J Nephrol DOI 10.1007/s40620-014-0163-z
ORIGINAL ARTICLE
Diagnostic and short-term prognostic utility of plasma proenkephalin (pro-ENK) for acute kidney injury in patients admitted with sepsis in the emergency department Rossella Marino • Joachim Struck • Oliver Hartmann • Alan S. Maisel • Miriam Rehfeldt • Laura Magrini • Olle Melander • Andreas Bergmann • Salvatore Di Somma
Received: 15 August 2014 / Accepted: 18 November 2014 Ó Italian Society of Nephrology 2014
Abstract Background Acute kidney injury (AKI) aggravates the prognosis of patients with sepsis. Reliable biomarkers for early detection of AKI in this setting are lacking. Enkephalins influence kidney function, and may have a role in AKI from sepsis. We utilized a novel immunoassay for plasma proenkephalin (pro-ENK), a stable surrogate marker for endogenous enkephalins, in patients hospitalized with sepsis, in order to assess its clinical utility. Methods In an observational retrospective study we enrolled 101 consecutive patients admitted to the emergency department (ED) with suspected sepsis. Plasma levels of pro-ENK and neutrophil gelatinase-associated lipocalin (NGAL) were evaluated at ED arrival for their association with presence and severity of AKI and 7-day mortality. Results pro-ENK was inversely correlated to creatinine clearance (r = -0.72) and increased with severity of AKI R. Marino L. Magrini S. Di Somma (&) Department of Medical Sciences and Translational Medicine, University of Rome Sapienza, Emergency Department Sant’Andrea Hospital, Via di Grottarossa 1035/1039, 00189 Rome, Italy e-mail: [email protected]; [email protected] J. Struck O. Hartmann M. Rehfeldt A. Bergmann Sphingotec GmbH, Hennigsdorf, Germany e-mail: [email protected] A. S. Maisel Veterans Affairs San Diego Healthcare System, San Diego, CA, USA O. Melander Department of Clinical Sciences, Lund University, Malmo¨, Sweden
as determined by RIFLE (risk, injury, failure, loss of function, end-stage renal disease) stages (p \ 0.0001; proENK median [interquartile range, IQR]) pmol/l: no AKI: 71 [41–97]; risk: 72 [51–120]; injury: 200 [104–259]; failure: 230 [104–670]; loss of function: 947 [273–811]. The majority of septic patients without AKI or at risk had pro-ENK concentrations within the normal range. While NGAL was similarly associated with AKI severity, it was strongly elevated already in septic patients without AKI. pro-ENK added predictive information to NGAL for detecting kidney dysfunction (added v2 10.0, p = 0.0016). Admission pro-ENK outperformed creatinine clearance in predicting 7-day mortality (pro-ENK: v2 13.4, p \ 0.001, area under curve, AUC 0.69; creatinine clearance: v2 4, p = 0.045, AUC: 0.61), and serial measurement improved prediction. Conclusions Use of pro-ENK in septic patients can detect the presence and severity of AKI. Moreover, pro-ENK is highly predictive of short-term mortality and could enable early identification of patients at risk of death. Keywords Enkephalin Acute kidney injury Sepsis RIFLE NGAL Mortality
Introduction Acute kidney injury (AKI) is a common complication among hospitalized patients. Its incidence has been increasing in recent years [1–3] and currently accounts for 20–23 % of all admitted patients. Moreover, the incidence of AKI is projected to further increase in developing countries as the population ages [1, 4]. It has been well demonstrated that AKI is associated with high mortality [5] and morbidity rates [5–7]. In septic patients AKI
