Abstract
Background: Acute kidney injury (AKI) during sepsis is associated with poor outcome. However, diagnosis of AKI with serum creatinine (SCr) level change is neither highly sensitive nor specific. Therefore, identification of novel biomarkers for early diagnosis of AKI is desirable. Aims: To evaluate the capacity of combining urinary netrin-1 and human kidney injury molecule type 1 (KIM-1) in the early diagnosis of septic AKI. Methods: We prospectively recruited 150 septic patients from Jun 2011 to Jun 2013 at Zhejiang Provincial People's Hospital, China. SCr, urinary netrin-1, and KIM-1 levels were recorded at 0, 1, 3, 6, 24, and 48 h of ICU admission and compared between AKI and non-AKI patients. In addition, we investigated the prognostic value of netrin-1 and KIM-1 between non-survivors and survivors in septic AKI patients. Results: SCr levels started to show elevation after 24 h of ICU admission. However, netrin-1 levels increased significantly as early as 1 h, peaked at 3–6 h and remained elevated up to 48 h of ICU admission in septic AKI patients. KIM-1 increased significantly by 6 h, peaked at 24 h and remained significantly elevated until 48 h of ICU admission. Furthermore, we observed significant higher urinary KIM-1 levels at 24 h and 48 h in non-survivors compared to survivors in AKI patients. Conclusions: Our results suggest that both netrin-1 and KIM-1 are clinically useful as early biomarkers in the diagnosis of septic AKI. In addition, persistent elevation of urinary KIM-1 level may be associated with poor prognosis.
Introduction
Sepsis and septic shock are the most common causes of morbidity and mortality among critically ill patients. They are also the most important causes of acute kidney injury (AKI), account for 50% or more of cases of AKI in intensive care unit (ICU).Citation1,Citation2 The mortality in septic AKI remains high during the past decades despite improvement of supportive therapy,Citation3 which may partially be explained by the fact that when the signs and symptoms of sepsis and septic shock appear, considerable renal damage might have already occurred.Citation4 Furthermore, recent evidence suggests that the pathophysiology of septic AKI is distinct from that of non-septic AKI,Citation5,Citation6 and therefore, important differences may exist in diagnosis, response to therapy, and clinical outcomes. Currently, the diagnosis of AKI relies on a decreased glomerular filtration rate (GFR), increased serum creatinine (SCr) with or without oliguria, described by two classification systems—the Acute Kidney Injury Network (AKIN) criteriaCitation7 and the RIFLE criteria (Risk, Injury, Failure, Loss, End-stage) kidney disease.Citation8 However, the SCr level displays poor specificity and sensitivity in the diagnosis of AKI.Citation9 Moreover, sepsis decreases production of SCr without major alterations in body weight, hematocrit, or extracellular fluid, and therefore creates further limitations on using changes in SCr levels as a reliable marker of AKI.Citation10 Hence, it is essential to develop novel biomarkers that enable early diagnosis and prediction of AKI, especially in septic patients.
Recent advance in technology made it possible to develop several encouraging biomarkers including Netrin-1,Citation11 kidney injury molecule-1 (KIM-1),Citation12 neutrophil gelatinase-associated lipocalin (NGAL),Citation13 N-acetyl-β-d-glucosaminidase (NAG)Citation14 and IL-18,Citation15 etc. They may represent promising new markers for early detection of septic AKI that, however, need to be further evaluated in the clinical settings. Netrin-1 is a laminin-related axon guidance molecule. KIM-1 is a transmembrane glycoprotein. Both of them are undetectable in the healthy kidney or urine and upregulated following kidney injury. Since they have been investigated in several animal models of AKI or a limited number of clinical studies, the translation to clinical application still need further validation. In addition, all biomarkers have individual strengths and weaknesses. Combination of multiple biomarkers to develop a biomarker panel would be an optimal way to detect AKI more efficiently and accurately. Here, we study to determine the utility of combining urine netrin-1 and KIM-1 in diagnosis of septic-AKI in the ICU.
