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ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 42-51

Serum and urinary neutrophil gelatinase-associated lipocalin in chronic liver disease patients and its value in detecting renal impairment


1 Department of Tropical Medicine, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

Date of Submission23-Dec-2019
Date of Decision06-Jan-2020
Date of Acceptance08-Jan-2020
Date of Web Publication20-Apr-2020

Correspondence Address:
MD Eman Elsayed Elshemy
MD Degree Tropical Medicine, Assistant Professor of Tropical Medicine Faculty of Medicine for Girls, Al-Azhar University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjamf.sjamf_110_19

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  Abstract 


Background Renal dysfunction is a common complication of liver cirrhosis. Renal dysfunction has a serious impact on the natural evolution of liver cirrhosis. Treatment and prognosis may be improved if an early diagnosis could be established and specific therapeutic interventions would be applied. Several new markers have become topics of research with studies mainly focused on cystatin c, kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin (NGAL).
Aim Evaluation of serum and urinary NGAL in chronic liver disease patients and its value in the detection of renal impairment.
Patients and methods It was performed on 45 patients with chronic liver disease and they were classified into three groups: group I included 15 patients with compensated chronic liver disease. Group II included 15 patients with decompensated liver disease and normal kidney functions. Group III included 15 patients with decompensated liver disease and impaired kidney functions.
Results There was highly significant increase in serum creatinine and urea level in group III in comparison to groups I and II and there was significant decrease in glomerular filtration rate in group II in comparison to group I and highly significant decrease in group III in comparison to groups I and II. There was significant decrease in serum Na in group II in comparison to group I and in group III in comparison to group II and highly significant decrease in group III in comparison to group I. Also, there was highly significant decrease in urinary Na in group II and group III in comparison to group I and significant decrease in group III in comparison to group II. As regard NGAL there was significant increase of serum and urinary NGAL in group III in comparison to group I and group II.
Conclusion Serum and urine NGAL are excellent markers for early detection of renal impairment in patients with chronic liver disease.

Keywords: acute kidney injury, HRS, neutrophil gelatinase-associated lipocalin in cirrhosis


How to cite this article:
Elshemy EE, Omar HM, Ahmed AS, Eltaweel EH. Serum and urinary neutrophil gelatinase-associated lipocalin in chronic liver disease patients and its value in detecting renal impairment. Sci J Al-Azhar Med Fac Girls 2020;4:42-51

How to cite this URL:
Elshemy EE, Omar HM, Ahmed AS, Eltaweel EH. Serum and urinary neutrophil gelatinase-associated lipocalin in chronic liver disease patients and its value in detecting renal impairment. Sci J Al-Azhar Med Fac Girls [serial online] 2020 [cited 2020 May 30];4:42-51. Available from: http://www.sjamf.eg.net/text.asp?2020/4/1/42/282860




  Introduction Top


Renal dysfunction has a serious impact on the natural evolution of liver cirrhosis. Treatment and prognosis may be improved if an early diagnosis could be established and specific therapeutic interventions would be applied [1].

Acute kidney injury (AKI) remains a major complication of decompensated liver cirrhosis with high morbidity and mortality rates [2].

Serum creatinine is still one of the main diagnostic criteria for AKI, although it has some disadvantages. In particular, a dynamic change in serum creatinine is a key criterion for cirrhosis-related AKI patients [3].

Neutrophil gelatinase-associated lipocalin (NGAL) is a new member of the lipocalin family. NGAL is expressed in injured renal tubules, can induce epithelial regeneration, enters the blood within 2 h after injury, and is excreted through the urine [4].

NGAL is emerging as an excellent standalone troponin-like biomarker in the plasma and urine for the prediction of AKI, monitoring clinical trials in AKI, and for the prognosis of AKI in several common clinical scenarios [5].


  Patients and methods Top


The study included 45 patients with chronic liver disease. After approval of local ethical committee, an informed consent was obtained from all patients before getting them involved in the study. The steps of the study, the aim, the potential benefits and hazards all discussed with patients. They were selected from those patients attending the inpatients of Hepatogastroenterology and Infectious Diseases Department and Intensive Care Unit of Al-Zahraa University Hospital from March 2017 to September 2017.

