Introduction
Sepsis is the systemic inflammatory response to infection, and has a high mortality rate despite the use of modern antibiotics and resuscitation therapies [
1], particularly in septic patients with associated acute kidney injury (AKI). Early diagnosis, appropriate classification and intervention in the initial periods of sepsis play a crucial role in decreasing mortality, and they are implemented in the emergency department (ED) [
2]. Although clinical scoring systems are useful tools to evaluate the severity and prognosis of sepsis, most of them are created for ICU patients and may not be applicable to the ED. Finding biomarkers and combining them with the clinical scoring systems for early diagnosis, risk stratification, and evaluation of prognosis of sepsis has great significance.
Neutrophil gelatinase-associated lipocalin (NGAL) is an endogenous bacteriostatic protein that is expressed and secreted by neutrophils, macrophages, hepatocytes, and renal tubular cells in various pathologic states [
3],[
4]. NGAL represents the activities of the neutrophils and comprises a critical component of innate immunity to bacterial infection [
5]. Matrix metalloproteinase-9 (MMP-9) and its inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), are promising novel biomarkers to predict the severity and outcome of sepsis [
6],[
7], and are involved in the pathogenesis of sepsis and septic shock [
8]. NGAL seems to positively modulate the activity of MMP-9 [
9]. It protects MMP-9 from proteolytic degradation and enhances its enzymatic activities by binding and forming the MMP-9/NGAL complex.
Some clinical studies have confirmed that serum NGAL, MMP-9 and TIMP-1 levels are increased in septic patients [
10],[
11]. However, most of the previous studies, which had small sample sizes, included ICU admission patients, and the relationship of these biomarkers could not be expounded. In this study, we questioned whether serum NGAL, MMP-9 and TIMP-1 concentrations are different according to the severity of sepsis, and compared their clinical value in early diagnosis, risk stratification and prognostic evaluation of sepsis with procalcitonin (PCT) and the mortality in emergency department sepsis (MEDS) score.
Discussion
During recent decades, the mortality of sepsis has remained unacceptably high. Early diagnosis and exact risk stratification, which are keys to initiating timely treatments, are significant predictors [
1]. Consequently, there is a demand for useful severity scoring systems and biomarkers to improve the outcomes in early diagnosis and risk assessment. Although it has disadvantages compared with the other severity scoring systems, the MEDS score is derived from septic patients in the ED and is more suitable for predicting mortality in this cohort [
16],[
17]. In the present study we chose MEDS score as a comparative variable to NGAL and TIMP-1. Meanwhile, in diagnosis, differential diagnosis, risk stratification and prognosis biomarkers play important roles in septic patients. The most widely used clinical biomarker is PCT, and while confirmed to be superior to other biomarkers in some studies, its prognostic value is controversial [
18],[
19].
Consistent with previous studies [
20],[
21], our study indicates the NGAL is elevated in the peripheral blood of sepsis patients. Human NGAL was originally identified as a novel protein isolated from secondary granules of human neutrophils and was subsequently demonstrated to be a 25-kDa protein covalently bound to neutrophil gelatinase [
3],[
22]. NGAL protein is mainly synthesized at the early-myelocyte stage of granulopoiesis during formation of secondary granules, and NGAL mRNA is normally expressed in many adult human tissues, such as bone marrow, uterus, prostate, salivary gland, stomach, colon, trachea, lung, liver and kidney [
23]. In addition, NGAL comprises a critical component of innate immunity to bacterial infection. Siderophores are synthesized by bacteria to scavenge iron from their surroundings, and use specific transporters to recover the siderophore-iron complex, ensuring their iron supply. The siderophore-chelating property of NGAL therefore renders it a bacteriostatic agent [
24]. NGAL expression has been studied in several normal tissues where it functions to modulate oxidative stress and to provide protection against bacterial infection, and it has been proved in animal experiments [
25]. Based on these two mechanisms, it is not surprising that circulating NGAL is increased in sepsis because of its structure and function, and that NGAL level increased according to the clinical severity of sepsis in our study.
