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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 3  |  Page : 778-783

Serum neutrophil gelatinase-associated lipocalin as an early biomarker in acute kidney injury in the pediatric ICU of Menoufia University


1 Pediatrics Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Medical Biochemistry Department, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Pediatrics Department, Awseem Central Hospital, Awseem, Egypt

Date of Submission30-Sep-2016
Date of Acceptance06-Nov-2016
Date of Web Publication15-Nov-2017

Correspondence Address:
Atef M Ibrahim
Awseem, Giza, 12511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.218289

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  Abstract 

Objective
The objective of the present study was to study the role of neutrophil gelatinase-associated lipocalin (NGAL) as an early biomarker of acute kidney injury (AKI) in the pediatric ICU (PICU) of Menoufia University.
Background
AKI represents a very devastating problem in critically ill children. Nephrologists are aware of the usefulness of serum NGAL as a biomarker of AKI.
Participants and methods
This study was carried out at the PICU of Menoufia University Hospital. This study included 80 participants. They were classified into group I (n = 20), including apparently healthy volunteers, and group II (n = 60), including children who were admitted to the PICU. All patients were subjected to full history and thorough clinical examination, routine investigations, and quantitative determination of serum NGAL levels.
Results
AKI developed in 18.3% of the cases. Cases had significantly higher levels of serum NGAL compared with the control group. There were significantly higher levels of serum NGAL among cases with AKI than in those without AKI. There were significantly higher levels of serum NGAL in septic AKI cases compared with septic non-AKI cases. The validity of serum NGAL for prediction of AKI, which documents the area under the curve, was 0.95 at a cutoff point of 155 ng/ml, sensitivity of 100%, specificity of 89.8%, positive predictive value of 68.8%, negative predictive value of 100%, and total accuracy of 91.7.
Conclusion
NGAL acts as a sensitive marker rather than a specific marker for AKI. At the same time, it presents a negative predictive value, more valuable than a positive predictive value, in detecting AKI.

Keywords: acute kidney injury, lipocalin-2, neutrophil gelatinase-associated lipocalin


How to cite this article:
El Gendy FM, Rizk MS, Saleh NY, Ibrahim AM. Serum neutrophil gelatinase-associated lipocalin as an early biomarker in acute kidney injury in the pediatric ICU of Menoufia University. Menoufia Med J 2017;30:778-83

How to cite this URL:
El Gendy FM, Rizk MS, Saleh NY, Ibrahim AM. Serum neutrophil gelatinase-associated lipocalin as an early biomarker in acute kidney injury in the pediatric ICU of Menoufia University. Menoufia Med J [serial online] 2017 [cited 2019 Nov 21];30:778-83. Available from: http://www.mmj.eg.net/text.asp?2017/30/3/778/218289


  Introduction Top


Acute kidney injury (AKI) is commonly defined as an abrupt decline in renal function, clinically manifesting as a reversible acute increase in nitrogen waste products, measured by blood urea nitrogen (BUN) and serum creatinine over the course of hours to weeks [1].

The incidence of AKI in children admitted to pediatric ICU (PICU) was about 5%, in children undergoing cardiac surgery for congenital heart disease showed incidence between 30 and 40%, and in children receiving bone marrow transplantation the incidence ranged from 15 to 34% [2].

The causes of AKI are classified into three general categories: prerenal, as an adaptive response to severe volume depletion and hypotension, with structurally intact nephrons; intrinsic, in response to cytotoxic, ischemic, or inflammatory insults to the kidney, with structural and functional damage; and postrenal, from obstruction to the passage of urine [3].

Deterioration of renal function may be discovered by a measured decrease in urine output. Often, it is diagnosed on the basis of blood tests for substances normally eliminated by the kidney: urea and creatinine. Both tests have their disadvantages. For instance, it takes about 24 h for the creatinine level to rise, even if both kidneys have ceased to function. A number of alternative markers has been proposed, such as neutrophil gelatinase-associated lipocalin (NGAL), KIM-1, IL-18, and cystatin C [4].

NGAL has been recently identified as a promising potential biomarker of AKI. This 25 kDa protein was initially found in activated neutrophils, as its name implies, and has a role as an innate antibacterial factor. However, it is also expressed in low amounts in many tissues, including kidney cells. Importantly, expression of NGAL is induced in injured tissues and its expression is quite rapid, usually within 2–4 h of injury. Fortunately, NGAL is very stable and easily detected in urine [5].