Materials and methods
Patient population
Patients who were admitted to the ICU at Zhejiang Provincial People's Hospital, China during the period of Jun 2011 to Jun 2013 and met the sepsis diagnosis criteria were enrolled in the study. The clinical diagnosis of sepsis requires finding a focus of infection as well as at least two signs of systemic inflammatory-response syndrome (SIRS) that includes abnormal body temperature (higher than 38°C or less than 36°C), heart rate >90 beats/min, respiration >20 breaths/min or arterial partial pressure of CO2 <32 mmHg, and deranged white blood cell (WBC) counts (>12 × 103/mm3, <4 × 103/mm3, or >10% bands).Citation16 Informed consent was obtained from subjects or surrogates. Patients were excluded if they had preexisting renal insufficiency, autoimmune diseases, malignancy, concomitant nephrotoxic drug use, and incomplete urine collections. According to the AKIN criteria, AKI was defined as a 50% increase in SCr from baseline within 48 h after ICU admission. Twenty healthy individuals matched for age and sex served as controls. The research protocol was approved by the Institutional Review Board of the hospital.
Specimen collection
For each patient, six urine samples and six blood samples were obtained that corresponded to times 0, 1, 3, 6, 24, and 48 h after ICU admission. Urine and blood sample were centrifuged at 3000 rpm for 10 min, and the supernatants stored in aliquots at −80°C for further detection.
Netrin-1 and KIM-1 quantitation by enzyme-linked immunosorbent assay
Fifty microliters of urine was used for quantitation of netrin-1 and KIM-1 as previously described and with some modifications.Citation17,Citation18 The assay done using an enzyme-linked immunosorbent assay kit (Netrin-1 from USCN Life Science, Wuhan, China, catalogue no. SEB827Hu; KIM-1 from Shanghai Kanu Biotechnology Co. Ltd, Shanghai, China). Briefly, netrin-1 or KIM-1 standard and samples were added to antibody-coated 96-well plates and incubated for 2 h at room temperature, followed by addition of biotin-conjugated polyclonal antibody specific for netrin-1 or KIM-1 and incubation for an additional 1 h. Plates were then washed and incubated with avidin conjugated to horseradish peroxidase for 1 h. Color was developed using tetramethylbenzidine substrate. Reaction was stopped by adding sulfuric acid to the plates. The color change was measured using a plate reader at a wavelength of 450 nm. The concentration of netrin-1 or KIM-1 in the samples was determined according to the standard curve.
Statistics
All data are assumed normal distribution and presented as mean ± SD. Descriptive statistics were computed to describe the demographic and clinical variables.
Receiver operating characteristic curves (ROC) and the area under the ROC curve (AUC) was calculated to analyze the predictive value of the biomarkers. Ninety-five percent confidence intervals (CI), sensitivities and specificities were reported for each time point. Statistical analyses for differences between group means were conducted by unpaired Student t test and two-way ANOVA followed by Bonferroni post hoc test. p-Value <0.05 was considered statistically significant. PRISM 5.0 (GraphPad Software, Inc., San Diego, CA ) and SPSS 13.0 (SPSS, Chicago, IL) softwares were used to analyze the data.
Results
Subject characteristics
During the period of enrollment, 150 septic patients were admitted in ICU at Zhejiang Provincial People's Hospital, China. According to the AKIN criteria, 49 patients (33%) showed at least 50% increase in SCr within 48 h of ICU admission and therefore, were diagnosed as AKI. No significant differences were noted between AKI and non-AKI groups with respect to age, gender and weight. The causes of sepsis include pneumonia, abdominal and biliary infection, UTI, cellulitis and others. No significant differences were found in the APACHE II (Acute Physiology and Chronic Health Evaluation II) score between AKI and non-AKI patients. However, ICU length of stay was longer in septic AKI group, and mortality rate in septic AKI and non-AKI groups were 59% and 20%, respectively (p < 0.001) ().
Table 1. Subjects’ characteristics.
Changes of SCr in septic AKI and non-AKI patients
shows the changes of SCr concentrations for patients who developed AKI and those who did not. SCr did not differ significantly between the two groups (AKI vs. non-AKI: 70.97 ± 25.13 vs. 71.53 ± 23.68) at baseline, which is close to that in healthy control group (69.85 ± 23.44). There were no obvious differences at 1, 3, and 6 h after ICU admission between the two groups. SCr levels in septic AKI patients were markedly increased at 24 h (138.67 ± 36.92 vs. 72.78 ± 19.96, p < 0.05) and 48 h (164.96 ± 49.33 vs. 77.17 ± 21.01, p < 0.05) of ICU admission.
