Study on the concentration of urinary neutrophil gelatinase associated lipocalin in patients occuring acute kidney failure in intensive care unit

Objective: To evaluate urinary neutrophil gelatinase-associated lipocalin (NGAL) concentration and its relation with causes, categories, stages and biochemical indexes of acute kidney injury (AKI) patients. Subjects and methods: A prospective, cross-sectional study in 121 patients with AKI who admitted to a general Intensive Care Unit (ICU), Trungvuong Hospital, Hochiminh City from 12 - 2013 to 01 - 2017 and a control group of 51 healthy people. Urinary NGAL had done in all 116 patients and healthy people.

STUDY ON THE CONCENTRATION OF URINARY NEUTROPHIL GELATINASE - ASSOCIATED LIPOCALIN IN PATIENTS

OCCURING ACUTE KIDNEY FAILURE

IN INTENSIVE CARE UNIT

Pham Ngoc Huy Tuan*; Nguyen Trung Kien**; Le Viet Thang**

SUMMARY

Objective: To evaluate urinary neutrophil gelatinase-associated lipocalin (NGAL) concentration and its relation with causes, categories, stages and biochemical indexes of acute kidney injury (AKI) patients Subjects and methods: A prospective, cross-sectional study in 121 patients with AKI who admitted to a general Intensive Care Unit (ICU), Trungvuong Hospital, Hochiminh City from 12 - 2013 to 01 - 2017 and a control group of 51 healthy people Urinary NGAL had done

in all 116 patients and healthy people Results: All of the AKI patients (100%) had urinary NGAL elevation The average concentration of urinary NGAL in our study group (434.06 ng/mL) was significantly higher than in control group (10.74 ng/mL) with p < 0.001 There was no significant difference in concentration of uNGAL in terms of causes The concentration of urinary NGAL was significantly higher in oliguria group than non-oliguria group (571.70 ng/mL compared to 355.95 ng/mL) with p < 0.005 Patients’ uNGAL concentration at the time of ICU admission was significantly related to their KDIGO stage (p < 0.001) Urinary NGAL had a moderate positive correlation with serum urea concentration (r = 0.567, p < 0.001) and a strong positive linear correlation with serum creatinine concentration (r = 0.850, p < 0.001) Conclusion: Urinary NGAL elevation was common in AKI patients The concentration of urinary NGAL depended on category and stage of AKI It had a moderate positive correlation with serum urea and strong positive correlation with creatinine concentration

* Keywords: Acute kidney injury; Urinary neutrophil gelatinase-associated lipocalin; Intensive care unit

INTRODUCTION

Acute kidney injury is a common and

devastating problem with in-hospital

mortality of 40% to 80% in the intensive

care setting [10] The traditional blood

(creatinine, blood urea nitrogen) and urine

markers of kidney injury (casts, fractional

excretion of sodium, urinary concentrating

ability) that have been used for decades

in clinical studies in diagnosis and

prognosis of AKI are insensitive and nonspecific and do not directly reflect injury to kidney cells Therefore, early recognition of renal injury is important and may help prevent further renal damage and functional impairment

Neutrophil gelatinase-associated lipocalin

is a small, 23 kDa protein that is an early biomarker for ischemic, septic, or nephrotoxic kidney injury It is normally

* 103 Military Hospital

** Trung Vuong Hospital

Corresponding author: Pham Ngoc Huy Tuan (bshuytuantv@yahoo.com.vn)

Date received: 03/07/2017 Date accepted: 18/07/2017

produced at low levels by the epithelial

cells of the kidney, but it is quickly

upregulated in the thick ascending limb

(TAL) of the loop of Henle and the

collecting ducts within three hours of

tubular epithelial injury

Urinary NGAL (uNGAL) has been

evaluated as an early biomarker of renal

tubular damage in a acute clinical settings

such as the operating room, ICU and

emergency department, and in high-risk

procedures such as cardiac surgery,

radio-contrast injection and after adult and

pediatric kidney and liver transplantation

[1, 6, 7, 8, 9] There is considerable

evidence that compared to increases in

serum creatinine, NGAL better detects

early or subclinical kidney injury, and

better predicts dialysis requirement and

mortality [1]

In Vietnam, there are lack of studies on

the role of uNGAL in AKI diagnosis and

prognogsis in patients who admitted to

general ICU Therefore, we conducted

this research for the aim: Evaluation of

the uNGAL concentration and its relation

with causes, categories, stages and some

biochemical indexes of AKI patients

SUBJECTS AND METHODS

1 Subjects

The study was conducted with a study

group of 121 AKI patients who admitted to

a general ICU, Trungvuong Hospital,

Hochiminh City from 12 - 2013 to 01 -

2017 and a control group of 51 healthy people

* Excluding criteria: patients with

chronic kidney failure, anuria, did not fit with diagnostic criteria, did not have enough test results or did not agree to participate in the study

2 Methods

* Study design: A prospective,

cross-sectional descriptive study

* Urinary NGAL measurement:

24-hour urine was collected After that, the volume of urine was measured before collecting 1 mL sample for testing purpose uNGAL was measured by the sandwich ELISA method using NGAL monoclonal antibody in the NGAL kit After that, the sample will be analyzed by Achitech System of Abbott, America to measure uNGAL concentration