NGAL can be used to predict the outcome of AKI [need for renal replacement therapy (RRT), length of stay, and mortality]. In addition, it can be used to monitor response to intervention and treatment [6].

The aim of our study was to determine the role of serum NGAL levels in early detection of AKI in the PICU of Menoufia University Hospital.


  Participants and Methods Top


This study was carried out at the PICU, Faculty of Medicine, Menoufia University Hospital.

This study included 80 participants. They were classified into two groups: group I (n = 20) included apparently healthy volunteers, and their ages ranged between 5 and 156 months with a median age of 44 months; and group II (n = 60) included children who were admitted to the PICU, and their ages ranged between 1 and 180 months with a median age of 40 months.

For group I participants, routine investigations such as liver and kidney function tests and complete blood count were carried out to confirm their healthy state.

For group II participants, serum creatinine and BUN were measured on the first day of admission, and then every other day during their stay in the PICU. This group of patients was further subdivided according to RIFLE criteria into two other categories: group A, patients who developed AKI, and group B, patients who did not develop AKI.

RIFLE criteria included the following: R, Risk; I, Injury; F, Failure; L, Loss of kidney functions; and E, End-stage kidney disease.

Patient selection

We included all patients admitted to the PICU.

Exclusion criteria

Patients under RRT and patients on renal dialysis were excluded from the study.

All patients were subjected to all of the following:

  1. Detailed history
  2. Physical examination
  3. General examination: Measurement of blood pressure, temperature, respiratory rate, and heart rate
  4. Local examination: Chest, abdominal, cardiac, and neurological examination
  5. Routine investigations: Urine analysis, serum creatinine (mg/dl) and creatinine clearance, serum urea (mg/dl), serum sodium and potassium, urine output in 24 h, and abdominopelvic ultrasound and radiography
  6. Special investigations: Serum NGAL (ng/ml).


Five milliliters of venous blood was collected under completely aseptic conditions from each participant.

The collected blood samples were divided as follows: 1 ml was transferred into EDTA-containing polypropylene tubes and then gently inverted, complete blood count was performed, and 4 ml was transferred into plain tubes for serum separation.

After clotting, samples were centrifuged at 3000 rpm for 5 min, and sera were separated and divided into two aliquots.

A fresh serum aliquot from each individual was used for serum creatinine, BUN, and other advised tests. The other aliquot was stored at −20°C until the NGAL assay. Hemolyzed samples were discarded, and repeated freezing and thawing were avoided.

Measurement of serum neutrophil gelatinase-associated lipocalin

BioVendor NGAL is an enzyme immunoassay [enzyme-linked immunosorbent assay (ELISA)] (BioVendor - Laboratorni medicina, Karasek, Brno, Czech Republic) used for the quantitative determination of NGAL levels in human serum.

In the BioVendor human lipocalin-2/NGAL ELISA, standards, quality controls, and samples are incubated in microplate wells precoated with polyclonal anti-human lipocalin-2 antibody. After 1 h of incubation and washing, biotin-labeled polyclonal anti-human lipocalin-2 antibody was added and incubated with captured lipocalin-2 for 1 h. After another washing, streptavidin–HRP conjugate was added. After 30 min of incubation and the last washing step, the remaining conjugate was allowed to react with the substrate solution (trimethoprim). The reaction was stopped by adding acidic solution, and absorbance of the resulting yellow product was measured. The absorbance was proportional to the concentration of lipocalin-2. A standard curve was constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples were determined using this standard curve [7].

Statistical analysis

Data were collected, tabulated, and analyzed by SPSS (Statistical Package for Social Science) version 17.0 on an IBM compatible computer. Two types of statistics were performed: descriptive statistics [e.g. percentage (%), mean (X), and SD] and analytic statistics, which included c2-tests, Fisher's exact test, Z-test, Mann–Whitney U-test, and Kruskal–Wallis test. Spearman's correlation (r) is a test used to measure the association between two not normally distributed quantitative variables or one quantitative and one qualitative ordinal variable. Receiver operating characteristic (ROC) curve is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true-positive rate against the false-positive rate at various threshold settings. The true-positive rate is also known as sensitivity. The false-positive rate is also known as the fallout and can be calculated as 1 − specificity. The ROC curve is thus the sensitivity as a function of fallout. In general, it was used to evaluate diagnostic accuracy of certain markers. A P value of less than 0.05 was considered statistically significant.


