Key words: neutrophil gelatinase-associated lipocalin, renal dysfunction, inflammation index.

Transcription

1 248 Available online at Annals of Clinical & Laboratory Science, vol. 45, no. 3, 2015 Corrected Neutrophil Gelatinase-Associated Lipocalin (NGAL) Level Adjusted by the Scoring System of an Inflammation Index for Screening Renal Dysfunction in Patients with Systemic Inflammation Jong Weon Choi 1, Tatsuyoshi Fujii 2, and Noriyoshi Fujii 3 1 Departments of Laboratory Medicine, College of Medicine, Inha University, Incheon, Korea, 2 School of Medicine, University of Tsukuba, Ibaraki, Japan, and 3 Department of Electrical Engineering and Bioscience, Waseda University, Tokyo, Japan. Abstract. This study investigated the use of corrected neutrophil gelatinase-associated lipocalin (cngal) values to screen renal dysfunction in patients with systemic inflammation. Plasma NGAL concentrations were measured using a fluorescent immunoassay in 259 patients with inflammatory diseases. An inflammation index was calculated using a scoring system based on high-sensitivity C-reactive protein (hscrp) and corrected erythrocyte sedimentation rates. The uncorrected NGAL (ungal) concentration increased with the inflammation index, but cngal increased in parallel with serum creatinine (scr) levels. After adjustment with the inflammation index, increased ungal concentrations returned to a level not significantly different from the control value. In a multivariate regression analysis, hscrp was more closely associated with ungal than cngal [r=0.513 (p<0.001) vs. r=0.201 (p=0.017)], but the scr level was more strongly linked to cngal than ungal (r=0.692 vs. r=0.583, respectively, p<0.001). In a receiver operating characteristics (ROC) curve analysis, the diagnostic accuracy of cngal, which identifies an increase of scr 0.3 mg/dl within 48 hours, was found to be superior to that of ungal [0.72 (95% CI, ) vs (95% CI, ), p=0.038]. In conclusion, cngal more accurately reflects renal dysfunction than ungal under inflammatory conditions. A measurement of cngal may provide helpful information for assessing patients with renal impairment, particularly in association with systemic inflammation. Key words: neutrophil gelatinase-associated lipocalin, renal dysfunction, inflammation index. Introduction Neutrophil gelatinase-associated lipocalin (NGAL) is a 25 kda glycoprotein, which was originally identified as a novel protein of a bacteriostatic agent isolated from secondary granules of activated human neutrophils [1,2]. NGAL is normally expressed in a variety of adult human tissues, including bone marrow, liver, kidney, lung, adipose tissue, smooth muscle, stomach, and colon [3]. Several of these tissues are prone to exposure of microorganisms and constitutively express the NGAL protein at low levels. Since NGAL is up-regulated shortly after ischemia or damage in renal tubular cells, it has received Address correspondence to Jong Weon Choi, MD, PhD; Department of Laboratory Medicine, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Incheon , Korea; phone: ; fax: ; e mail: jwchoi@inha.ac.kr growing attention as a potential parameter for early detection of various renal injuries [4]. In contrast to serum creatinine (scr), NGAL is specifically induced in the damaged nephron and then released into blood and urine, where it can be readily measured [5]. However, NGAL is also highly induced in a variety of inflammatory conditions [6]. Systemic inflammation contributes to the development of acute kidney injury in critically ill patients, which is associated with pathogenesis, such as hypoperfusion, microvascular thrombosis, infiltration of immune cells, and apoptosis [7,8]. Although NGAL has been known as an early predictor for acute kidney injury, it is difficult to interpret whether NGAL plays a more important role as a marker of acute kidney injury than as an inflammatory protein, particularly in patients with both systemic inflammation and renal dysfunction /15/ by the Association of Clinical Scientists, Inc.