* Diagnostic criteria: KDIGO definition

and classification of AKI [5]

* Diagnostic criteria for AKI: serum

creatinine ≥ 136.5 µmol/L within 48h

* AKI degree:

+ AKI stage I: serum creatinine from 136.5 - 220 µmol/L

+ AKI stage II: serum creatinine from

220 - 353.6 µmol/L

+ AKI stage III: ≥ 353.6 µmol/L

* Statistical analysis:

Statistical analyses were conducted using SPSS 20.0

RESULTS AND DISCUSSIONS

Table 1: Urinary NGAL concentration in study group

Urinary NGAL

(ng/mL)

The average concentration of urinary

NGAL in our study group was 434.06 ng/mL,

which was significantly higher than in control

group (10.74 ng/mL) with p < 0.001 The

maximum and minimum concentration of

urianay NGAL was 1292.38 and 69.63 ng/mL

respectively With the refference range of

urinary NGAL from 43.62 to 114.66 ng/mL,

all of the AKI patients (100%) had urinary

NGAL elevation Study of Au V also showed

that the mean immediate postoperative

urinary NGAL levels in patients who

developed sustained AKI were 204.8 ng/mL,

and significantly higher than those who had

normal renal function (31.9 ± 113 ng/mL)

with p < 0.001 [1] This result was similar to

other studies of Geus H.R, Makris K,

Zappitelli M: there was a significant higher

of urinary NGAL concentration in patients

who submitted AKI compare to non-AKI

patients with p < 0.05 [6, 7, 8] These

differences in uNGAL concentration are

expected because kidney injury associated

with primary renal insults may be more

severe than that in most patients included

in our study But our patients were probably

more severely ill, with a higher proportion

having sepsis than healthy people

In current clinical practice, the gold

standard for identification and classification

of AKI is dependent on serial serum

creatinine measurements, which are

especially unreliable during acute changes in kidney function We identified uNGAL as one of the most upregulated genes in the kidney soon after ischemic injury NGAL protein was also markedly induced in kidney tubule cells and easily detected in the plasma and urine in animal models of ischemic and nephrotoxic AKI The expression of uNGAL protein was also dramatically increased in kidney tubules

of humans with ischemic, septic, and post-transplant AKI Importantly, NGAL in the urine was found to be an early predictive biomarker of AKI in a variety of acute clinical settings Emerging experimental and clinical evidence indicated that in the early phases of AKI from diverse etiologies, NGAL accumulates within two distinct pools, namely, a renal and a systemic pool Gene expression studies in AKI had clearly demonstrated rapid and massive upregulation of NGAL mRNA in the thick ascending limb of Henle's loop and the collecting ducts, with resultant synthesis of NGAL protein in the distal nephron (the renal pool) and secretion into the urine where it comprises the major fraction of urinary NGAL

This finding also confirms the need for future research to evaluate uNGAL in different renal disease subgroups, in order to understand fully how best to use uNGAL to diagnose AKI

Table 2: Relation between urinary NGAL concentration and the causes of AKI

In our study, sepsis was the most

common causes with the proportion of

57.8% There was no significant difference

between these causes with p > 0.05 Our

result was similar to study of Vaidya D.S:

there was no significant difference between

urinary NGAL concentration and several

causes of AKI in these studies (p > 0.05)

[10], but was different with other studies

of Di Nardo M and Geus H.R (there was a

significant higher concentration of urinary

NGAL in septic AKI patients than

non-septic AKI patients with p < 0.001 [4, 6]

Lipoproteins also have strong affinity for

Toll-like receptors (TLRs) that trigger an

innate immune response Therefore, it

could be postulated that these circulating

ligands which are linked to tubular epithelial TLR activation are responsible for the increased uNGAL concentrations that we observed in patients who had sepsis, but showed no increases in their serum creatinine levels However, recent studies in patients with sepsis, septic shock, and systemic inflammatory response syndrome had reported contradictory findings A possible explanation for this difference is the variability of the subject inclusion time (up to 48 h after ICU admission) Intensive resuscitation and the administration of antibiotics may have already occurred before study inclusion, therefore most likely inducing rapid

changes of uNGAL values

Table 3: Relation between urinary NGAL concentration and AKI category

In our study, category of anuria occupied in 36.2% of all AKI patients The concentration of urinary NGAL was significantly higher in anuria group than non-anuria group (571.70 ng/mL compared with 355.95 ng/mL) with p < 0.005 Our findings highlight the mechanistic insights of NGAL levels based on the specimens being measured

Urine NGAL is proposed to derive predominantly from local renal synthesis of NGAL in the thick ascending limb of the loop of Henle and the collecting ducts when under inflammatory and oxidative stress Therefore, the concentration of urinary NGAL was directly related to the renal tubule injury in AKI patients as well as urine excretion ability

Table 4: Relation between serum NGAL concentration and stage of AKI

Following to the KDIGO classification,

the stage 1 AKI in our study made up the

highest proportion (69.8%) Stage 2 and 3

occupied smaller proportion (22.4% and

7.8%, respectively) Our results also pointed

that patients’ uNGAL concentrations at

the time of ICU admission were significantly

related to their KDIGO stage (p < 0.001)

