Role of Urinary Neutrophil Gelatinase-Associated Lipocalin

(NGAL) to Predict Severity Congenital Anomalies of the

Kidney and Urinary Tract (CAKUT)

Oke Rina Ramayani1*, Kiking Ritarwan2, Putri Chairani Eyanoer3 Rosmayanti S

Siregar1, Rafita Ramayati1
Pediatric Department1, Neurology Department2, Public Health Department3
Medicine Faculty, University of Sumatera Utara, Medan, Indonesia

* Email: oke_rina@yahoo.com

Abstract. The prevalence of chronic kidney diseases (CKD) in children has risen rapidly in
worldwide due to congenital anomalies of the kidney and urinary tract (CAKUT). Urine
NGAL has become a biomarker which has been demonstrated to be associated with poor
prognosis of CKD. The aim of this study was to analyzed urine NGAL on long term CKD in
patients CAKUT. This is a cohort study, involving 120 patients with CAKUT, who were
divided into two groups: the obstructive type and non obstructive type. The primary follow-up
end-point was a composite of halving of eGFR or recurrent urinary tract infection. Secondary
follow-up end-point was eGFR of less than 15 mL/min or death caused by renal. The mean
age of the patients was 7.5 ± 4.1 years and 60% were boys. Sixty two point five percents of
patients had obstructive CAKUT. In bivariate analysis there was strong association between
concentration of urine NGAL and progressivity of CKD (p=.0.001) After regression, urin
NGAL continued to be associated with progressivity of CKD (AUC 74%). Therefore, we
suggest that urine NGAL could be considered in predicted severe CKD in CAKUT patients.

1. Introduction
These CAKUT anomalies are the spectrum of malformations that occur in the kidney and urinary tract,
result from defective development of the kidney and urinary tract.[1,2] The most common post natal
manifestation of CAKUT include palpable abdominal mass or decreased urinary output (obstructive
type), meanwhile in those without urinary tract symptoms (non obstructive type), detection of CAKUT
is not easy, as an incidental finding during a routine examination or while the child is undergoing
investigation for an unrelated complaint. [3,4] Almost all infants and children with CAKUT have
higher chance to develop urinary tract infection (UTI) and or obstruction that are risk to renal
impairment (chronic kidney disease).
Most CAKUT cases in developing countries lack of ante/post natal screening, delay treatment
and progress from chronic kidney disease (CKD) to end stage renal disease (ESRD). In CAKUT with
CKD, the tubulointerstitial disease is an important step that leads to nephron loss and in turn leads to
interstitial inflammation and fibrosis.[1,5,6] In response to injury, tubular epithelial cells produce
cytokines, such as NGAL. The production of urin NGAL is increased occurring in states of tubular
injury [7,8] Aim of this article is to describe role of urin NGAL to predict severity of CAKUT .

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2. Material and Methods
This was a cohort study carried out in the Department of Pediatric Nephrology, University of
Sumatera Utara, Medan, Indonesia. This study was done over a period of 4 years from February 2014-
February 2017. After written informed consent and ethical clearance from University of Sumatera
Utara, patients with CKD due to CAKUT with stable kidney function for at least for 6 months were
enrolled in the study. All the new cases aged between 1 and 17 years with CKD stage II-IV were
enrolled as per KDIGO (Kidney Disease: Improving Global Outcomes) guidelines [9]
A total of 122 subjects were enrolled in the study, 120 subjects completed the study up to end-
point and 2 subjects dropped out from the study. After enrollment these patients were followed for 18
months. The diagnostic criteria of CAKUT were defect of the renal parenchyma and or urinary tract
malformation documented by renal ultrasonography and voiding cystoureterogram. In our study
disease progression was decided on the basis of declining eGFR or progression of CKD stage, ie,
progression from stage II to stage III or stage III to stage IV. The Schwartz formula was used to
calculate the eGFR.[10] The staging criteria for CKD were defined as: stage II renal damage with
eGFR of 60–89 mL/min per 1.73 m2; stage III eGFR of 30–59 mL/min per 1.73 m2 and stage IV
eGFR of 15–29 mL/min per 1.73 m2 [9] Patients with non-syndromic vesicoureteral reflux (VUR),
malignant renal tumor, sepsis, massive proteinuria, being treated with steroids or immunosuppressive
agents were excluded from the study. Because of CAKUT is life-long disease, so chronologic age was
used as the index date instead. To ensure the validity of the index date, the patients were followed-up
for at least 6 months and those with incomplete observation were not included in this study.
Biochemistry, urinalysis, and urine protein measurements were performed as per study protocols.

