPN excretion pattern in the patient group did not exhibit significant differences through the six time points; similarly, there was no significant difference between perioperative and postoperative (1-year) samples (Fig. 2). On the first postoperative day, urine from the obstructed kidney exhibited increased concentrations for all four proteins compared with urine from the nonobstructed kidney and with urine from controls (Table 2). This was also the case for uNGAL and uCyC in the perioperative samples. We used ROC curve analyses to test the ability of uNGAL and uβ2-M perioperatively measured in urine from the obstructed kidney to distinguish children with UPJO from healthy children. The area under the uNGAL ROC curve (AUC) was 0.923 [95 % confidence interval (CI) 0.837– 1.000], and the best cutoff value was 20.57 ng/mg Cr, which yielded 82 % sensitivity and 100 % specificity. The uβ2-M ROC AUC was 0.811 (95 % CI 0.661–0.952), and the best cutoff value was 191.8 ng/mg Cr, which yielded 68 % sensitivity and 92 % specificity.

Table 2 Urinary concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), β2-microglobulin (β2-
CyC (ng/mg Cr)

β2-M (ng/mg Cr)

OPN (ng/mg Cr)

11.9 (0–46.1)

99.0 (43.4–163.3)

109.8 (9.2–256.2)

1,664.9 (578.6–4,676.3)

73.3*, ** (5.3–591.3) 24.0* (0–1201.2)

122.3** (0–232.3) 116.2 (27.1–184.1)

207.5* (11.7–1,346.8) 172.5* (16.1–2,216.7)

1,218.9 (0–5,946.9) 1,439.3 (0–5,884.3)

Patients 1. Preoperative 2. Perioperative Obstructed kidney Contralateral kidney 3. Postoperative (1 day) Obstructed kidney Contralateral kidney 4. Postoperative (3 weeks) Obstructed kidney Bladder urine (both kidneys) 5. Postoperative (3 months) 6. Postoperative (1 year) Controls

130.8*, ** (14.6–12006.3) 151.2*, ** (60.1–6283.6) 267.6*, ** (32.4–9,287.3) 2,131.7*, ** (85.6–16,337.9) 27.2* (2.2–281.2) 67.9 (0–257.7) 81.8 (16.7–626.9) 769.0 (0–3,823.0) 81.9* (19.2–969.7) 71.3* (15.4–287.9) 6.0 (0–116.2) 10.7 (0–646.7) 8.1 (3.4–19.6)

111.5 (56.0–344.0) 110.2 (39.2–174.4) 85.2 (4.2–763.7) 74.2 (0–172.6) 99.5 (13.5–215.6)

152.4* (4.8–604.6) 131.7 (0–303.3) 100.2 (0–891.4) 99.5 (0–452.2) 113.6 (1.6–219.2)

1,282.0 1,168.8 1,295.9 1,409.4 1,139.7

(378.2–5,708.6) (173.6–4,325.5) (145.4–15,496.2) (415.7–2,761.3) (89.5–3,052.7)

Values normalized to urinary creatinine (Cr) and presented as medians, with ranges in parenthesis *p<0.05, patients compared with controls, **p<0.05, urine from obstructed kidney compared with nonobstructed kidney at the same time point

Pediatr Nephrol (2012) 27:2099–2106

2103

Fig. 1 Urinary concentrations of neutrophil gelatinaseassociated lipocalin (NGAL), cystatin C (CyC), β2microglobulin (β2-M), and osteopontin (OPN) in patients and controls. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. The error bars above and below the box indicate the 90th and 10th percentiles. In order to make the medians comparable, the perioperative and postoperative (1 day) concentrations are calculated as an average of the concentrations in urine from the obstructed and the contralateral kidney. The postoperative (3 weeks) concentrations are from bladder urine. *p<0.05, patients compared with controls. a NGAL. Patients: Kruskal– Wallis test, chi-squared055.138, df05, p00.0001. b CyC. Patients: Kruskal–Wallis test, chi-squared011.266, df05, p0 0.0464. c β2-M. Patients: Kruskal–Wallis test, chi-squared0 22.696, df05, p00.0009. d OPN. Patients: Kruskal–Wallis test, chi-squared02.302, df05, p00.8060