Exclusion criteria

Diabetic patients, hypertensive patients, urinary tract infection, chronic parenchymal kidney disease, patients on chronic hemodialysis, obstructive uropathy, and malignancy.

Inclusion criteria

This study included 45 patients with chronic liver disease and they were divided into three groups: group I included 15 patients with compensated chronic liver disease as a control group. Group II included 15 patients with decompensated liver disease and normal kidney functions. Group III included 15 patients with decompensated liver disease and impaired kidney functions.

All patients and control group were subjected to the following:
  1. Informed consent had been taken from all patients.
  2. Full history.
  3. Clinical examination including:
    1. General examination
    2. Local abdominal examination and systemic examination.
  4. Imaging part of the work: abdominal ultrasonography.


Using Toshiba SSA-340A machine with a 3.5 MHz curved convex probe to exclude chronic parenchymal kidney disease, obstructive uropathy, and malignancy.
  1. Laboratory part of the work includes the following. Specimen collection: patients should be fasting for at least 8 h prior to sample collection. Venous blood is collected in plane tubes for biochemical measurement (alanine aminotransferase, aspartate aminotransferase, albumin, bilirubin, serum Na, serum K, fasting blood sugar, urea, creatinine, and urinary Na). The creatinine clearance was assessed using the Cockcroft and Gault formula: creatinine clearance=[140–age (y)]×weight (kg)]/[72×serum creatinine (mg/dl)] (multiply by 0.85 for women). Plane tubes were used for measurement of serum NGAL in which blood samples were centrifuged and serum was collected and stored at −20°C until tested for NGAL. Plane tube for measurement of urine NGAL in which samples were stored at −20°C until tested for NGAL.
  2. Biochemical tests: alanine aminotransferase, aspartate aminotransferase, albumin, bilirubin, serum Na, serum K, fasting blood sugar, urea, creatinine, and urinary Na, which were done on Cobas c311 autoanalyzer (Germany) using Roche reagent kits.
  3. Measurement of NGAL: the kit uses a double-antibody sandwich enzyme-linked immunosorbent assay to assay the level of human NGAL in samples.


Assay procedure

(a) Standard dilution: this test kit supplied will be the one with the original standard reagent, please dilute it yourself according to the instructions (1600, 800, 400, 200, and 100 ng/ml). (b) The quantity of the plates depends on the quantities of to-be-tested samples and the standard. It is suggested to duplicate each standard and blank well. Every sample shall be made according to your required quantity and try to use the duplicated well as possible. (c) Inject samples: (i) blank well: do not add samples and NGAL-antibody labeled with biotin, streptavidin–HRP, only chromogen solution A and B, and stop solution are allowed; other operations are the same. (ii) Standard wells: add standard 50 µl, streptavidin–HRP 50 µl (since the standard already has combined biotin antibody, it is not necessary to add the antibody. (d) To be test wells: add sample 40 µl, and then add both NGAL-antibody 10 µl and streptavidin–HRP 50 µl. Then seal the sealing membrane and by gently shaking, incubated 60 min at 37°C. (e) Coinfection: dilute 30 times the 30× washing concentrate with distilled water as standby. (f) Washing: remove the membrane carefully, and drain the liquid, shake away the remaining water. (g) Add chromogen solution A 50 µl, then chromogen solution B 50 µl to each well. Gently mixed, incubate for 10 min at 37°C away from light. (h) Stop: add stop solution 50 µl into each well to stop the reaction (the blue changes into yellow immediately). (i) Final measurement: take blank well as zero, measure the optical density (OD) under 450 nm wavelength which will be carried out within 15 min after adding the stop solution. (j) According to standards’ concentration and the corresponding OD values, calculate out the standard curve linear regression equation, and then apply the OD values of the sample on the regression equation to calculate the corresponding sample’s concentration. It is acceptable to use different kinds of software to make calculations.