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complicates approximately 5–7 % of hospital admissions and up to 30 % of admissions to intensive care units (ICU). AKI occurs in 19 % of patients with moderate sepsis, 23 % with severe sepsis and 51 % with septic shock [8]. Moreover, septic AKI is associated with 70 % mortality compared to 45 % mortality in patients with non-septic AKI [9]. Upon presentation to the hospital, a rapid diagnosis of AKI is difficult. Kidney damage may occur before, during, or after loss of kidney function is clinically manifest and the tools needed for differentiation are not yet available [10]. This may lead to a dramatic underestimation of the incidence of AKI [11]. According to the international guidelines Kidney Disease: Improving Global Outcomes (KDIGO), the diagnosis of AKI requires serial assessment of laboratory tests over a period of several days and is based mainly on serum creatinine (sCr) [12]. This need for repeated sCr evaluations and monitoring of urinary output after admission could result in the delay of appropriate therapy. Thus, the use of biomarkers to detect acute kidney damage could be of great utility in the emergency department (ED) in order to distinguish AKI from other pathological and chronic kidney conditions [13]. Furthermore, these biomarkers could contribute to the diagnosis of AKI by identifying a subgroup with ‘subclinical AKI’ where injury could be present even in the absence of an increased sCr [14, 15]. Among damage biomarkers of AKI, a large body of evidence for the detection of AKI exists for both urine and plasma neutrophil gelatinase-associated lipocalin (NGAL) [16, 17]. However, NGAL has limitations, especially in critically ill patients, where inflammation, even in the absence of AKI, can lead to a rise in NGAL levels [16, 18]. Enkephalins are well known as small endogenous opioid peptides. However, their involvement in regulating renal function has not been thoroughly investigated. The corresponding gene PENK is expressed in the central nervous system, but also in multiple non-neuronal tissues including the kidney, heart and muscle [19]. Proteolytic processing of the primary translational product of PENK, preproenkephalin A (267 amino acids), involves first removal of the signal sequence giving rise to a propeptide (243 aa), which is further cleaved leading to several small bioactive peptides, namely four copies of methionine-enkephalin (5 aa) and one copy each of leucine-enkephalin (5 aa), methionine-enkephalin-Arg-Gly-Leu (8 aa), methionine-enkephalin-Arg-Phe (7 aa) and enkelytin. Another peptide generated in this process is the larger and stable pro-ENK (amino acids 119–159 of proenkephalin A; 41 aa). Clearance mechanisms of these peptides are largely unknown. However, due to their low molecular weight all above mentioned cleaved peptides must be assumed to be freely filterable through the glomerulus.
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While methods for measuring plasma met-enkephalin have been reported, due to the small size and instability of met-enkephalin and the complex assay procedure the results obtained with these methods must be considered with caution. Using such assay, enkephalin levels have been described to be elevated in chronic renal failure [20] and in uremic patients under dialysis [21]. pro-ENK has been established as a reliable surrogate marker for enkephalins [22]. It is stable for at least 48 h in plasma, whereas met- and leu-enkephalin, possess a halflife time in human plasma of less than 15 min [22]. The assay for pro-ENK exhibits ideal accuracy characteristics, indicating that pro-ENK is freely circulating [22]. Recently, plasma pro-ENK was found to be associated with adverse outcome in acute myocardial infarction patients, and a strong association with renal dysfunction was noted [23]. In the present study we assessed the association of plasma pro-ENK in sepsis patients with presence and severity of AKI as well as with short-term mortality.