2 Corrected NGAL level adjusted by an inflammation index 249 Table 1. Corrected NGAL values by a scoring system using hscrp and cesr levels in patients with inflammatory diseases. Assigned No. Age Male (%) Mean±SD or median (range) score of subjects (years) Inflammation Uncorrected Corrected Serum (n=259) index NGAL level NGAL level creatinine (ng/ml) (ng/ml) (mg/dl) hscrp (mg/dl) < (21 82) 49(52.1) 1.15± ( ) 68.3 ( ) 1.18± (20 87) 40 (47.6) 1.91± (23 980) a 62.9 ( ) 1.34± (24 81) 23 (63.8) 2.64± ( ) a,b 74.3 ( ) 1.37±1.09 > (23 79) 29 (64.4) 3.46± ( ) a,c ( ) c 2.25±2.63 c cesr (mm/hr) < (22 81) 65(53.3) 1.27± ( ) 66.0 ( ) 1.16± (25 80) 40 (63.4) 2.07± ( ) a 64.5 ( ) 1.35± (20 84) 25 (45.5) 2.99± ( ) a,b 87.3 ( ) 1.39±1.19 > (26 87) 11 (57.9) 3.63± ( ) a,c ( ) c 2.55±3.42 c Abbreviations: hscrp, high-sensitivity C-reactive protein; cesr, corrected erythrocyte sedimentation rate; a Significant (p<0.05) vs. corrected NGAL levels. b Significant (p<0.05) vs. hscrp ( mg/dl) and cesr ( mm/hr) of the corresponding groups. c Significant (p<0.05) vs. hscrp ( mg/dl) and cesr ( mm/hr) of the corresponding groups. Table 2. Uncorrected- and corrected-ngal levels in relation to an inflammation index in patients with and without renal impairment. NGAL levels Percent Serum Inflammatory parameters (ng/ml) difference creatinine hscrp cesr (95% CI) (mg/dl) (mg/dl) (mm/hr) uncorrected corrected Patient group (n=259) Without renal impairment (scr <1.2 mg/dl, n=170) Inflammation 63(27 534) 63(27 534) NS 0.88± ( ) 4.9 ( ) index 1.0 (n=60) Inflammation 121(15 844) a,b 62.4( ) 49.8 ( ) 0.87± ( ) a,b 20.5 ( ) a,b index >1.0 (n=110) With renal impairment (scr 1.2 mg/dl, n=89) Inflammation 154(63 432) a 154 (63 432) a NS 1.72±0.69 a 0.08( ) 5.2( ) index 1.0 (n=11) Inflammation index >1.0 (n=78) 423( ) a,b 160.8( ) a 57.9( ) 2.65±2.46 a,b 6.19( ) a,b 31.2( ) a,b Healthy controls (n=51) 60(18 156) 60(18 156) NS 0.85± ( ) 4.8( ) Abbreviations: hscrp, high-sensitivity C-reactive protein; cesr, corrected erythrocyte sedimentation rate; a Significant (p<0.05) vs. healthy controls; b Significant (p<0.05) vs. patients with inflammation index 1.0 of the corresponding group.

