Urine neutrophil gelatinase–associated lipocalin predicts outcome and renal failure in open and endovascular thoracic abdominal aortic aneurysm surgery

Urine neutrophil gelatinase–associated lipocalin (uNGAL) has been evaluated as a biomarker for AKI detection and adverse outcome in open and endovascular thoracoabdominal aortic aneurysm surgery. This observational, retrospective study included 52 patients. UNGAL was measured peri-operatively (48 h) and correlated with AKI requiring dialysis, tracheotomy and adverse outcome. Mean patients’ age was 64.5 years. A total of 26.9% (n = 14) developed AKI, and 21.1% (n = 11) required dialysis, tracheotomy rate was 19.2% (n = 10) and in-hospital mortality rate was 7.6% (n = 4). uNGAL levels were related to AKI requiring dialysis at ICU (p = 0.0002), need for tracheotomy at baseline and admission on ICU (p = 0.0222, p = 0.0028, respectively), as well as adverse discharge modality (p = 0.0051, p = 0.0048, respectively). Diagnostic quality was good for uNGAL levels at admission to ICU regarding AKI requiring dialysis (sensitivity: 81.8% [48.2–97.7]; specificity: 87.8% [73.8–95.9]; area under the curve (AUC): 0.874 [0.752–0.949]). The diagnostic quality of uNGAL was favorable for the prediction of tracheotomy (sensitivity: 70.0% [34.8–93.3]; specificity: 83.3% [68.6–93.0]; AUC: 0.807 [0.674–0.903]) and adverse discharge (sensitivity: 77.8% [40.0–97.2]; specificity: 83.7% [69.3–93.2]; AUC: 0.817 [0.685–0.910]). uNGAL may be valuable as an post-operative predictor of AKI and adverse outcome after open and endovascular TAAA repair.

have been identified after impairment of kidney function, and NGAL seems to have a protective effect on cells that may be related to its ability to scavenge iron and to induce cell growth [18][19][20] . In murine models, NGAL is the most rapidly induced protein of nephrotoxic and ischemic AKI and is detectable 3 h following an initial kidney injury 21,22 . Furthermore NGAL has been described as biomarker of adverse outcome in different cardiovascular settings 23,24 .
Here we investigated the intriguing potential of urine NGAL (uNGAL) as a marker of AKI and adverse outcome in the context of complex endovascular and open TAAA surgery.
Of the 13.4% (n = 7) who developed sepsis, six cases were related to pneumonia and one case to small intestine ischemia following embolization during open type III repair. Two patients (3.8%) developed spinal cord ischemia, one after an endovascular type 2 TAAA repair and one patient after an open type 3 TAAA repair.
The in-hospital mortality rate was 7.6% (n = 4), and the total mortality rate during the follow-up (mean follow-up, 13.2 months (±5.3, [2-20 months]) was 9.6% (n = 5). Of the latter, there were two cases of pneumonic sepsis, one each of cerebral bleeding and small intestine ischemia associated with pancreas necrosis and peritonitis, and one thoracic aortic rupture at 19 post-operative weeks after type IV repair.

Correlation of uNGAL and biomarkers, clinical scoring systems, and outcome parameters.
We observed an increasing correlation between uNGAL and serum creatinine over time. Furthermore, a significant correlation between uNGAL and the APACHE-II score was observed for all time points (ƍ = 0.457 admission to ICU; ƍ = 0.364 24 h after admission to ICU; ƍ = 0.439 48 h after admission to ICU). Additionally, a significant correlation for all time points of uNGAL and urine output could be assessed (ƍ = −0.320 admission to ICU; ƍ = −0.349 24 h after admission to ICU; ƍ = −0.559 48 h after admission to ICU).
Looking at a correlation of non-repeated factors with uNGAL at ICU, a significant correlation was found for length of ICU stay (ƍ = 0.390; p = 0.0046), as well as for the duration of dialysis (uNGAL: ƍ = 0.543; p < 0.0001). The ventilation time and in-hospital stay showed low to moderate correlations with uNGAL but were not significant (uNGAL: ventilation, ƍ = 0.272; p = 0.0557; in-hospital stay: ƍ = 0.265; p = 0.0598) ( Table 2).