Materials and methods Study population We enrolled 101 consecutive patients arriving in the ED of our university hospital (Sant’Andrea Hospital, University La Sapienza, Rome, Italy) with the diagnosis of sepsis, severe sepsis or septic shock between December 2011 and April 2012. Sepsis was diagnosed according to the international guidelines of the Surviving Sepsis Campaign [24]. Exclusion criteria were age \18 years and the patient’s inability to give informed consent. All patients gave the informed written consent according to the declaration of Helsinki. The Ethical committee of Sant’Andrea Hospital approved the study. Upon arrival in the ED, each patient underwent recording of anamnestic data, physical examination, electrocardiogram, laboratory tests and radiologic exams. Parameters including temperature, arterial blood pressure, heart rate and respiratory rate were recorded daily, as well as arterial blood gases, daily monitoring of fluid balance and creatinine clearance. RIFLE (risk, injury, failure, loss of function, end-stage renal disease) criteria [25] were applied to determine the acute kidney status of the patients. The patients’ survival status was determined over 7 days post admission. The Acute physiology and chronic health evaluation II (APACHE II) score was calculated as a measure of organ damage severity. Blood samples for measurement of pro-ENK and NGAL were obtained from a peripheral vein or central venous line, collected in ethylenediamine tetraacetic acid (EDTA)
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tubes, centrifuged at 4,000g for 15 min, and plasma was stored at -40 °C until batch measurement. NGAL assay Plasma NGAL levels were measured using an NGAL enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (Kit 036; BioPorto Diagnostics, Gentofte, Denmark). pro-ENK assay An assay for stable pro-ENK (amino acids 119–159 of proenkephalin A) has been previously reported [22] and was modified as recently described [23]. In brief, two mouse monoclonal anti-pro-ENK antibodies were developed by immunization with pro-ENK peptide (amino acids 119–159 of proenkephalin A). One antibody (2 lg) was used to coat polystyrene tubes. The other antibody labelled with methyl-acridinium ester served as the detector antibody. Standards (pro-ENK peptide; amino acids 119–159 of proenkephalin A) and samples (50 ll) were incubated in tubes with the detector antibody (150 ll). After equilibration, tubes were washed and bound chemiluminescence was detected with a luminometer LB952T/16 (Berthold, Bad Wildbad, Germany). The lower detection limit of the assay was 5.5 pmol/l. Intra- and interassay coefficients of variation were, respectively, 6.4 and 9.5 % at 50 pmol/l, and 4.0 and 6.5 % at 150 pmol/l. The normal range for plasma pro-ENK was determined in a general population cohort (Malmo¨ Diet and Cancer Study [26]; n = 4,643): The mean ± standard deviation (SD) was 46.6 ± 14.1 pmol/l, the median [range] was 45 [9–518] pmol/l. The 99th percentile of the normal distribution was 80 pmol/l. Statistical analysis Values were expressed as medians and interquartile ranges (IQR), or counts and percentages, as appropriate. Group comparisons of continuous variables were performed using the non-parametric Kruskal–Wallis test. Biomarker data were log-transformed. Spearman’s correlation coefficient was used to determine the correlation between continuous variables. Multivariate linear regression analysis was performed for pro-ENK (log-transformed) including the variables: age, gender, mean arterial pressure (MAP), concomitant diseases, creatinine clearance, procalcitonin (PCT) and APACHE II score. Logistic regression models were used to evaluate and compare the predictive performance of the biomarkers for predicting kidney disease. Cox proportional hazards regression models were used to analyze the effect of risk
factors on survival. The assumptions of proportional hazard were tested for all variables. For both regression models, the predictive value of each model was assessed by the model likelihood ratio Chi square statistic, and the concordance index (c index or area under the curve, AUC) was given as an effect measure. To demonstrate independence and to test for differences in the predictive value of the biomarkers under investigation, we used the likelihood ratio Chi square test for nested models to assess whether one biomarker adds predictive value to a clinical model with another biomarker and vice versa. Survival curves plotted by the Kaplan–Meier method were used for illustrative purposes. All statistical tests were 2-tailed and a two-sided p value of 0.05 was considered as significant. The statistical analyses were performed using R version 2.5.1 and Statistical Package for the Social Sciences (IBM SPSS) version 22 (SPSS Inc., Chicago, IL, USA).