3 250 Annals of Clinical & Laboratory Science, vol. 45, no. 3, 2015 Table 3. Multivariate regression analysis of corrected- and uncorrected-ngal as a dependent variable in patients with inflammatory diseases. Univariate model Multivariate model a Standard β P values Standard β P values Corrected NGAL Kidney function Serum creatinine < <0.001 egfr < <0.001 Inflammatory markers hscrp < cesr Uncorrected NGAL Kidney function Serum creatinine < <0.001 egfr < <0.001 Inflammatory markers hscrp < <0.001 cesr < <0.001 Abbreviations: egfr, estimated glomerular filtration rate; hscrp, high-sensitivity C-reactive protein; cesr, corrected erythrocyte sedimentation rate; a Adjusted for age, systolic and diastolic blood pressure, and body mass index, which were associated with plasma NGAL concentrations in a univariate analysis. Previous studies for NGAL have largely focused on its role as an indicator of acute kidney injury and diabetic nephropathy [5,9,10]. There have been no studies which have closely examined an adjusted value for plasma NGAL concentrations in patients with inflammatory diseases. In the present study, we evaluated a new parameter of corrected NGAL (cngal) levels which are amended with an inflammation index using a scoring system. We also tested the ability of cngal to predict worsening kidney function in patients with inflammatory diseases, particularly in comparison with that of uncorrected NGAL (ungal) levels. Materials and Methods A total of 259 patients under clinical investigation of systemic inflammation were tested by measuring NGAL, scr, estimated glomerular filtration rates (egfr), highsensitivity C-reactive protein (hscrp), and corrected erythrocyte sedimentation rates (cesr). Their ages ranged from 20 to 87 years (median age, 55 years) and 141 patients were males (54.4%). Their medical records were reviewed for clinical and demographic data. As a control group, age- and gender-matched healthy individuals (n=51) who had no history of recent infection or renal dysfunction, were enrolled. This study was approved by the Institutional Review Board of Inha University Hospital. Patients with cardiovascular diseases (n = 4), diabetic nephropathy (n=2), stroke (n=2), and multiple trauma (n=1) were excluded in this study because these conditions may influence plasma NGAL levels. Subjects who had missing values (n=5), a recent operation (n=3), or administration of anti-inflammatory drugs (n=2) were also excluded from analysis. The subject cohort was classified into 4 groups, based on hscrp and cesr levels, respectively. Scores (0.5, 1, 1.5, and 2) were assigned to each group in relation to the degree of hscrp and cesr, as listed in Table 1, according to the following scheme: patients with hscrp <0.3 mg/dl (score 0.5), mg/dl (score 1.0), mg/dl (score 1.5), and > 10.0 mg/dl (score 2.0); patients with cesr <15.0 mm/hr (score 0.5), mm/hr (score 1.0), mm/hr (score 1.5), and >60.0 mm/hr (score 2.0). The plasma NGAL level was measured by fluorescence immunoassay using the Triage NGAL assay (Alere, Inc., San Diego, CA, USA), which can rapidly analyze plasma NGAL with a measurable range from 15 ng/ml to 1300

4 Corrected NGAL level adjusted by an inflammation index 251 Table 4. Grade of renal failure according to the SOFA score and the positive rates in relation to the cutoff limits of ungal and cngal in patient populations. Grade of renal failure No. of subjects Increase of scr Positive rates in relation to Inflammation Median (range; ng/ml) Renal scr level (n=259, %) 0.3 mg/dl cutoff limits (n, %) index > 1.0 ungal cngal score (mg/dl) within 48h ungal > cngal > (n, %) of SOFA (n=25, %) 150 ng/ml 135 ng/ml 0 < (65.2) 3(1.8) 45(26.6) a 13(7.6) 110(65.1) 84(15 936) 62.2( ) (22.7) 10(16.9) 33(55.9)a 22(37.2) 50(84.7) 183( ) b 91.5( ) (6.2) 6(37.5) 16(100.0) 13(81.2) 13(81.2) 512( ) b 267.4( ) (3.1) 5(62.5) 8(100.0) 8(100.0) 8(100.0) 784( ) b 307.6( ) 4 >5.00 7(2.7) 1(14.3) 7(100.0) 7(100.0) 7(100.0) 1300( ) b 433.1( ) Abbreviations: scr, serum creatinine; ungal, uncorrected NGAL; cngal, corrected NGAL; a Significant (p<0.05) vs. positive rates based on cngal >135 ng/ ml, respectively; b Significant (p<0.05) vs. median cngal levels of each group, respectively. ng/ml. The intra-assay CVs (n=20) for three samples (mean NGAL, ng/ml) were %; the inter-assay CVs calculated from duplicate results in 10 subsequent assays were %. A medical decision point was regarded as 150 ng/ml [5]. The cngal was calculated using the following formula: cngal (ng/ml)=ungal (ng/ml)/inflammation index. The inflammation index was obtained from the sum of the scores, which were given to each patient in association with the degree of hscrp and cesr levels. Positive rates were recalculated using the cngal concentration (>135 ng/ml), which was the provisional cutoff point, based on the 95 percentile of cngal in the healthy controls. Plasma NGAL concentrations were measured at admission to the hospital, and scr levels were tested daily over the following 3 days. ESR was determined by the Westergren sedimentation technique using StaRRsed Auto-Compact (Mechatronics