Discussion We collected pre-, peri-, and postoperative urine samples from 24 children who underwent surgery to treat UPJO and compared them with urine samples from healthy children. Data showed significantly increased concentrations of NGAL and β2-M in urine from obstructed kidneys compared with contralateral kidneys and controls. The urinary biomarker discovery field in UPJO is challenged by the biological variability (both intra- and interindividual) in the composition of urine, the fact that the potential biomarkers are nonspecific to UPJO, and the multifactorial status of the disease, which features different etiologies and varying severity. All of these challenges came into play in the study. We established strict criteria for inclusion and exclusion, which contributed to a more homogenous study group compared with many other reported studies. Patients for whom surgical treatment is indicated due to either frequent pyelonephritis or asymptomatic massive hydronephrosis were

not included. Our inclusion criteria resulted in the creation of two groups of patients: one with reduced DRF (n06) and one with symptoms of hydronephrosis consisting of pain (n018). Results from the two groups were compared and analyzed independently without any significant differences between groups being identified. Therefore, we believe it is reasonable to present data from this collective study sample. There were significantly increased concentrations of NGAL in the perioperative and postoperative (1 day) urine samples compared with preoperative urine and controls. At those time points, NGAL concentration in urine from the obstructed kidney was found to be significantly increased compared with urine from the contralateral kidney. This corresponds well to a study by Kuwabara et al., who reported increased NGAL concentrations in urine from a dilated pelvis in a mouse model with obstructive nephropathy [4]. In a clinical case–control study, Wasilewska et al. reported a significantly greater preoperative uNGAL level in children with severe hydronephrosis compared with

2104

Pediatr Nephrol (2012) 27:2099–2106

Fig. 2 Individual urinary concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), β2microglobulin (β2-M), and osteopontin (OPN) in patients and controls. Perioperative values are concentrations in urine from the obstructed kidney. Only patients who attended the 1-year follow-up evaluation are presented (n018) ,together with controls (n013)

controls, a finding our study did not confirm [11]. Preoperative uNGAL in their study was twofold higher than our measured preoperative uNGAL, and in contrast to our findings, uNGAL from healthy controls was almost fourfold lower. Both patient and control groups in the study of Wasilewska et al. were considerably younger (median age 2.2 and 5.0 years, respectively) than the two groups in our study (median age 8.0 and 8.3 years, respectively), which may partially explain the discrepancy between concentration levels. We did not observe a significant correlation between age and uNGAL, but there was a tendency toward a negative correlation. Ichino et al. reported very high uNGAL concentrations in healthy children younger than 1 year, with a rapid decline within the following 1–2 years [22]. Their results emphasize the necessity of age adjustment regarding interpreting uNGAL concentrations in children. Corresponding to NGAL, β2-M concentrations in the perioperative and postoperative (1 day) urine samples were significantly increased compared with preoperative urine and controls. Bartoli et al. studied 28 children referred for

surgical treatment due to UPJO and compared their preoperative uβ2-M to that of healthy children [16]; surprisingly, we observed uβ2-M concentrations approximately threefold lower than the concentrations reported in their study. Nevertheless, the two studies are not completely comparable, as Bartoli et al.’s study group consisted of both unilateral and bilateral UPJO, as well as cases with recurrent urinary infections. These discrepancies may all have contributed to their higher uβ2-M concentrations. Our study showed a negative correlation between age and uβ2-M, and this may partially explain the higher uβ2-M concentrations in Bartoli et al.’s study, as their report included five infants. Comparison of preoperative patient samples with controls demonstrated no significant differences in uOPN and uCyC. At the first postoperative day, OPN and CyC in urine from the obstructed kidney were significantly increased compared with urine from the nonobstructed kidney and controls, which was probably a response to the surgical trauma and the presence of the stent. Urinary excretion patterns of CyC and OPN in ureteral obstruction have not