Figure 1. Changes of serum creatinine (SCr) levels at various time points after ICU admission in septic AKI and non-AKI patients. Two-way ANOVA followed by Bonferroni post hoc test was used here. Note: *p < 0.05.
Changes of urinary netrin-1 in septic AKI and non-AKI patients
To explore the possibility of using novel biomarkers for early detection of AKI in septic patients, we first determined netrin-1 levels in urine. Distinct from the elevation of SCr levels which only started to show after 24 h of ICU admission, urinary netrin-1 had increased significantly as early as 1 h after ICU admission (). The values peaked at 3–6 h and remained significantly elevated until 24 h. The peak levels of netrin-1 at 3 h increased approximately 7-fold (19.03 ± 7.25 vs. 2.50 ± 1.46). By contrast, netrin-1 in patients who did not develop AKI remained baseline at various time points, which were similar to that in the healthy control group (2.32 ± 1.09).
Figure 2. Changes of urinary netrin-1 levels at various time points after ICU admission in septic AKI and non-AKI patients. Two-way ANOVA followed by Bonferroni post hoc test was used here. Note: *p < 0.05.
Changes of urinary KIM-1 in septic AKI and non-AKI patients
Besides netrin-1, we simultaneously measured urinary KIM-1 levels. As showed in , urinary KIM-1 started showing elevation at 6 h after ICU admission, which is also much earlier than the changes of SCr concentrations (24 h). KIM-1 values peaked at 24 h and remained increase until 48 h of ICU admission. The peak level of KIM-1 at 24 h displayed approximately 45-fold increase (40.57 ± 16.30 vs. 0.90 ± 0.34). By contrast, KIM-1 in non-AKI patients remained baseline at various time points, which were similar to that in the healthy control group (0.85 ± 0.37).
Figure 3. Changes of human kidney injury molecule type 1 (KIM-1) levels at various time points after ICU admission in septic AKI and non-AKI patients. Two-way ANOVA followed by Bonferroni post hoc test was used here. Note: *p < 0.05.
Diagnostic ability of urinary netrin-1 and KIM-1
The ability of netrin-1 and KIM-1 in diagnosing septic AKI is illustrated in . Immediately after ICU admission, the AUCs for the diagnosis of septic AKI using netrin-1 were 0.279 and 0.299 for KIM-1, respectively. The AUC at different time points increased to 0.858 at 3 h for netrin-1 and 0.916 for KIM-1 at 24 h. The sensitivities and specificities for netrin-1 and KIM-1 are also listed in .
Table 2. Comparative diagnostic performance characteristics of netrin-1 and KIM-1 for the septic AKI using the area under the curve.
SCr, urinary netrin-1 and KIM-1 levels between non-survivors and survivors in septic AKI patients
We further observed the prognostic value of netrin-1 and KIM-1 levels between non-survivors and survivors in septic AKI patients (). As mentioned above, the mortality rate in septic AKI patients is 59% (29 out of 49) in our study. There were no significant differences in SCr and netrin-1 during 48 h of follow-up between non-survivors and survivors. However, we observed significant higher urinary KIM-1 levels at 24 h (46.89 ± 15.27 vs. 35.67 ± 11.34, p = 0.0075) and 48 h (21.53 ± 6.29 vs. 16.66 ± 5.07, p = 0.0061) in non-survivors compared to survivors.
Table 3. SCr, urinary netrin-1 and KIM-1 levels between non-survivors and survivors.
Discussions
In this study, we investigated the changes of two novel biomarkers netrin-1 and KIM-1 in septic AKI patients. We reported that septic patients developing AKI displayed significantly increased urinary netrin-1 levels within the first 1 h, and KIM-1 within the first 6 h of ICU admission, both preceding the rise of SCr by 24 h. Furthermore, persistent elevation of KIM-1 level at 24 h and 48 h may be associated with poor prognosis.