Results pro-ENK and kidney disease Patient characteristics are presented in Table 1. pro-ENK was elevated in patients with severe sepsis or shock compared to patients with sepsis (median 205 [87–485] vs. 73 [47–123] pmol/l, p \ 0.001); 47 % of all patients were above the upper 99th percentile of the normal range (80 pmol/l). pro-ENK levels were inversely correlated to creatinine clearance (r = -0.72, p \ 0.0001) (Fig. 1). At multivariate linear regression analysis, creatinine clearance was the strongest determinant for pro-ENK (partial r = 0.52, followed by r = 0.16 for concomitant cardiovascular disease and r = 0.13 for MAP; all other variables including age and gender were non-significant). Overall, 49 (49 %) patients showed signs of AKI. The concentration of pro-ENK was associated to severity of acute renal dysfunction: pro-ENK increased with severity of RIFLE stages (global p \ 0.0001) (Fig. 2): median [IQR] concentration (pmol/l) of pro-ENK for patients without AKI (n = 52) was 71 [41–97], comparable to that for patients at risk for AKI (n = 15): 72 [51–120]. Patients with kidney injury (n = 8) showed a median pro-ENK of 200 [104–259] pmol/l, those with failure (n = 24) had a median of 230 [104–670], and those with loss of kidney function 947 [273–811] pmol/l. Post-hoc comparisons demonstrated significant differences between stages of failure and no AKI, as well as between failure and patients at risk (both p \ 0.05). For NGAL, the separation between RIFLE classes was comparable to that for pro-ENK (Fig. 2): median [IQR] concentration was 0.5 [0.3–0.8], 0.4 [0.3–0.8], 0.8
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J Nephrol Table 1 Patient characteristics Variable
All patients (n = 101)
Demographics Gender, male (%)
61 (60 %)
Age (years), median [IQR]
78 [72–83]
Concomitant diseases Cardiovascular, n (%)
26 (26 %)
Hypertensive, n (%)
47 (47 %)
Diabetes, n (%) Hepatic cancer, n (%)
35 (35 %) 1 (1 %)
Lung cancer, n (%)
8 (8 %)
Gastric cancer, n (%)
2 (2 %)
Colon cancer, n (%)
2 (2 %)
Antihypertensive drugs ARBs, n (%)
18 (18 %)
ACE inhibitors, n (%)
29 (29 %)
Routine laboratory tests Creatinine clearance (ml/min), median [IQR]
48 [23–77]
PCT (ng/ml), median [IQR]
2.8 [0.6–10.7]
Blood cultures Performed, n (%)
35 (35 %)
Positive, n (% of performed)
25 (71 %)
Gram- bacteria, n (% of all positives)
11 (44 %)
Gram? bacteria, n (% of all positives) Site of infection
14 (56 %)
Skin, n (%)
2 (2 %)
Lung, n (%)
46 (46 %)
Abdomen, n (%)
10 (10 %)
Urinary tract, n (%)
33 (33 %)
Other APACHE II score (points), median [IQR]
16 [13–21]
Final diagnosis, severe sepsis or shock
29 (29 %)
Days hospitalized, median [IQR]
5 [2–7]
Deaths within 7 days after admission
28 (28 %)
IQR interquartile range, ARBs angiotensin receptor blockers, ACE angiotensin converting enzyme, PCT procalcitonin, APACHE II acute physiology and chronic health evaluation II
[0.6–1.4], 1.3 [0.6–3.2] and 2.7 [2.5–2.8] lg/ml for patients without AKI, at risk, with kidney injury, failure and loss, respectively (global p \ 0.0001). Post-hoc comparisons showed significant differences between the stage of failure and no AKI, as well as between failure and patients at risk (both, p \ 0.05). The majority of patients without AKI or at risk, i.e. 40 out of 67 (60 %) for both groups, had pro-ENK concentrations below the 99th percentile of the normal range (80 pmol/l), despite septic inflammation. For patients with AKI (injury, failure or loss), 27 out of 34 patients (79 %) were above the normal range. In contrast, for NGAL, all
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Fig. 1 Correlation of creatinine clearance and pro-ENK. Creatinine clearance and plasma pro-ENK concentrations were determined for all patients on admission. The Spearman correlation coefficient was r = -0.72, p \ 0.0001. pro-ENK proenkephalin