Manufacturing BV, Zwaag, the Netherlands). The cesr was calculated, based on a normal hematocrit of 45%, from the following formula: cesr (mm/hr)=(subject s hematocrit/45) xesr (mm/hr). Blood urea nitrogen, scr, and hscrp levels were analyzed with a chemical analyzer (Hitachi 7600; Hitachi, Tokyo, Japan). To assess the degree of renal failure, the renal score of SOFA was used [11], and the patients were categorized into 5 groups (grade 0 to grade 4) on the basis of the scr levels as follows: scr <1.20 mg/dl (grade 0), mg/dl (grade 1), mg/dl (grade 2), mg/dl (grade 3), and >5.00 mg/dl (grade 4). An impaired renal function was defined as a scr level 1.20 mg/dl [11]. The egfr was calculated using the Modification of Diet in Renal Disease (MDRD) formula: egfr=186x[scr (mg/dl)] x[age (years)] A correction factor of was used for women, and 90.0 ml/min/1.73 m 2 was considered normal [12]. The progression of renal impairment was defined as an abrupt (within 48 hours) decrease in kidney function, showing an absolute increase in scr of more than or equal to 0.3 mg/dl, which was based on the AKIN criteria [13]. Patients were divided into 2 groups according to the decrease in kidney function: progression group (n=25) and non-progression group (n=234). Data were expressed as mean ± standard deviation (SD) if normally distributed and as median (range) if nonnormally distributed. Normality of data was determined by the Kolmogorov-Smirnov test. Categorical variables were listed as frequencies and proportions. A

5 252 Annals of Clinical & Laboratory Science, vol. 45, no. 3, 2015 levels were significantly higher than the cngal levels in patients with elevated hscrp and cesr levels; however, no significant difference was observed between ungal and cngal concentrations in patients with non-elevated hscrp and cesr levels. The ungal level was 2 times higher in patients with cesr ( mm/hr) than in those with cesr ( mm/hr), but there were no significant differences in cngal and scr levels between the two groups (Table 1). Figure 1. The trends for ungal, cngal, and scr according to an inflammation index. The ungal concentration shows a steep elevation with an increase in the inflammation index; however, the cngal concentration exhibits a gradual increase in parallel with scr level. Abbreviation: ungal, uncorrected NGAL; cngal, corrected NGAL; scr, serum creatinine. Mann-Whitney U test and a Student t-test were used to analyze data between the two groups. Differences between multiple groups were analyzed by ANOVA, followed by Bonferroni s test for normally distributed values and by the Kruskal-Wallis test for nonparametric values. A multivariate regression analysis of ungal and cn- GAL was conducted for the dependent variable. Adjustment for potential confounders, including age, body mass index (BMI), and blood pressure was performed for independent variables. A receiver operating characteristics (ROC) curve was analyzed to compare the diagnostic accuracy of corrected- and uncorrected- NGAL to identify any increase of scr 0.3 mg/dl within 48 hours. Accuracy was established by using the 95% confidence interval (CI) for the difference rate between cngal and ungal levels. A data analysis was done using SPSS software (version 14.0, SPSS Inc., Chicago, IL, USA). All p values <0.05 were considered statistically significant. Results The baseline characteristics of the study population and the assigned scores in relation to inflammation index are summarized in Table 1. The ungal Of the 259 subjects included in this study, 188 (72.6%) were patients with an inflammation index >1.0, and 89 (34.4%) showed renal dysfunction based on the renal score of SOFA. Among the patients without renal impairment, subjects with an inflammation index >1.0 showed significantly higher levels in ungal, hscrp, and cesr than the healthy controls; however, there were no significant differences in cngal and scr levels between the two groups. The percent difference between un- GAL and cngal levels, after adjusting to the inflammation index, was 57.9% (95% CI, %) in patients with renal impairment and 49.8% (95% CI, %) in patients without renal impairment (Table 2). In a multivariate regression analysis, the scr and egfr levels were more closely associated with the cngal concentration (r=0.692 and r=-0.615, p<0.001) than with the ungal concentration (r=0.583 and r=-0.560, p<0.001), after adjustment for potential confounders. In contrast, the hscrp and cesr levels were more strongly correlated with the ungal concentration (r=0.513 and r=0.439, p<0.001) than with the cngal concentration [r=0.201 (p=0.017) and r=0.159 (p=0.062)] (Table 3). An increase in the ungal concentration above the medical decision point (150 ng/ml) was found in 64 (71.1%) of 90 patients with renal failure (SOFA score; grade 1 to grade 4), and was found in 45 (26.6%) of 169 patients without renal failure (SOFA score; grade 0). Conversely, an increase in the cn- GAL concentration above the cutoff point (135 ng/ ml) was observed in 50 (55.6%) and 13 (7.6%) of the corresponding groups, respectively (Table 4).