Statistical analysis

Data were collected, revised, coded, and entered to the Statistical Package for Social Sciences (IBM SPSS, Chicago, Illinois, USA), version 23. The quantitative data were presented as mean, SDs, and ranges while qualitative data were presented as number and percentages. The comparison between two independent groups with qualitative data was done by using c2 test and/or Fisher’s exact test only when the expected count in any cell was found to be less than 5. The comparison between more than two independent groups with quantitative data and parametric distribution was done by using analysis of variance followed by post-hoc analysis using LSD test. Spearman’s correlation coefficients were used to assess the correlation between two quantitative parameters in the same group. Receiver operating characteristic curve was used to assess the best cutoff point for serum NGAL and urinary NGAL in prediction of patients with renal impairment. The confidence interval was set to 99% and the margin of error accepted was set to 1%. So, the P value was considered significant as the following: P value more than 0.05: nonsignificant. P value less than 0.05: significant. P value less than 0.001: highly significant ([Table 1],[Table 2],[Table 3],[Table 4]).
Table 1 Results of renal functions in the studied groups

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Table 2 Results of serum Na, K, and urinary Na in the studied groups

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Table 3 Results of serum and urinary neutrophil gelatinase-associated lipocalin among the studied groups

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Table 4 Correlation study between serum neutrophil gelatinase-associated lipocalin and other studied parameters in the studied groups

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  Results Top


Group II

Group III

The univariate analysis shows that serum urea, serum NGAL, and urinary NGAL was found in early detection of renal impairment in hepatic patients while there was no statistically significant association found between serum creatinine and early detection of renal impairment ([Table 5],[Table 6],[Table 7],[Table 8]). Also, multivariate analysis shows that the best predictor for patients with renal impairment was serum NGAL followed by urinary NGAL ([Table 9], [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11],[Figure 12],[Figure 13],[Figure 14],[Figure 15],[Figure 16],[Figure 17],[Figure 18],[Figure 19],[Figure 20]).
Table 5 Correlation between urinary neutrophil gelatinase-associated lipocalin and serum neutrophil gelatinase-associated lipocalin in all studied groups

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Table 6 Correlation between glomerular filtration rate and serum creatinine in all studied groups

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Table 7 Correlation between serum creatinine and urinary Na in all studied groups

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Table 8 Logistic regression analysis for early prediction of renal impairment in hepatic patients

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Table 9 Diagnostic performance of urinary, serum neutrophil gelatinase-associated lipocalin, and combination

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Figure 1 Comparison between groups as regards serum urea.

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Figure 2 Comparison between groups as regards serum creatinine.

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Figure 3 Comparison between groups as regards GFR. GFR, glomerular filtration rate.

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Figure 4 Comparison between groups as regards serum Na.

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Figure 5 Comparison between groups as regards serum K.

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Figure 6 Comparison between groups as regards urinary Na.

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Figure 7 Comparison between groups as regards serum NGAL. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 8 Comparison between groups as regards urinary NGAL. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 9 Negative correlation between serum NGAL and serum albumin. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 10 Negative correlation between serum NGAL and serum Na. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 11 Negative correlation between serum NGAL and serum albumin. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 12 Negative correlation between serum NGAL and serum Na. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 13 Negative correlation between serum NGAL and urinary Na. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 14 Negative correlation between serum NGAL and GFR. GFR, glomerular filtration rate; NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 15 Positive correlation between serum NGAL and serum creatinine. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 16 Positive correlation between serum NGAL and serum urea. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 17 Positive correlation between urinary NGAL and serum NGAL in all studied groups. NGAL, neutrophil gelatinase-associated lipocalin.

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Figure 18 Negative correlation between serum creatinine and GFR in all studied groups. GFR, glomerular filtration rate.

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Figure 19 Negative correlation between serum creatinine and urinary Na in all studied groups.

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Figure 20 Diagnostic performance of urinary, serum NGAL and combination. NGAL, neutrophil gelatinase-associated lipocalin.

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  Discussion Top


The diagnosis of AKI is based on an increase in serum cretanine and oliguria [6]. However, serum creatinine is not a reliable marker during early acute-phase kidney dysfunction as its concentrations may be not elevated until about 50% of renal function has been lost [7].

Increased levels of NGAL can be identified in the plasma and urine starting 2–4 h after a kidney injury, resulting from alterations in glomerular filtration and tubular reabsorption and also by increased secretion in tubular epithelial cells [8].

In our study, there was significant decrease in glomerular filtration rate (GFR) in group II in comparison to group I and highly significant decrease in group III in comparison to groups I and II.