but three patients (two without AKI and one at risk) had concentrations above the upper normal limit (0.1 lg/ml); the mean NGAL concentration in plasma from healthy donors has been reported as 0.063 lg/ml (range 0.037–0.106) with the NGAL Rapid ELISA Kit (Kit 036; BioPorto Diagnostics, Hellerup, Denmark) (for more detail, a brochure can be downloaded at http://www.bio porto.com/products/bioporto_diagnostics/ngal_elisa_kits/ ngal_rapid_elisa_kit_ruo) [27]. The diagnostic accuracy of pro-ENK for detecting kidney disease within the investigated population (no AKI or at risk vs. all other RIFLE categories) was comparable to that of NGAL (pro-ENK: v2 34.9, p \ 0.001, AUC 0.815 vs. NGAL: v2 31.3, p \ 0.001, AUC 0.800). The predictive information of pro-ENK was independent from that of NGAL: the combination of both biomarkers provided additional predictive information for detecting kidney dysfunction (added v2 10.0, p = 0.0016 for the additive effect of pro-ENK to NGAL). pro-ENK and short-term outcome Within 7 days post admission, 28 (28 %) patients died. Both admission pro-ENK and NGAL were elevated in those early deaths, while PCT and creatinine clearance was not (Table 2). Despite the high correlation between proENK and creatinine clearance, pro-ENK was a better predictor of mortality within 7 days (pro-ENK: v2 13.4, p \ 0.001, AUC 0.69 vs. creatinine clearance: v2 4, p = 0.045, AUC 0.61). pro-ENK was independent from creatinine clearance and provided superior prognostic information (added v2 11.2, p \ 0.001). NGAL performed
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Fig. 2 Concentration of pro-ENK (a) and NGAL (b) in patients according to the RIFLE criteria. Plasma levels of both markers were determined for all patients on admission and are shown here for patient subgroups according to presence and severity of AKI using the RIFLE criteria (box 25th–75th percentile, whiskers 10th–90th
percentile). The upper normal limits are 80 pmol/l (pro-ENK) and 0.1 lg/ml (NGAL). pro-ENK proenkephalin, NGAL neutrophil gelatinase-associated lipocalin, RIFLE risk, injury, failure, loss of function, end-stage renal disease, AKI acute kidney injury
Table 2 Selected variables by all-cause mortality within 7 days after admission Variable median [IQR]
All (n = 101)
No. of deaths within 7 days (n = 28)
7-day survivors (n = 73)
PCT (ng/ml) pro-ENK (pmol/l)
p value
2.8 [0.6–10.7]
4.1 [1.3–13.0]
2.2 [0.6–9.0]
0.102
87 [50–205]
209 [77–499]
75 [47–124]
\0.001 \0.001
NGAL (lg/ml)
0.6 [0.4–1.2]
1.3 [0.5–2.1]
0.6 [0.3–0.8]
Creatinine clearance (ml/min)
48 [23–77]
33 [15–69]
56 [29–81]
0.071
APACHE II score (points)
16 [13–21]
23 [18–27]
14 [12–18]
\0.001
Values are median and inter-quartile range (IQR); p value from non-parametric Kruskal–Wallis test PCT procalcitonin, pro-ENK proenkephalin, NGAL neutrophil gelatinase-associated lipocalin, APACHE II acute physiology and chronic health evaluation II
comparably to pro-ENK (v2 14.2, p \ 0.001, AUC 0.69), while the APACHE II score was by far the strongest predictor in our population (v2 21.3, p \ 0.001, AUC 0.75). PCT was not predictive of mortality within 7 days (v2 2.1, p = 0.144, AUC 0.60). Serial measurement of pro-ENK further improved its predictive performance: the c index (AUC) increased from 0.69 for pro-ENK measured at baseline to 0.90 for the 49 patients still alive and hospitalized at day 4 post admission. To illustrate the dynamic and added value of serial proENK measurement, we applied a cutoff at [100 pmol/l to define patients at risk. This cut-point was at the 75th percentile of patients without AKI, and is slightly above the upper normal range. It also appears to offer the best distinction between groups. At baseline, 43 patients were at risk. Their 7-day survival rate was 56 % (Fig. 3, left). Of those, 5 (12 %) dropped below our risk cutoff as early as one day post admission, with an observed survival rate of 100 % (Fig. 3, right).