2.1 Urine NGAL examination

Urine samples were analyzed for NGAL using an established and validated enzyme-linked
immunosorbent assay. A clean, morning midstream urine sample (25 mL) was collected into a sterile
test tube and centrifuged at 5,000 rpm for 15 minutes. The supernatant was transferred to an
Eppendorf tube and stored at -80°C until assessment for urine NGAL. A monoclonal antibody specific
for human Lipocalin-2 has been pre coated onto a microplate (R&D Systems Europe). All subsequent
steps were performed at room temperature. Standard and samples are pipetted into the wells and any
Lipocalin-2 present is bound by the immobilized antibody. After washing away any unbound
substances, an enzyme linked monoclonal antibody specific for human Lipocalin 2 is added to the
wells. Following a wash to remove any unbound antibody enzyme reagent, a substrate solution is
added to the wells and color develops in proportion to the amount of Lipocalin-2 bound. TMB
substrate was added for color development, which was read after 30 minutes at 450 nm with a
microplate reader.
2.2 Follow-up and end-points
Baseline renal function tests for all patients were recorded at the time of enrollment. All of the patients
had follow-up visits at 6-month intervals. The primary follow-up end-point was a composite of
halving of eGFR or recurrent urinary tract infection. Secondary follow-up end-point was eGFR of less
than 15 mL/min (ESRD) or death caused by renal.
2.3 Statistical analysis
Continuous variables were expressed as mean ± standard deviation. Student’s t test was applied for
comparison of data having normal distribution and Mann-Whitney U test for non-Gaussian
distribution between groups. Chi square test was used to compare categorical variables. Receiver
operator curve analysis was done for log NGAL and identifying the optimal NGAL cut-off values for
predicting progression of CKD. Statistical significance was set at P<0.05.

3. Result and Discussion

3.1 Basic demographic data.
The basic characteristics of CAKUT patients are presented in Table 1. There mean age of the patients
were 7.5 ± 4.1 years. There were 45 (37.5%) non obstructive and 75 (62.5) obstructive CAKUT.

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 Table 1. Baseline demographic data
All CAKUT Non Obstructive Obstructive
(n=120) (n= 45) (n=75)
Gender (Males, n) 72 20 52
Age, years (SD) 7.5(4.1) 7.7(3.8) 8.1(4,3)
GFR (n)
Stage II 32 9 23
Stage III 18 5 13
Stage IV 70 31 39

3.2 Follow-up data

Urinary NGAL is a protein produced by kidney tubular cells and can reflect damage in renal tubules.
The increasing production of urine NGAL occurred if increased of cell tubular injury, such as in
obstructed kidneys. In this situation, NGAL was highly expressed in distal tubule and accumulated in
the urine collected from the pelvis renalis. Obstruction type of CAKUT, showed the highest level of
urin NGAL (Figure 1 and Table 2). Kuwabara et al, found that in the unilateral uterine rat model
obstruction (UUO), urinary NGAL levels in rat urine experienced the highest levels [11]

Table 2. Urine NGAL in Progressivity CKD

Sensor Event p
(n=46) (n=74)
Urin 439.5 1206.5 0.001
NGAL
(ng/ml )
median

Figure 1. Urin NGAL and stage CKD

Findings from this study (indicate that NGAL represents a novel risk marker of CKD progression.
Urinary NGAL showed predictive power progression CKD into ESRD. In adult CKD patients, urinary
NGAL just not a simple surrogate index of baseline GFR, because after adjust to GFR, urinary NGAL
still have power to predict CKD progression.[12] Urinary NGAL might be particularly useful in the
evaluation of kidney recovery in patients with low-grade proteinuria. Proteinuria is an important direct
mediator of tubular epithelial cell injury and urinary NGAL increased in paralel with degree of
proteinuria [13]. In CAKUT, commonly found low grade proteinuria and after treatment of CAKUT
also found the same situation. In these circumstances, urine NGAL continues to increase especially in
CAKUT with CKD stage 3 and 4.
In CAKUT children it is necessary to prolong the follow up after adolescence because risk of
severe CKD and ESRD (Figure 2 and Table 3). The ItaliKid study showed that children with moderate
and severe CKD, had risk to developing ESRD estimated at 70 and 97%, respectively.[14] This data
showed that predict to severe CKD is warranted. Children from milder CKD had risk to developing

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ESRD. Role of urin NGAL to predict severity CKD in CAKUT patients found to be established, so
patient at risk severe CKD could be identified. This study had several limitations which may have
influenced the results. We included various types of CAKUT disease with varying severity of GFR.
Patients were administered according to obstructive or non-obstructive types of CAKUT but these
factors may affect the number of patients achieving “recovery” to evaluate the predictive value of
urine NGAL from the beginning of treatment.

Table 3. Area Under the Curve

Area SE p 95%CI
0.744 0.047 0.001 0.652-0.835

Figure 2. Urin NGAL to predict severe CKD in CAKUT

4. Conclusions
Urinary NGAL might be particularly useful in the evaluation of severe CKD in CAKUT patients.
Acknowledgements.
The author gratefully acknowledge that the present research is supported Ministry of Research and Technology and Higher
Education Republic of Indonesia. The support is under the research grant DRPM 2018. Contract Number
144/UN5.2.3.1/PPM/KP-DRPM/2018.

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