  Results Top


Of 18.3% of cases affected by AKI 9.1% were divided into the risk group, 54.5% into the injury group, and 36.4% into the failure group. Two cases out of 11 (18.2%) needed RRT [Table 1].
Table 1: Distribution of the studied cases according to AKI by RIFLE criteria

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There were significantly higher rates of elevated BUN, decreased UOP, and decreased creatinine clearance among cases with AKI than those without AKI. However, there were nonsignificant differences regarding serum creatinine between the same groups [Table 2].
Table 2: Comparison between AKI and non-AKI cases with regard to laboratory data

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Demonstrates that cases had significantly higher levels of serum NGAL compared with controls (P < 0.001) [Table 3].
Table 3: Comparison between cases and controls regarding serum NGAL

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The level of serum NGAL was significantly higher among cases with AKI than those without AKI [Table 4].
Table 4: Comparison between AKI and non-AKI cases with regard to serum NGAL

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The levels of serum NGAL increased with increasing severity of kidney disease [Table 5].
Table 5: Relationship between serum NGAL and degree of AKI

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There was no significant difference between septic and nonseptic cases with regard to serum NGAL, whereas both septic and nonseptic cases had significantly higher levels of serum NGAL compared with the control group; significantly higher levels of serum NGAL were found in septic AKI cases than in septic non-AKI cases [Table 6].
Table 6 Comparisons between serum NGAL in septic patients, nonseptic patients, and controls and serum NGAL in septic AKI patients and septic non-AKI patients

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There was a significant difference between patients who died and survivors with regard to serum NGAL [Table 7].
Table 7: Comparison between serum NGAL in survivors and nonsurvivors

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The validity of serum NGAL in predicting AKI, which documented that the area under the curve (AUC) was 0.95 at a cutoff point of 155, the sensitivity was 100%, specificity was 89.8%, positive predictive value (PPV) was 68.8%, negative predictive value (NPV) was 100%, and total accuracy was 91.7.


  Discussion Top


AKI, formerly known as acute renal failure, continues to represent a very common and potentially devastating problem in critically ill children and adults. The reported incidence of AKI in this population varied greatly because of the lack of a standard consensus definition. The reported incidence rate of AKI in children admitted to PICUs ranged from 8 and 89% [8].

Unfortunately, the mortality and morbidity associated with AKI remain unacceptably high (up to 80% mortality in critically ill children and adults) [9].

The current clinical practice is to use serum creatinine levels as a marker of kidney function, in spite of it being a relatively poor indicator of the early stage of AKI. Serum creatinine may also vary with age, sex, muscle mass, medications, and hydration status [10]; in addition, serum creatinine levels may stay unchanged until ~50% of kidney function is lost [11].

NGAL is a small protein (25 kD) mainly known for its capacity to bind siderophores, which are small hydrophobic molecules containing iron, transporting them inside cells to activate cytoplasmic iron-dependent pathways, thus protecting the same cell from oxidative stress. NGAL is emerging as an excellent biomarker in the urine and plasma for several processes such as early prediction of AKI, monitoring clinical trials related to AKI, and for the prognosis of AKI in several common clinical scenarios [12].

We studied the value of serum NGAL for early detection of AKI in the PICU of Menoufia University Hospital, including 60 children (43 males and 17 females) admitted to the PICU of Menoufia University and 20 apparently healthy children matched for age and sex (11 males and nine females) as a control group. We found a nonsignificant difference between studied cases and controls with regard to age and sex. This was in agreement with the study by Youssef et al. [13].

Our study showed that 11 out of 60 critically ill patients (18.3%) developed AKI. These 11 patients were divided according to RIFLE criteria into the following: one (9.1%) as risk, six (54.5%) as injury, and four (36.4%) as failure. Only two out of 11 AKI patients (18.2%) needed RRT.

Zapitelli et al. [14] analyzed a group of 140 children admitted to PICU; AKI developed in 75.7%, with 35.7% being at the risk stage, 22.1% at the injury stage, and only 17.9% at the failure stage.

According to Washburn et al. [15] in a population of 137 children, AKI was noted in 75.2% of patients, with the risk stage seen in 36.5% of cases, the injury stage in 20.4%, and failure stage only in 18.3%. Youssef et al. [13] reported that AKI developed in 13 out of 60 (21.7%) critically ill children, and five of these critically ill children (8.3%) had greater severity and needed RRT.