Discussion In the present study we demonstrate that plasma pro-ENK in sepsis patients is independently associated with the presence and severity of AKI as well as with short-term mortality. As opposed to NGAL, plasma pro-ENK in septic patients without kidney dysfunction was essentially in the normal range. Approximately 2 million people worldwide die each year due to AKI, and hospitalized patients with AKI have a post-discharge mortality rate twice as high as those without AKI [5]. Moreover, the increased risk of death rises incrementally with the severity of AKI [11]. Recent data indicate that even transient perturbations in kidney function in hospitalized patients increase the risk of death [28]. Therefore it is important to diagnose AKI early, and to apply prompt and specific treatment strategies in order to reduce the risk. As a consequence, there has been a growing interest in the discovery of new biomarkers for
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Fig. 3 Illustration of added value of serial measurement of pro-ENK using a cutoff at 100 pmol/l. On the left, Kaplan–Meier plot showing survival rate of patients with plasma pro-ENK concentration \100 vs. [100 pmol/l on admission. On the right, Kaplan–Meier plot showing
survival rate of a subgroup of patients with plasma pro-ENK concentration on admission \100 pmol/l and which, on the next day, remained below vs. rose above 100 pmol/l
early diagnosis of AKI. Recently pro-ENK, a stable surrogate marker for endogenous enkephalins, has emerged as a novel candidate biomarker strongly associated with kidney dysfunction in acute myocardial infarction [22, 23]. Endogenous opioids mediate some of the circulatory and renal excretory responses to plasma volume expansion, and the delta opioid receptor evokes changes in renal excretory function [29]. Opioids have a strong influence on renal function. One mechanism is the accumulation of oxidative stress factors in the kidney during pathological processes like sepsis, which in turn increase pro-ENK expression [30]. During sepsis, an internal redistribution of blood flow occurs to the cortex and away from the medulla, which leads to a massive collapse of the renal and tubular cells, leading to the loss of kidney function [31]. Another possible mechanism is the interplay of opioids and pain proprioception with pathological processes that take place in the kidney [32]. These integrated neural and hormonal mechanisms within the central nervous system and kidneys may culminate in alterations in urine output and urine sodium excretion [33]. It is well known that clearance of creatinine has limitations in detecting early changes in renal function, including its dependency on factors independent of AKI, including muscle mass, liver function, fever, age, sex, race, protein intake and hydration status [25, 34]. Our data suggest that pro-ENK is more useful as an early biomarker of AKI than sCr upon entrance to the ED. Analysis in a general population cohort has revealed measures of kidney function as by far the strongest determinants of plasma
pro-ENK (Melander et al., unpublished). Levels of proENK were essentially not influenced by underlying comorbidities such as diabetes. Even though the present study includes too few patients to draw definite conclusions, we could not find any indication of comorbidities impairing pro-ENK’s capacity to diagnose AKI. We compared pro-ENK to NGAL on the basis of RIFLE stages and demonstrated that for patients without AKI proENK levels were essentially within the normal range, despite the presence of inflammation, while pro-ENK concentration was increased in patients with AKI. On the contrary, NGAL was elevated above the normal range for almost all patients without AKI, confirming that NGAL is influenced by inflammation. This is supportive of our recent observations that plasma NGAL could be higher in the occurrence of AKI in the ED in patients with sepsis compared to those free from infections [17]. Thus, we conclude that pro-ENK is more specific than NGAL for assessing AKI in septic patients. In our study, we did not collect urine samples, so that a comparison of plasma pro-ENK and NGAL with other potential AKI biomarkers to be measured in urine was not possible. Such comparison should be considered for further studies in a larger cohort of patients. Since AKI aggravates the prognosis of sepsis patients, it is notable that baseline pro-ENK is superior to creatinine clearance and PCT in predicting death. In addition, proENK proved a dynamic marker: the 24-h reassessment significantly improved risk stratification. In conclusion, in patients admitted for sepsis, plasma pro-ENK is an early biomarker useful for promptly