GFR may decrease before an increase in serum creatinine is detected. This is even more pronounced in patients with cirrhosis who have compromised hepatic conversion of creatinine, lower muscle mass, and increased renal tubular secretion of creatinine [9].

In our study, there was highly significant decrease in serum Na in group III in comparison to group I and significant decrease in group II in comparison to group I.

This result was in agreement with Borroni et al. [10] who concluded that hyponatremia was found in 57 (29.80%) out of 191 admissions.

In our study, there was highly significant decrease in urinary Na in groups II and III in comparison to group I.

This result was in agreement with Alsaad and Wadei [11], who approved that the majority of ESLD patients with renal dysfunction of unknown etiology or duration have a low 24-h urinary sodium excretion and an FeNa less than 1% irrespective of renal pathology on kidney biopsy.

In our study, urinary and serum NGAL were significantly higher in group III in comparison to groups I and II.

El-Bassat et al. [12] proved that uNGAL and pNGAL in cirrhotic patients with and without renal impairment including and excluding infection were significantly higher in cirrhotic patients with impaired kidney function either with or without infection compared with those with normal kidney function. Gungor et al. [13] found that patients with type 1 and type 2 hepatorenal syndrome (HRS) had significantly higher plasma and urine NGAL levels compared with patients with stable cirrhosis and controls.

In our study, multivariate analysis showed that the best predictor for hepatic patients with renal impairment was serum NGAL followed by urinary NGAL.

This was in agreement with Gungor et al. [13] who made a prospective observational study to investigate the level of plasma and urine NGAL in the prediction of mortality in patients with hepatorenal syndrome. Cox regression analysis showed that plasma NGAL and MELD-Na scores were independent predictors of mortality. Also, in agreement with Alhadad et al. [14] it has been proved that pNGAL level was a predictor of GFR below 60 ml/m and serum creatinine level correlated positively with MELD and MELD-Na scores and negatively with the GFR, so it is still a useful marker of the kidney function.

In our study, there was positive correlation between serum and urine NGAL with serum creatinine and Child–Pugh score and this explains that NGAL can predict mortality in patients with end-stage liver disease.

This result was in agreement with Zhang et al. [15] who concluded that plasma NGAL and urine NGAL were positively correlated with blood creatinine, MELD score, Child–Pugh score, and ascites.

In our study, the best cutoff value of serum NGAL between groups I and II and group III was more than 275 ng/ml, area under the curve=0.79, with a sensitivity of 80% and specificity of 73.33%.

This result was in agreement with Fodor et al. [16] who concluded that the best cutoff value to predict early-stage AKI was determined. For plasma NGAL this value was 252 ng/ml, with a sensitivity of 87% and a specificity of 83%.

In our study, the best cutoff value of urinary NGAL between groups I and II and group III was more than 343 ng/ml, area under the curve=0.78, with a sensitivity of 93.33% and a specificity of 53.33%.

In a study conducted by Zappitelli et al. [17] the highest and lowest cutoff points for sensitivity of urine NGAL varied from 54 to 85% and for specificity they varied from 97 to 44%. In their study, the area under the curve value for prediction of AKI was 0.79, which was higher than our study. Another study performed by Wagener et al. [18] suggested the cutoff point of urinary NGAL to be 213 ng/ml with a sensitivity and specificity of 73 and 78%, respectively.

In our study serum NGAL correlated inversely with GFR.

This was in agreement with Woo et al. [19] who concluded that the urinary NGAL level showed a significant inverse correlation with GFR. This was in contrast with Alhadad et al. [14] who found that pNGAL positively correlated with the GFR despite being of weak statistical correlation.


  Conclusion Top


Serum and urine NGAL are excellent markers for early detection of renal impairment in patients with chronic liver disease. The best cutoff value of serum NGAL between groups I, II and group III was more than 275 ng/ml, with sensitivity of 80%, specificity of 73.33%, positive predictive value of 60%, and negative predictive value of 88%. Also, the best cutoff value of urinary NGAL between groups I, II and group III was more than 343 ng/ml, with a sensitivity of 93.33%, specificity of 53.33%, positive predictive value of 50%, and negative predictive value of 94.1%. When using both serum and urinary NGAL, with a sensitivity of 93.33%, specificity of 76.67%, positive predictive value of 66.7%, and negative predictive value of 95.8%.