Our results showed 33% of septic patients develop AKI in ICU, which is similar as previous reported 11–60% of septic patients with concomitant AKI.Citation2 Development of AKI during sepsis is associated with poor outcome and independently predicts increased mortality. In clinical practice, AKI is often diagnosed late since rise of SCr and oliguria during sepsis often appear after the window of opportunity for effective therapy already passed.Citation3 Therefore, identification of novel biomarkers facilitating early diagnosis of AKI is desirable. One important criterion for such biomarkers is that they should be kidney-specific and highly sensitive for acute kidney injury. Urine samples may serve as ideal noninvasive sources for biomarkers to diagnose and classify kidney diseases.Citation9
Netrin-1, a multifunctional laminin-related, neuronal guidance protein, is widely expressed in various tissues, including the kidney.Citation19 However, the role of netrin-1 in kidney pathophysiology is still not fully understood. As reported by Grenz et al.Citation20 and Wang et al.Citation21 that netrin-1 may exert renal protective functions. Netrin-1 deficient mouse showed aggravated AKI while over expression of netrin-1 suppressed ischemia-reperfusion induced apoptosis and increased tubular epithelial cell proliferation. Urinary netrin-1 excretion was detected to increase dramatically in various AKI animal models as well as in AKI patients with different causes including ischemic AKI, radio-contrast-induced AKI, sepsis-induced AKI and drug-induced AKI.Citation22 In our study, no changes of netrin-1 level were detected in healthy volunteer urine samples. Urinary netrin-1 levels were significantly elevated as early as 1 h after ICU admission, while SCr levels elevated 24 h later, which indicated that urinary netrin-1 is a useful biomarker for the early diagnosis of AKI in septic patients. Our results were in consistent with previous reportsCitation23 that in patients undergoing cardiopulmonary by-pass (CPB), subjects developing AKI displayed significantly increased urinary netrin-1 levels within the first 2 h of the initiation of CPB. Early detection of AKI will provide a window of opportunity to implement potential therapeutic interventions before any detectable changes in SCr would occur. Therefore, researchers have proposed that the utility of netrin-1 may be of particular importance due to early detection of AKI and allow for appropriate and timely interventions which may significantly decrease morbidity and mortality related to AKI in the future.Citation24
Given the multifactorial etiologies of AKI,Citation25 any single biomarker is unlikely to be sensitive enough to reflect the multiple pathways such as ischemic and inflammatory involved in the pathogenesis of AKI. Simultaneous examination of other biomarkers may provide additional information. Therefore, in addition to netrin-1, we simultaneously examined urinary KIM-1 levels in our patient populations. KIM-1 is transmembrane glycoprotein. KIM-1 gene or protein expression is undetectable in the healthy kidney or urine. We observed KIM-1 levels elevated as early as 6 h and maintained high level up to 48 h of ICU admission in septic patients, which is similar to previous studies that KIM-1 peaked within 1 d after cardiac surgery and maintained 2 d later in AKI patients.Citation12 In our study, we further compared the urinary SCr, netrin-1 and KIM-1 levels in septic AKI patients between non-survivors and survivors. We reported that KIM-1 levels were significantly higher at 24 and 48 h of ICU admission in non-survival AKI patients, which indicated the importance prognosis of KIM-1 in AKI patients. Consistently, Liangos et al.Citation26 also reported that high urinary KIM-1 expression was associated with adverse clinical outcome (death and need for dialysis) in patients with AKI.
The current study has several limitations. First, it is a single center pilot study comparing non-AKI and AKI septic patients. The number of subjects is relatively small, and thus the β-error is relatively high. Therefore, these results will need to be validated in a larger population. Second, our study only enrolled patients with normal kidney function, and it will be important to confirm our findings in documented high-risk settings, such as patients with preexisting kidney dysfunction, diabetes mellitus, and concomitant nephrotoxic drug use. Third, we only measured urinary netrin-1 and KIM-1 level. As mentioned above, all biomarkers have individual strengths and weaknesses. Combination of more biomarkers to develop a biomarker panel may represent an optimal way to detect AKI more efficiently and accurately.
In summary, our results suggest that both netrin-1 and KIM-1 are clinically useful as early biomarkers for the diagnosis of septic AKI. In addition, persistent elevation of urinary KIM-1 level may be associated with poor prognosis. As AKI remains a complex disease, the utility of combination with netrin-1, KIM-1, and other biomarkers reflecting different biologic pathways in the early diagnosis and determining the prognosis remains an important area for further study.
Declaration of interest
This work was supported by the grants from Science and Technology Department of Zhejiang Province, China (2011R50018-14) and Zhejiang Provincial Health Department, China (2012KYB021 and 2013RCA004).
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Acknowledgements
The authors thank Dr. Ziqiang Zhu (National Institutes of Health, USA) for the critical review of the manuscript.
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