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detecting AKI and, in contrast to NGAL, it is not influenced by inflammation. Thus, the use of pro-ENK in septic patients could allow physicians to immediately assess the presence and severity of AKI. This would enable adequate treatment to be started promptly to prevent ongoing kidney failure, or the search for and removal of any offending agent. Moreover, pro-ENK is highly predictive of shortterm mortality and could enable an early identification of patients at risk of death. Acknowledgments We would like to thank Janine Sachse and Julia Weber for their excellent technical assistance. Conflict of interest University La Sapienza Rome, Sant’Andrea Hospital, received a research grant from Sphingotec GmbH for this study. Joachim Struck, Oliver Hartmann, Miriam Rehfeldt and Andreas Bergmann are employed by Sphingotec GmbH, and Andreas Bergmann holds shares in Sphingotec GmbH. Sphingotec GmbH holds patent rights related to the pro-ENK assay. Alan S. Maisel is a consultant for Sphingotec GmbH.
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ORIGINAL ARTICLE
Diagnostic and short-term prognostic utility of plasma proenkephalin (pro-ENK) for acute kidney injury in patients admitted with sepsis in the emergency department Rossella Marino • Joachim Struck • Oliver Hartmann • Alan S. Maisel • Miriam Rehfeldt • Laura Magrini • Olle Melander • Andreas Bergmann • Salvatore Di Somma
Received: 15 August 2014 / Accepted: 18 November 2014 Ó Italian Society of Nephrology 2014
Abstract Background Acute kidney injury (AKI) aggravates the prognosis of patients with sepsis. Reliable biomarkers for early detection of AKI in this setting are lacking. Enkephalins influence kidney function, and may have a role in AKI from sepsis. We utilized a novel immunoassay for plasma proenkephalin (pro-ENK), a stable surrogate marker for endogenous enkephalins, in patients hospitalized with sepsis, in order to assess its clinical utility. Methods In an observational retrospective study we enrolled 101 consecutive patients admitted to the emergency department (ED) with suspected sepsis. Plasma levels of pro-ENK and neutrophil gelatinase-associated lipocalin (NGAL) were evaluated at ED arrival for their association with presence and severity of AKI and 7-day mortality. Results pro-ENK was inversely correlated to creatinine clearance (r = -0.72) and increased with severity of AKI R. Marino L. Magrini S. Di Somma (&) Department of Medical Sciences and Translational Medicine, University of Rome Sapienza, Emergency Department Sant’Andrea Hospital, Via di Grottarossa 1035/1039, 00189 Rome, Italy e-mail: [email protected]; [email protected] J. Struck O. Hartmann M. Rehfeldt A. Bergmann Sphingotec GmbH, Hennigsdorf, Germany e-mail: [email protected] A. S. Maisel Veterans Affairs San Diego Healthcare System, San Diego, CA, USA O. Melander Department of Clinical Sciences, Lund University, Malmo¨, Sweden
as determined by RIFLE (risk, injury, failure, loss of function, end-stage renal disease) stages (p \ 0.0001; proENK median [interquartile range, IQR]) pmol/l: no AKI: 71 [41–97]; risk: 72 [51–120]; injury: 200 [104–259]; failure: 230 [104–670]; loss of function: 947 [273–811]. The majority of septic patients without AKI or at risk had pro-ENK concentrations within the normal range. While NGAL was similarly associated with AKI severity, it was strongly elevated already in septic patients without AKI. pro-ENK added predictive information to NGAL for detecting kidney dysfunction (added v2 10.0, p = 0.0016). Admission pro-ENK outperformed creatinine clearance in predicting 7-day mortality (pro-ENK: v2 13.4, p \ 0.001, area under curve, AUC 0.69; creatinine clearance: v2 4, p = 0.045, AUC: 0.61), and serial measurement improved prediction. Conclusions Use of pro-ENK in septic patients can detect the presence and severity of AKI. Moreover, pro-ENK is highly predictive of short-term mortality and could enable early identification of patients at risk of death. Keywords Enkephalin Acute kidney injury Sepsis RIFLE NGAL Mortality