Recommendations

Further studies on larger groups of hepatic patients to clarify the diagnostic and prognostic values of plasma and urinary NGAL in these patients is recommended. Further validations of suggested cutoff values for urinary and serum NGAL in differentiation AKI types in cirrhotic patients is advised. Further studies are required to determine the impact of NGAL measurement in differentiation between AKI types in cirrhotic patients on patient’s survival and progression.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Firu SG, Streba CT, Firu D, Tache DE, Rogoveanu I. Neutrophil gelatinase associated lipocalin (NGAL) − a biomarker of renal dysfunction in patients with liver cirrhosis: do we have enough proof?. J Med Life 2015; 8:15–20.  Back to cited text no. 1
    
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Ostermann M, Philips BJ, Forni LG. Clinical review: biomarkers of acute kidney injury: where are we now. Crit Care 2012; 16:233.  Back to cited text no. 8
    
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Sherman D, Fish D, Teitelbaum I. Assessing renal function in cirrhotic patients: problems and pitfalls. Am J Kidney Dis 2003; 41:269–278.  Back to cited text no. 9
    
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Borroni G, Maggi A, Sangiovanni A, Cazzaniga M, Salerno F. Clinical relevance of hyponatraemia for the hospital outcome of cirrhotic patients. Dig Liver Dis 2000; 32:605–610.  Back to cited text no. 10
    
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Alsaad AA, Wadei HM. Fractional excretion of sodium in hepatorenal syndrome: clinical and pathological correlation. World J Hepatol 2016; 8:1497–1501.  Back to cited text no. 11
    
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El-Bassat H, Ziada DH, Taha A, Alm-Eldin R. Urinary neutrophil gelatinase-associated lipocalin as a biomarker for the diagnosis of hepatorenal syndrome in cirrhotic patients. Tanta Med J 2013; 41:346–352.  Back to cited text no. 12
    
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Gungor G, Ataseven H, Demir A, Solak Y, Gaipov A, Biyik M et al. Neutrophil gelatinase-associated lipocalin in prediction of mortality in patients with hepatorenal syndrome: a prospective observational study. Liver Int 2014; 34:49–57.  Back to cited text no. 13
    
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Alhadad OM, Alsebaey A, Amer MO, El-Said HH, Salman TA. Neutrophil gelatinase-associated lipocalin: a new marker of renal function in c-related end stage liver disease. Gastroenterol Res Prac 2015; 2015:815484.  Back to cited text no. 14
    
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Zhang Z, Wu L, Chen X, Chen L, Wang G, Yan H. Effect of neutrophil gelatinase-associated lipocalin on prognosis of patients with type 2 hepatorenal syndrome. Zhonghua Gan Zang Za Zhi 2015; 23:449–453.  Back to cited text no. 15
    
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Fodor R, Grigorescu B, Veres M, Orlandea M, Badea J, Hlavathy K, Cioc A. Plasma neutrophil gelatinase associated lipocalin (NGAL) − early biomarker for acute kidney injury in critically ill patients. J Cri Care Med 2015; 1:154–161.  Back to cited text no. 16
    
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Zappitelli M, Washburn KK, Arikan AA, Loftis L, Ma Q, Devarajan PP. Urine neutrophil gelatinase-associated lipocalin is an early marker of acute kidney injury in critically ill children: a prospective cohort study. Crit Care 2007; 11:R84.  Back to cited text no. 17
    
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Wagener G, Jan M, Kim M, Mori K, Barasch JM, Sladen RN, Lee HT. Association between increases in urinary neutrophil gelatinase-associated lipocalin and acute renal dysfunction after adult cardiac surgery. Anesthesiology 2006; 105:485–491.  Back to cited text no. 18
    
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Woo KS, Choi JL, Kim BR, Kim JE, An WS, Han JY. Urinary neutrophil gelatinase-associated lipocalin levels in comparison with glomerular filtration rate for evaluation of renal function in patients with diabetic chronic kidney disease. Diabetes Metab J Aug 2012; 36:307–313.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]



 

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