Correlation of uNGAL levels and AKI requiring dialysis.
Starting with admission to ICU, uNGAL levels differed significantly among patients suffering from AKI requiring dialysis and those with non-impaired renal function. With the exception of baseline uNGAL levels, each time point showed a significant correlation. A ROC analysis for uNGAL with respect to AKI requiring dialysis treatment showed a good diagnostic quality for the time points 'baseline' and 'ICU' (baseline: Se = 81.8% [48.2-97.7 Table 1. Patient Characteristics According to favorable and adverse discharge. Examination of different patient characteristics separated by the discharge modality "favorable" and "adverse. " If data were missing, the sample size included is reported for the corresponding parameter. All variables are described as absolute frequencies n (%), mean ± SD or median (Q1, Q3), and ranges. ORs with 95% CIs are reported for dichotomous variables together with the p value.
Investigating uNGAL at each time point, we identified a significant difference between patients with a favorable and an adverse discharge modality at all time points (details in Fig. 3 and Supplemental Table 5-6).
Coincidence of AKI requiring dialysis, tracheotomy, and discharge modality. The probability of an adverse discharge in dependence of a tracheotomy and AKI requiring dialysis was assessed to evaluate treatment dependencies in association with patient outcomes. We observed a complete or quasi-complete separation of the patients (p = 0.0242, p = 0.0012, Table 3), i.e., an adverse discharge modality was observed only if a tracheotomy was performed. Only one patient who had a tracheotomy had a favorable outcome, and this patient also had suffered from AKI requiring dialysis. Of those patients with adverse outcome, two died without requiring dialysis, and their uNGAL levels decreased within 48 h (details in Table 3 and Supplemental Table 7).

Repeated measures analysis of uNGAL.
In a longitudinal analysis, a significant impact of the baseline value of uNGAL on the post-surgical measured levels of uNGAL within the first 48 h after admission to ICU (Table 4; base: p = 0.0011) was observed. Overall, uNGAL did not change significantly over time (base: p = 0.6935). The repeated factors urea, urine, and serum creatinine were also identified as significantly related to uNGAL (Table 4; urea: p = 0.0462; urine: p = 0.0044; serum creatinine: p = 0.0018). Nevertheless, the slope estimate of urine was close to zero. Analyzing patients and surgery characteristics in the single parameter model, sex influenced uNGAL levels significantly (p = 0.0337) while age did not show a significant effect (p = 0.2754). Judging from the estimates, the highest regression slopes were related to the sex (0.7529 ± 0.3445), chronic kidney disease (CKD) (0.5158 ± 0.4384), and serum creatinine levels (0.4636 ± 0.1451). Although the CKD impact was not significant, it had a large impact on data variation. Furthermore, the multivariable model confirmed a significant influence of baseline uNGAL value, serum creatinine, and sex on uNGAL (baseline uNGAL: p = 0.0010; serum creatinine: p = 0.0153; sex: p = 0.0365), but uNGAL levels still did not differ significantly over time (time: p = 0.8628). For the multivariable model, the largest slope estimate still corresponded to sex (0.7149 ± 0.3324) and serum creatinine (0.3548 ± 0.1440).
Looking at the estimate for sex, men showed increased uNGAL values in contrast to women. Nevertheless, discharge modalities did not differ significantly between women and men (p = 1.0000; Table 1).