MMP-9 is one of the family of MMPs, and is induced by many inflammatory factors, including interleukin (IL)-1b, IL-8, and tumor necrosis factor-α. It is stored in the tertiary granules of polymorphonuclear leukocytes, which are key effectors in acute inflammatory diseases such as sepsis [
26]. MMPs are not expressed during normal conditions but are expressed and activated increasingly during inflammation, and their main function has been considered to be the degradation and removal of extracellular matrix molecules from the tissue [
27]. It is reported that MMP-9 and gelatinase activity increased significantly after sepsis, and TIMP-1, an MMP-9 inhibitor, blocked these activities, as well as the ensuing septic shock. Furthermore, with the development of sepsis, these results are consistent with MMP-9-induced caspase-3 activation in response to infection, which increases TIMP-1 level and thereby inhibits MMP-9, in turn decreasing transforming growth factor-β1 and caspase-3 signaling pathways and improving survival in septic rats [
28]. The integrity of tissue architecture is closely dependent on the delicate balance between the activation of MMPs and their inhibition by TIMPs. Any alteration in this balance is linked to tissue damage. Elevated levels of MMP-9 and TIMP-1 were reported in septic patients, and higher TIMP-1 levels at the beginning of severe sepsis were predictive of death [
29].
In humans, NGAL was originally identified as a 25-kDa protein covalently linked to MMP-9 in human neutrophils, which normally provide the main cellular source of circulating NGAL. By forming the MMP-9/NGAL complex, NGAL protects MMP-9 from proteolytic degradation, increasing the enzymatic activity of MMP-9 and subsequently enhancing tumoral invasiveness and diffusion [
30]. In the present study, MMP-9 was higher with increased severity of sepsis in the groups with SIRS, sepsis and severe sepsis (
P <0.01), but the level decreased according to severity of septic shock. MMP-9 elevation has become a useful marker of the severity of sepsis [
10], but a multicenter study by Lorente
et al. [
31] found a non-significant increase in MMP-9. MMP/TIMP ratios may mirror the balance between the proteolytic activity of MMPs and their tissue inhibitors, and the importance of reduced MMP-9/TIMP-1 ratio as a predictive biomarker of severity in septic patients [
31]. In our study, TIMP-1 levels significantly increased on admission according to the severity of sepsis. Therefore, the variation in MMP-9 was not only related to the change in TIMP-1, which is the specific inhibitor of MMP-9, but also to the variation of NGAL.
The ED often serves as the first location for initial evaluation and risk stratification, which is essential to the timely management of septic patients. Our research also focuses on the early prognostic evaluation of septic patients. Our study suggests that the prognostic value of NGAL, MMP-9 or TIMP-1 is superior to PCT and MEDS score, and that NGAL, TIMP-1, MMP-9 or MEDS score, but not PCT, are robust independent predictors of 28-day death in patients with sepsis. We found that the difference in NGAL, TIMP-1 and MMP-9 levels between non-survivors and survivors was significant. From the ROC analysis of a large sample of septic patients at ED admission, we found that NGAL and TIMP-1 were superior to PCT and showed higher sensitivity, specificity, PPV, and NPV in the early diagnosis of 28-day mortality. Taken together, these results suggest that NGAL and TIMP-1 may be useful to identify septic patients at risk of short-term mortality in the ED.
The clinical practice scoring systems are useful tools to evaluate the severity and prognosis of septic patients. While severity scoring systems and biomarkers have their own advantages and disadvantages in the prognosis of sepsis, both can be considered objective and accurate. Researchers have attempted to combine severity scoring systems with biomarkers to improve the accuracy of risk stratification and prognosis. In our study, the prognostic ability of the combination of NGAL or TIMP-1 with MEDS score was superior to either biomarker or MEDS score alone, and also superior to the combination of PCT with MEDS score. The combination of severity scoring systems and biomarkers is more effective in risk stratification and prognosis, as indicated in the present study. These results demonstrate that NGAL and TIMP-1 enhanced the ability of the MEDS score in the risk stratification and prognosis evaluation.
AKI in ICUs is frequently complicated with sepsis, and AKI and sepsis increase mortality synergistically. NGAL is an emerging biomarker for AKI. Its performance in early detection of renal damage has been valuable in several AKI cohorts [
21],[
32],[
33]. In the present study, 89 (24.7%) septic patients developed AKI, according to the AKIN criteria. NGAL was significantly higher in septic AKI patients than in the other AKI patients and non-AKI patients (
P <0.01). From the AUC of NGAL for septic AKI, we discovered that the prognostic ability of NGAL was superior to other biomarkers. The present study proved that NGAL was a biomarker for AKI, particularly in septic patients.
Limitations
Some limitations merit consideration in this study. It was a single-center study and did not compare other severity score systems and biomarkers. Sepsis was established according to criteria for sepsis as defined by ACCP/SCCM as it was difficult to obtain pathogen samples. The dynamic changes of biomarkers were not observed. We did not have information on patient treatment preferences that could introduce confounding and bias.
Competing interests
The authors declare that they have no competing interests.