6 Corrected NGAL level adjusted by an inflammation index 253 The trends for the ungal, cngal, and scr concentrations according to the inflammation index are illustrated in Figure 1. The ungal concentration displayed a steep elevation with an increase in the inflammation index but the cngal concentration gradually increased in parallel with scr levels. As shown in Figure 2, a regression analysis revealed that the scatter plots of cngal converged more toward the correlation line than did the scatter plots of ungal after adjusting to the inflammation index. The correlation coefficient of the cngal concentration was higher than that of un- GAL (r 2 =0.525, p<0.001 vs. r 2 =0.424, p<0.001). The diagnostic values of cngal and ungal concentrations to detect an increase of scr 0.3 mg/dl within 48 hours were investigated. In an ROC curve analysis, the area under the curve (AUC) of the cngal concentration was significantly larger than that of ungal [0.72 (95% CI, ) vs (95% CI, ), p=0.038]. The cutoff point of the cngal concentration to identify an increase of scr 0.3 mg/dl within 48 hours was 217 ng/ml, where the sensitivity and specificity of the cngal concentration were 62.0% and 83.5%, respectively (Figure 3). Discussion Figure 2. Regression analysis of ungal (a) and cngal (b) versus serum creatinine levels in patients with inflammation. Scatter plots of cngal are more centralized into the regression line compared to those of ungal. The correlation coefficient of cngal is higher than that of ungal after adjustment to the inflammation index (r 2 =0.525, p<0.001 vs. r 2 =0.424, p<0.001). In this study, a new parameter, cngal, was determined using a scoring system, and the parameter was used to screen for renal impairment in patients with systemic inflammation. The results suggest that the new parameter more accurately reflects renal impairment than the ungal concentration in inflammatory diseases because the value was obtained by adjusting the plasma NGAL concentrations with an inflammation index. In this study, patients with an inflammation index >1.0 had 1.9 to 2.7 fold higher ungal levels than those with an inflammation index 1.0. However, no significant difference was observed in cngal levels between the two groups. Interestingly, the increased ungal concentration observed in patients with an inflammation index >1.0, who had no renal dysfunction, decreased to a level not significantly different from the healthy controls, after amending to the inflammation index. These results imply that NGAL is produced in response to an inflammatory reaction, irrespective of kidney function, and the elevated NGAL levels return to baseline values after correction using our inflammatory index. Our data are in partial agreement with the results of previous studies, which demonstrated that NGAL originates from non-renal tissues, particularly in sepsis, causing elevated NGAL levels in the absence of acute kidney injury [14,15]. These observations corroborate the results of Smertka et al [16], which disclosed that increased serum NGAL values are not solely a marker of acute kidney injury, and more significantly represent the severity of inflammatory status. Serum NGAL levels are increased in children with bacterial infection [17]. Recently, it has been reported that NGAL is required for an immune defense against pulmonary infection by E. coli or

7 254 Annals of Clinical & Laboratory Science, vol. 45, no. 3, 2015 a hscrp concentration <0.3 mg/dl and with a cesr level <15.0 mm/h, the sum of the scores in the subjects who were within the reference range in hscrp and cesr becomes 1 (0.5 plus 0.5). Thus, in patients without laboratory evidence of inflammation, the cngal levels are the same values as the initial ungal concentrations. The corrected value using our scoring system seems to reduce the impact of the inflammatory condition on the plasma NGAL concentration, particularly in patients with renal dysfunction in conjunction with inflammatory diseases. Figure 3. The diagnostic accuracies of cngal and un- GAL to identify an increase of scr 