Pediatr Nephrol (2012) 27:2099–2106

previously been studied. Animal experiments demonstrate increased renal OPN expression in ureteral obstruction and that OPN facilitates macrophage recruitment to sites of inflammation; however, OPN also suppresses apoptosis and may function as a survival factor for renal tubulointerstitial cells [18, 19]. Our study did not show a high uOPN, as would have been predicted by the animal studies mentioned. This might be explained by the dual actions of OPN. It is well known that surgery leads to specific endocrine, immunologic, and metabolic changes and effects various organ functions [23]. In comparison with open surgery, a laparoscopic procedure reduces surgical stress; however, the establishment of pneumoperitoneum alters certain physiologic functions [24]. The increase in abdominal pressure affects the renal system and is associated with a decrease in renal blood flow, glomerular filtration, and urinary output [24]. It is important to consider the physiological effects of the laparoscopic procedure when interpreting the results of this study. The observed increases in urinary concentrations of NGAL and β2-M might simply be a response to surgical stress. However, comparisons between concentrations in urine from the obstructed and nonobstructed kidneys do not support this as the only explanation, as concentrations in urine from the obstructed kidney were significantly higher. One weakness of this study is the lack of functional patient data. An absolute kidney function (glomerular filtration rate; GFR) would have been desirable, but this category of patients is not routinely assessed for GFR. Instead, we had the opportunity to correlate our results with the DRF and AP diameter, but this was limited by the fact that only six patients had a DRF < 40 %. Another limitation is the control group. The parents were interviewed regarding the health status of the child, but none of the children underwent a renal ultrasonography to rule out hydronephrosis. Furthermore, controls were not exposed to surgical trauma, which, in any case, would not be ethically justifiable. The study was also limited by a small sample size and thus a lack of statistical power. In addition, it only represents results from a single institution. In conclusion, this study confirmed increased concentrations of NGAL and β2-M in urine from obstructed kidneys when compared with the contralateral kidneys and controls. With the significant decline in concentrations of uNGAL, uCyC, and uβ2-M after the first postoperative day, these proteins may be potential markers of convalescence, indicating success of the surgical treatment. In addition, the diagnostic profiles of uNGAL and uβ2-M in identifying children with UPJO from healthy children were good. Future studies should be designed with strict patient group definitions (e.g., children with different degrees of UPJO, with nonobstructive hydronephrosis, and healthy children) in order to investigate the discriminative ability

2105

of the biomarker. An essential challenge is the establishment of reference values, as they are likely to depend on the methods of analysis but also on the biological variation between individuals and probably the age of the child. Therefore, the cutoff values of NGAL and β2-M presented in our study cannot be directly generalized to other patient groups. Larger clinical studies with a longer follow-up are required to determine reference ranges that may need to be individualized. Acknowledgements The authors thank the technicians at the Water and Salt Research Center, Aarhus University Hospital, Denmark, for expert technical assistance, and Professor Jørgen Thorup (Department of Pediatric Surgery, Rigshospitalet, Copenhagen University Hospital, Denmark) for inclusion of patients into a pilot study

References 1. Cortes D, Jorgensen TM, Rittig S, Thaarup J, Hansen A, Andersen KV, Thorup J, Jorgensen C, Sogaard K, Eskild-Jensen A, Frokiaer J, Horlyk A, Jensen F (2006) Prenatal diagnosed hydronephrosis and other urological anomalies. Ugeskr Laeger 168:2544–2550 2. Madsen MG, Norregaard R, Frokiaer J, Jorgensen TM (2011) Urinary biomarkers in prenatally diagnosed unilateral hydronephrosis. J Pediatr Urol 7:105–112 3. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P (2003) Identification of neutrophil gelatinaseassociated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14:2534–2543 4. Kuwabara T, Mori K, Mukoyama M, Kasahara M, Yokoi H, Saito Y, Yoshioka T, Ogawa Y, Imamaki H, Kusakabe T, Ebihara K, Omata M, Satoh N, Sugawara A, Barasch J, Nakao K (2009) Urinary neutrophil gelatinase-associated lipocalin levels reflect damage to glomeruli, proximal tubules, and distal nephrons. Kidney Int 75:285–294 5. Ding H, He Y, Li K, Yang J, Li X, Lu R, Gao W (2007) Urinary neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for renal tubulointerstitial injury in IgA nephropathy. Clin Immunol 123:227–234 6. Devarajan P (2008) Emerging urinary biomarkers in the diagnosis of acute kidney injury. Expert Opin Med Diagn 2:387–398 7. Mishra J, Mori K, Ma Q, Kelly C, Yang J, Mitsnefes M, Barasch J, Devarajan P (2004) Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 15:3073–3082 8. Brunner HI, Mueller M, Rutherford C, Passo MH, Witte D, Grom A, Mishra J, Devarajan P (2006) Urinary neutrophil gelatinaseassociated lipocalin as a biomarker of nephritis in childhood-onset systemic lupus erythematosus. Arthritis Rheum 54:2577–2584 9. Bolignano D, Donato V, Coppolino G, Campo S, Buemi A, Lacquaniti A, Buemi M (2008) Neutrophil gelatinase-associated lipocalin (NGAL) as a marker of kidney damage. Am J Kidney Dis 52:595–605 10. Bolignano D, Coppolino G, Campo S, Aloisi C, Nicocia G, Frisina N, Buemi M (2007) Neutrophil gelatinase-associated lipocalin in patients with autosomal-dominant polycystic kidney disease. Am J Nephrol 27:373–378 11. Wasilewska A, Taranta-Janusz K, Debek W, Zoch-Zwierz W, Kuroczycka-Saniutycz E (2011) KIM-1 and NGAL: new markers of obstructive nephropathy. Pediatr Nephrol 26:579–586