Discussion
Based on the results of this study, we could confirm that post-operatively measured uNGAL could be used as a postoperative biomarker for AKI requiring dialysis and as a predictive biomarker for needing tracheotomy and for adverse discharge modality (namely discharge from weaning ward or death) after open and endovascular TAAA repair. Furthermore, pre-interventional measured uNGAL levels correlated significantly with the need for tracheotomy and an adverse discharge modality. To our knowledge, no study to date has evaluated uNGAL as a biomarker for patient outcome and prolonged ventilation after cardiovascular surgery.
NGAL has been successfully used in different settings for the diagnosis and prognosis of AKI. Although several studies have investigated the value of NGAL to predict AKI in pediatrics, nephrology, and heart surgery, only limited evidence exists for its predictive abilities after TAAA surgery [25][26][27][28] . Chang et al. described the use of new biomarkers such as NGAL for AKI detection after endovascular aortic repair 29 . Kalimeris et al. emphasized the value of elevated NGAL levels as predictors of AKI after repair of abdominal aortic aneurysm 30 . These findings support our results on the predictive ability of uNGAL baseline measurement.
Comparing the value of serum NGAL (sNGAL) and uNGAL in the aortic aneurysm setting, Kokot et al. identified an increased predictive value for uNGAL compared with sNGAL regarding the probability of AKI 31 . In  the present study, we observed a significant correlation of uNGAL with serum creatinine levels and a significant negative correlation with urine volume. These findings underline a direct relation of uNGAL and impaired renal function after TAAA repair. Of interest, two of the patients with elevated uNGAL levels who died post-operatively did not develop AKI. In both cases, uNGAL dropped within 48 h to a normal level. The pre-operative elevation of uNGAL in these patients may be a consequence of an assumed function of NGAL as a stress response protein, indicating a reduced general condition, which could influence postoperative outcomes 20 . Furthermore the course of uNGAL in these cases may correlate with a reported decreased specificity of uNGAL as biomarker for AKI 32 .
Only a study including a larger cohort of patients favorably in a multicentric setting could answer this question appropriately. The function and meaning of NGAL as a stress response protein cannot clearly be discriminated in the setting of this study from its relevance as an early biomarker of AKI. It would have been favorable to correlate the course of uNGAL with further biomarkers of the innate immune response such as Interleukin 6 (IL-6), which has been described as potential predictor of the post-interventional inflammation status after major surgery 33 . Yet, the survey of IL-6 during the first days on ICU was fragmentary, so a potential correlation between uNGAL and IL-6 could not be assessed in the reported setting. In this context, Abella et al. described a regulatory role for NGAL in the innate immune response. NGAL is involved in a multitude of physiological and pathophysiological processes, such as apoptosis, infection and inflammation 34   induced by tumor necrosis factor (TNF) and lipopolysaccharide (LPS), is activated by inflammation and infection 35 . Thus elevated baseline levels of NGAL as in the current study may be related to an altered immune status of the patients, which may be in turn related to adverse outcome 36 . Accordingly, the results regarding the predictive role of baseline uNGAL in this study may be consistent with the results of Lindberg et al., who described Plasma-NGAL as independent predictor of all-cause mortality and major adverse cardiovascular event in general population 37 . Finally, this thesis could not be validated by further findings in our study.  Nevertheless, while an increasing correlation between uNGAL and serum creatinine levels as well as urinary extraction could be assessed within the first 48 h after open and endovascular TAAA surgery, the initial assumption of uNGAL as a predictor of AKI still stands.
The multivariate analysis of uNGAL highlighted the influence of the baseline level of uNGAL and serum creatinine with post-surgical measurements of uNGAL, which strengthens the predictive power of uNGAL regarding postoperative AKI with the need for dialysis. With regard to the sex-specific differences of uNGAL, our results support the findings of Thrailkill et al., who identified significantly increased uNGAL levels in male patients with diabetes 38 . These results together may indicate a different applicability of uNGAL for men and women. Even if the outcome for men and women had not differed significantly in the present study, this finding seems important regarding a potential implementation of uNGAL as a urinary biomarker.
uNGAL levels and adverse discharge correlated significantly, and every patient who died or was discharged via the weaning ward showed already elevated uNGAL levels at baseline. Furthermore, the trend increased after admission to ICU. In agreement, Siew et al. found uNGAL as a prognostic biomarker for outcome of patients suffering from AKI 39 . The observed correlation of elevated uNGAL levels with the APACHE II score also underscores its ability to predict patient outcome after complex aortic surgery, and an association of post-surgical AKI and the APACHE II score has been described before 40,41 . Post-operative AKI after complex aortic surgery is directly related to adverse outcome, so the consistent results regarding the predictive power of uNGAL seem conclusive 4,40 .
Tracheotomy was conducted in this study if a prolonged artificial respiration was required. With regard to the correlation of elevated uNGAL levels and tracheotomy or adverse discharge, an almost complete concordance was found. Except for one patient who suffered from post-operative AKI and tracheotomy but was not discharged via the weaning ward, all patients who received a tracheotomy had an adverse discharge modality. In agreement, other authors also have described a correlation of tracheotomy with postoperative AKI and adverse outcome 42,43 .
In terms of the elevated NGAL-levels and their correlation with patients' outcome, an early detection of elevated NGAL-levels by use of rapid testing kit may enable early medical and interventional treatment options such as dialysis therapy 44 .
Certain limitations of the present study must be taken into account while evaluating the discussed results: A prospective, multicenter study including more homogenous patients treated by open or endovascular means would have improved the quality of the assessed data. Otherwise, TAAA is a rare disease, and few vascular surgery centers perform open and endovascular treatment regularly. A discrimination of the function of NGAL as a stress response protein vs. its function as a kidney injury marker would be helpful. Currently, no panel of biomarkers associated with inflammation or kidney injury is available, which would enable a clear separation of uNGAL as a biomarker for inflammation or AKI in the described setting. The assessment of further biomarkers for the immune response was not possible during this study, hence a potential correlation of uNGAL and those biomarkers was not conducted. An exclusion of patients with pre-existing chronic renal failure would be useful regarding the homogeneity of this patient cohort. Still, although no significant influence regarding pre-existing CKD could be observed, the presented findings emphasize the relevance of uNGAL as a postoperative biomarker of AKI and predictor of adverse outcomes after open and endovascular TAAA repair. Although our analysis was conducted in a hypothesis-generating manner, the results are concordant with what has been reported in the literature.