In our study, there was a significantly higher rate of elevated BUN, decreased UOP, and decreased creatinine clearance among cases with AKI than those without AKI. However, there was a nonsignificant difference regarding serum creatinine between the same groups. This is explained by the fact that AKI is diagnosed when serum creatinine increases by at least 1.5 times of its baseline value within 48 h (which may still be within the normal range) and not mainly by the absolute value of elevated serum creatinine [16].

Raluca et al. [17] reported that there is no statistically significant difference between patients who developed AKI and those who did not develop AKI at 48 h regarding serum creatinine and creatinine clearance. Monika [18] reported that the majority of patients who developed AKI (10 out of 16) still showed normal UOP values.

Our results demonstrated that cases had significantly higher levels of serum NGAL than controls. The levels of serum NGAL were highly significant among cases with AKI than in those without AKI. Bailey et al. [19] reported that there was a significant difference in serum NGAL between healthy children and critically ill children.

Merrikhi et al. [20] reported that serum NGAL levels significantly increased in patients who developed AKI compared with those who did not.

Youssef et al. [13] reported that there was a significant difference in serum NGAL between critically ill children and controls. Cruz et al. [11] reported that NGAL levels were significantly increased in ICU patients with severe illness compared with healthy adults and increased in AKI patients compared with non-AKI patients.

Our results showed that serum NGAL significantly increased in critically ill children with sepsis as compared with the control group, and also was significantly increased in septic patients with AKI than septic patients without AKI. One hypothesis to explain this observation is that sepsis induces greater injury to the kidneys compared with other contributing factors [14].

Wheeler et al. [21] reported that serum NGAL was significantly increased in children with septic shock compared with both healthy controls and critically ill children; there was a highly significant difference in serum NGAL between septic patients who developed AKI and septic patients who did not develop AKI. Bagshaw et al. [22] found that NGAL levels were higher in septic AKI patients than in nonseptic AKI patients.

Our study showed that there was a significant difference between survivors and nonsurvivors with regard to serum NGAL. Kumpers et al. [23] found that serum NGAL was highly sensitive to discriminate between survivors and nonsurvivors. In addition, Ridder et al. [24] found that plasma and urine NGAL concentrations at ICU admission are significantly higher in nonsurviving patients compared with concentrations in surviving patients. Therefore NGAL measured at admission might be an early indicator of mortality. On the other hand, Wheeler et al. [21] reported that there was no significant difference in serum NGAL concentrations between survivors and nonsurvivors.

Our study showed the validity of serum NGAL in predicting AKI, which documented that the AUC was 0.95 at cutoff point of 155 ng/ml, the sensitivity was 100%, specificity was 89.8%, PPV was 68.8%, NPV was 100%, 95% confidence interval (CI) was 0.89–1.0, and total accuracy was 91.7%.

Akcan-Arikan et al. [9] have shown that at a cutoff of 139 ng/ml the ROC curve for NGAL revealed an AUC of 0.677 with 95% CI of 0.557–0.786. The sensitivity of NGAL was 86%, specificity was 39%, PPV was 39%, and NPV was 94%.

Youssef et al. [13] detected the ability of serum NGAL to detect AKI earlier in critically ill children and to evaluate its sensitivity and specificity in AKI detection. They found that NGAL acts as a sensitive marker rather than a specific one for AKI. At the same time, it presents as a NPV, more valuable than a PPV, in detecting AKI (AUC = 0.63, CI 95%, cutoff point 89.5 ng/ml, sensitivity 84.6%, specificity 59.5%, PPV 36.7%, and NPV 68.4%).

Wheeler et al. [21] showed the ROC curve of serum NGAL for the prediction of AKI, AUC = 0.667, 95% CI = 0.557–0.786, with an optimal cutoff point of 139 ng/ml, sensitivity of 86%, specificity of 39%, PPV of 39%, and NPV of 94%.


  Conclusion Top


We found that NGAL acts as a sensitive marker rather than a specific one for AKI. At the same time, it presents a NPV, more valuable than a PPV, in detecting AKI.

Our results of early predictive, sensitive, nonspecific serum NGAL, although of clear statistical significance, will certainly need validation in a larger trial, including patients with pre-existing chronic kidney disease and comorbid conditions that normally accumulate with impaired renal function. The ability of biomarkers such as NGAL to discern both the onset and resolution of AKI will further validate their use in the clinical setting and greatly enhance our understanding of AKI in the pediatric population.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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