2106 12. Nickolas TL, O'Rourke MJ, Yang J, Sise ME, Canetta PA, Barasch N, Buchen C, Khan F, Mori K, Giglio J, Devarajan P, Barasch J (2008) Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury. Ann Intern Med 148:810–819 13. Koyner JL, Vaidya VS, Bennett MR, Ma Q, Worcester E, Akhter SA, Raman J, Jeevanandam V, O'Connor MF, Devarajan P, Bonventre JV, Murray PT (2010) Urinary biomarkers in the clinical prognosis and early detection of acute kidney injury. Clin J Am Soc Nephrol 5:2154–2165 14. Bartoli F, Gesualdo L, Paradies G, Caldarulo E, Infante B, Grandaliano G, Monno R, Leggio S, Salzillo F, Schena FP, Leggio A (2000) Renal expression of monocyte chemotactic protein-1 and epidermal growth factor in children with obstructive hydronephrosis. J Pediatr Surg 35:569–572 15. Miyata T, Jadoul M, Kurokawa K, de Van Ypersele SC (1998) Beta-2 microglobulin in renal disease. J Am Soc Nephrol 9:1723–1735 16. Bartoli F, Penza R, Aceto G, Niglio F, D'Addato O, Pastore V, Campanella V, Magaldi S, Lasalandra C, Di BG, Gesualdo L (2011) Urinary epidermal growth factor, monocyte chemotactic protein-1, and beta2-microglobulin in children with ureteropelvic junction obstruction. J Pediatr Surg 46:530–536 17. Xie Y, Sakatsume M, Nishi S, Narita I, Arakawa M, Gejyo F (2001) Expression, roles, receptors, and regulation of osteopontin in the kidney. Kidney Int 60:1645–1657

Pediatr Nephrol (2012) 27:2099–2106 18. Ophascharoensuk V, Giachelli CM, Gordon K, Hughes J, Pichler R, Brown P, Liaw L, Schmidt R, Shankland SJ, Alpers CE, Couser WG, Johnson RJ (1999) Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis. Kidney Int 56: 571–580 19. Yoo KH, Thornhill BA, Forbes MS, Coleman CM, Marcinko ES, Liaw L, Chevalier RL (2006) Osteopontin regulates renal apoptosis and interstitial fibrosis in neonatal chronic unilateral ureteral obstruction. Kidney Int 70:1735–1741 20. Diamond JR, Kreisberg R, Evans R, Nguyen TA, Ricardo SD (1998) Regulation of proximal tubular osteopontin in experimental hydronephrosis in the rat. Kidney Int 54:1501–1509 21. Fernbach SK, Maizels M, Conway JJ (1993) Ultrasound grading of hydronephrosis: introduction to the system used by the Society for Fetal Urology. Pediatr Radiol 23:478–480 22. Ichino M, Kusaka M, Kuroyanagi Y, Mori T, Morooka M, Sasaki H, Shiroki R, Shishido S, Kurahashi H, Hoshinaga K (2010) Urinary neutrophil-gelatinase associated lipocalin is a potential noninvasive marker for renal scarring in patients with vesicoureteral reflux. J Urol 183:2001–2007 23. Ure BM, Suempelmann R, Metzelder MM, Kuebler J (2007) Physiological responses to endoscopic surgery in children. Semin Pediatr Surg 16:217–223 24. Grabowski JE, Talamini MA (2009) Physiological effects of pneumoperitoneum. J Gastrointest Surg 13:1009–1016