Methods
Ethics approval and consent to participate. The local ethics committee approved this study (University Hospital Aachen EK004/14). This study was performed in accordance with the Declaration of Helsinki in its actual form. Written informed consent was obtained preoperatively from all subjects.
Inclusion and exclusion criteria. Patients suffering from TAAA larger than 6 cm, defined according to the Crawford classification, were eligible for inclusion 45 . Exclusion criteria were age below 18 years, pregnancy, chronic kidney disease requiring permanent dialysis treatment. No emergency procedures were included.
Clinical and laboratory data collection. In this retrospective study, data on demographics, medical history, and admission diagnosis as well as daily physiological variables, surgical interventions, need for dialysis, and any kind of adverse event were collected from medical records and electronic bedside flow charts (IntelliSpace Critical Care and Anesthesia; Philips Healthcare, Andover, Massachusetts, USA). During the surgical treatment, according to standardized and consistent operations, blood and urine samples were collected from patients at five predefined time intervals: pre-interventionally, at admission to the intensive care unit (ICU), and at 12 h, 24 h, and 48 h after admission to ICU. AKI was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria 46 . In this study the serum creatinine criteria were used to define AKI, whereat urine volume was not applied for AKI determination. Baseline creatinine was the lowest pre-intervention value. AKI was defined by a reduction in kidney function with an increase in serum creatinine (>26.4 µmol/L) or percentage increase of serum creatinine above 50%, as recommended in current guidelines 47 . In this pilot study we focused on AKI requiring dialysis. Surgical protocol. Open surgery. The operative protocol for open TAAA repair has been published in detail before 48,49 . Briefly, it includes intubation with a double lumen endotracheal tube, cerebrospinal fluid drainage, perioperative monitoring of Motor Evoked potentials (MEP), sequential aortic clamping if possible, extracorporeal circulation with distal aortic perfusion, selective visceral perfusion and mild hypothermia of 32 °C to 33 °C 50 . Custodiol ® (Dr. Franz Köhler Chemie, Austria) at 44 °C was used for renal perfusion instead of blood perfusion. This approach had been described to protect the kidneys from ischemic organ damage 51 . Thoracolaparotomy through the fifth to eighth intercostal spaces depending on the extent of the aneurysm was used for surgical access as well as a groin cut-down to the femoral vessels for placement of the extracorporeal circulation cannulas. Endovascular surgery. The procedure was performed under general anesthesia, and the same protocol was used for the neurological monitoring. The detailed procedure of fenestrated endovascular aortic aneurysm repair has been described before 52 . In cases of endovascular procedure, renal perfusion was not interfered with directly. Radiation and iodinated contrast solution were used conservatively to reduce the toxic effects on the kidneys. Furthermore, a reduced contrast solution dose (one fourth of the standard dose) for the selective angiography of the renal arteries was used because it has been described as protective regarding acute kidney failure 53 . Endpoints. Levels of uNGAL and their correlation with AKI following open and endovascular aortic repair was the primary endpoint. Correlations of uNGAL levels and tracheotomy as well as adverse discharge modality were assessed as secondary endpoints as these dichotomous events are related to poor outcome. Furthermore, correlations with different parameters and scoring systems such as APACHE II and Lactate have been observed.