0.3 mg/dl within 48 hours in patients with an inflammation index >1.0. The area under the curve (AUC) for cngal was significantly larger than that for ungal [0.72 (95% CI, ) vs (95% CI, ), p=0.038]. Klebsiella pneumonia [18,19]. Wang et al. [20] found that there was a strong positive association between NGAL concentration and hscrp, independent of age, sex, and adiposity, indicating that NGAL is an inflammatory marker. Xu et al. [21] assert that NGAL was significantly correlated with CRP, and determination of NGAL was more specific and sensitive than CRP in the distinction between viral and bacterial infection. In our study, ungal concentrations had increased in parallel with hscrp and cesr levels; however, the cngal concentration was not significantly different with respect to the change in hscrp and cesr levels. The scr levels had increased with cn- GAL, but not with ungal. Furthermore, ungal was more strongly correlated with hscrp than with scr. However, cngal was more closely linked to scr than to hscrp. On the basis of these findings, it is assumed that cngal is more significantly implicated with kidney function than ungal, particularly under inflammation conditions. The inflammatory status, probably from bacterial or viral infection, is one of the most common problems in clinical practice. Inflammation is recognized as a risk factor for acute kidney injury [22]. In the current study, our scoring system was mainly focused on patients with inflammation. Because the score of 0.5 is given to each of the subjects with Aydogdu et al. [23] reported that plasma NGAL should be used with caution as a marker of acute kidney injury because it can increase in the absence of tubular damage in critically ill patients. In our study, among 169 patients without renal impairment, 45 (26.6%) were positive in the ungal test with a cutoff limit of 150 ng/ml. However, the positive rate was decreased to 7.6% (n=13) when the new parameter of cngal (>135 ng/ml) was applied as a decision point. Interestingly, only 3 (1.8%) individuals of the 169 patients without renal impairment showed a progression of disease, yielding an increase of scr 0.3 mg/dl within 48 hours. These results suggest that ungal levels may be falsely elevated in a considerable number of patients with inflammation, regardless of the progression of disease. Our data stress that plasma NGAL levels should be interpreted after adjusting to an inflammation index. In the present study, the diagnostic ability of cn- GAL and ungal to predict a worsening of renal function was investigated. The AUC of the ROC curve for cngal was significantly larger than that for ungal. These observations indicate that cn- GAL is superior to ungal in diagnostic accuracy, and that cngal may be a more reliable predictor than ungal to identify an aggravation of renal dysfunction in patients with systemic inflammation. This study has several limitations. We did not conduct multiple measurements of plasma NGAL levels in combination with the change in scr levels. As in any observational study, there are possibly unmeasured confounders for which we did not adjust during our statistical analysis. Despite these

8 Corrected NGAL level adjusted by an inflammation index 255 limitations, our study maintains some significance. To the best of our knowledge, there have been no studies that investigated the corrected value for NGAL using a scoring system in patients with inflammatory diseases. These findings may have important implications for the clinical management of patients with systemic inflammation, who had concurrent renal dysfunction. However, the present findings obtained in a single-center uncontrolled study may need to be validated in larger randomized prospective trials. In conclusion, this study tested a new parameter, the cngal level, to screen for renal dysfunction, especially in patients with inflammatory diseases. Our study shows that plasma NGAL levels need to be adjusted to assess renal impairment in patients with systemic inflammation. 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