This result was similar to the study of

Geus H.R (p < 0.0001) and Zapittelli M (p

< 0.0002) when research on the relation

between uNGAL and RIFFLE stage [6, 8]

NGAL fulfills a central role in regulating

epithelial neogenesis, and in iron chelation and delivery after ischemic or toxic insults to the renal tubular epithelium After kidney injury, NGAL is rapidly expressed on the apical epithelial membranes of the distal nephron NGAL is excreted in the urine through exocytosis and has local bacteriostatic and proapoptotic effects Therefore, uNGAL concentration had a positive relation with the level of renal damage which exhibited throughout the high stage

of KDIGO classification

Table 5: Correlation between serum NGAL and urea, creatinine concentration

Serum NGAL Indexes

In our study, urinary NGAL had a

moderate positive correlation with serum

urea concentration (r = 0.567, p < 0.001)

and a strong positive linear correlation

with serum creatinine concentration

(r = 0.850, p < 0.001) The correlation

equation was created (uNGAL = 18,89*Urea

+ 165.63; uNGAL = 2.63*creatinine - 142.30) Boglignano D also pointed that a significant correlation was also found between serum creatinine and uNGAL (r = 0.399, p < 0.001) [2] NGAL has mainly been studied in the setting of acute renal failure Patients who experienced

acute renal dysfunction showed a marked

increase in urinary NGAL levels, which

preceded the increase in serum creatinine

by a day In a single case of acute tubular

necrosis due to heart failure induced

hypotension, NGAL tubular expression

was reported to be strongly increased [3]

Hence, measurements of NGAL may

serve as a very early marker of worsening renal function Urinary (or plasma) NGAL levels could therefore be used to adjust therapy, to anticipate and possibly prevent expected renal injury, even before

a peak in serum creatinine occurs This potential of NGAL needs to be explored further in future studies

uNGAL = 18.89*Urea + 165.63

0 500 1000 1500 2000

Urea

Chart 1: Correlation between urinary NGAL and urea concentration

uNGAL = 2,63*creatinine - 142.30

0 500 1000 1500 2000 2500

Creatinine

Chart 2: Correlation between urinary NGAL and creatinine concentration

CONCLUSIONS

In our study, all of the AKI patients

(100%) had urinary NGAL elevation The

average concentration of urinary NGAL in

our study group (434.06 ng/mL) was

significantly higher than that in control

group (10.74 ng/mL) with p < 0.001

There was no significant difference in

concentration of uNGAL in terms of

causes The concentration of urinary

NGAL was significantly higher in oliguria

group compared to non-oliguria group (571.70 ng/mL compared to 355.95 ng/mL) with p < 0.005 Patients’ uNGAL concentrations at the time of ICU admission were significantly related to their KDIGO stage (p < 0.001) Urinary NGAL had a moderate positive correlation with serum urea concentration (r = 0.567,

p < 0.001) and a strong positive linear correlation with serum creatinine concentration (r = 0.850, p < 0.001)

REFFERENCES

1 Au V et al Urinary neutrophil

gelatinase-associated lipocalin distinguishes sustained

from transient acute kidney injury after

general surgery KI reports 2016, 1 (1),

pp.3-9

2 Bolignano D.et al Neutrophil

gelatinase-associated lipocalin as a marker of kidney

damage American Journal of Kidney

Diseases 2008, 52 (3), pp.595-605

3 Damman K et al Urinary neutrophil

gelatinase associated lipocalin (NGAL), a

marker of tubular damage, is increased in

patients with chronic heart failure European

Journal of Heart Failure 2008, 10 (10),

pp.997-1000

4 Di Nardo M et al Impact of severe

sepsis on serum and urinary biomarkers of

acute kidney injury in critically Ill children: An

observational study Blood Purification 2013,

35 (1-3), pp.172-176

5 Disease, K Improving global outcomes

(KDIGO) acute kidney injury work group:

KDIGO clinical practice guideline for acute

kidney injury Kidney Int Suppl 2012, 2, pp.1-138

6 Geus H R H D et al Neutrophil

admission predicts for acute kidney injury in

adult patients American Journal of Respiratory

and Critical Care Medicine 2011, 183 (7), pp.907-914

7 Makris K et al Urinary neutrophil

gelatinase-associated lipocalin as an early marker of acute kidney injury in critically ill multiple trauma patients Clinical Chemistry and Laboratory Medicine 2009, p.79

8 Zappitelli M et al Urine neutrophil

gelatinase-associated lipocalin is an early marker of acute kidney injury in critically ill children: a prospective cohort study Critical

Care 2007, 11 (4), p.R84

9 Chertow G.M et al Acute kidney injury,

mortality, length of stay, and costs in hospitalized patients J Am Soc Nephrol

2005, 16 (11), pp.3365-3370

10 Vaidya V.S et al Urinary biomarkers

for sensitive and specific detection of acute kidney injury in humans Clin Transl Sci 2008,

1 (3), pp.200-208