Biomarkers.
Tracheotomy was indicated in case of extended artificial ventilation. In our hospital, a tracheotomy is performed after a minimum 96 h of ventilation. Adverse discharge modality was defined as discharge via weaning ward or death. As no patient was discharged from ICU, this modality of discharge was not defined as adverse discharge modality. All patients who survived the first 30 days were contacted between December 2015 and January 2016.

Statistical analyses.
For description, all continuous variables are expressed as mean values ± standard deviation (SD) or 95% confidence intervals (CIs). For heavily skewed distributions, the median, the 0.25-0.50 quantile (Q1), and the 0.75 quantile (Q3) were used instead. Categorical variables are expressed as absolute frequencies and percentages. Correlations between continuous parameters were assessed by the Spearman correlation coefficient for each time point ("rho", ƍ). Some measurements in the data are missing completely at random; a systematic bias could not be detected.
The uNGAL level distribution was skewed, so exact Wilcoxon signed-rank tests were chosen to compare uNGAL for different factors between single time points. Comparisons between frequencies were conducted using a χ² test or Fisher's exact test in cases of small cell frequencies (≤5). For patient characteristics with respect to their discharge, exact odds ratios (ORs), corresponding 95% (penalized) likelihood Cis, and p values are given in Table 1.
Linear models with repeated measures were applied to evaluate the impact of certain metabolic factors on uNGAL. As a base model, we considered the fixed time effect (repeated factor), preoperative NGAL value (baseline value), and a random intercept. All further linear models are extensions of the base model. The response parameter uNGAL was log transformed to meet the model requirements. A Kenward-Rogers adjustment was used to account for the small sample size. For the covariance, an AR (1) structure was assumed. Model fit was evaluated with the help of residual plots.
Given the small sample size, we focused on analyzing the influence of single parameters (univariate analysis). Therefore, we considered the clinical factors of age and sex and all metabolic factors that showed interesting correlations or were considered relevant according to literature and experience, separately as additional covariables for the base model. Furthermore, we performed a multivariable analysis for uNGAL modeling in addition to the base model for the impact of sex, creatinine levels in the serum, and AKI/dialysis simultaneously because they were the most promising predictors within the univariate analysis. Considering these parameters simultaneously improved the model according to the AIC (Akaike's information criterion) value. At first, tracheotomy also was considered, but because it worsened the model fit (AIC) and did not have a significant influence, we omitted it from the model. We report for all fixed-effect covariables the (slope) estimate, its standard error, degrees of freedom (DF), the value of the test statistic (t-value), and the p value. Because we considered more than two time points, the p value of the overall F-Test (type 3) is reported for the overall time effect together with the two DFs (Num DF/Den DF) and the value of the F-statistic (F-value). We assessed any effect in the statistical models as significant if the corresponding p value fell below the 5% margin. No alpha adjustment was carried out because this study was considered exploratory.
A receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic property of uNGAL with respect to each patient's direct discharge category (favorable/adverse), tracheotomy, and AKI requiring dialysis. Sensitivity (Se), specificity (Sp), likelihood ratios (LR±), area under the curve (AUC), and the Youden-optimal cut-off (maximize Se + Sp − 1) are reported together with the plotted ROC curves.
Boxplots were chosen to present the data distribution of uNGAL for selected patient characteristics over time. Spaghetti plots were used to illustrate the concrete course of uNGAL over time for patients with adverse direct discharge.

Conclusion
uNGAL may be used as a postoperative biomarker of AKI requiring dialysis, tracheotomy and adverse discharge modality after open and endovascular TAAA repair.
Availability of data and materials. The datasets used and analyzed during the current study are available from the corresponding author upon request.