Introduction Acute kidney injury (AKI) is a common complication among hospitalized patients. Its incidence has been increasing in recent years [1–3] and currently accounts for 20–23 % of all admitted patients. Moreover, the incidence of AKI is projected to further increase in developing countries as the population ages [1, 4]. It has been well demonstrated that AKI is associated with high mortality [5] and morbidity rates [5–7]. In septic patients AKI
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complicates approximately 5–7 % of hospital admissions and up to 30 % of admissions to intensive care units (ICU). AKI occurs in 19 % of patients with moderate sepsis, 23 % with severe sepsis and 51 % with septic shock [8]. Moreover, septic AKI is associated with 70 % mortality compared to 45 % mortality in patients with non-septic AKI [9]. Upon presentation to the hospital, a rapid diagnosis of AKI is difficult. Kidney damage may occur before, during, or after loss of kidney function is clinically manifest and the tools needed for differentiation are not yet available [10]. This may lead to a dramatic underestimation of the incidence of AKI [11]. According to the international guidelines Kidney Disease: Improving Global Outcomes (KDIGO), the diagnosis of AKI requires serial assessment of laboratory tests over a period of several days and is based mainly on serum creatinine (sCr) [12]. This need for repeated sCr evaluations and monitoring of urinary output after admission could result in the delay of appropriate therapy. Thus, the use of biomarkers to detect acute kidney damage could be of great utility in the emergency department (ED) in order to distinguish AKI from other pathological and chronic kidney conditions [13]. Furthermore, these biomarkers could contribute to the diagnosis of AKI by identifying a subgroup with ‘subclinical AKI’ where injury could be present even in the absence of an increased sCr [14, 15]. Among damage biomarkers of AKI, a large body of evidence for the detection of AKI exists for both urine and plasma neutrophil gelatinase-associated lipocalin (NGAL) [16, 17]. However, NGAL has limitations, especially in critically ill patients, where inflammation, even in the absence of AKI, can lead to a rise in NGAL levels [16, 18]. Enkephalins are well known as small endogenous opioid peptides. However, their involvement in regulating renal function has not been thoroughly investigated. The corresponding gene PENK is expressed in the central nervous system, but also in multiple non-neuronal tissues including the kidney, heart and muscle [19]. Proteolytic processing of the primary translational product of PENK, preproenkephalin A (267 amino acids), involves first removal of the signal sequence giving rise to a propeptide (243 aa), which is further cleaved leading to several small bioactive peptides, namely four copies of methionine-enkephalin (5 aa) and one copy each of leucine-enkephalin (5 aa), methionine-enkephalin-Arg-Gly-Leu (8 aa), methionine-enkephalin-Arg-Phe (7 aa) and enkelytin. Another peptide generated in this process is the larger and stable pro-ENK (amino acids 119–159 of proenkephalin A; 41 aa). Clearance mechanisms of these peptides are largely unknown. However, due to their low molecular weight all above mentioned cleaved peptides must be assumed to be freely filterable through the glomerulus.
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While methods for measuring plasma met-enkephalin have been reported, due to the small size and instability of met-enkephalin and the complex assay procedure the results obtained with these methods must be considered with caution. Using such assay, enkephalin levels have been described to be elevated in chronic renal failure [20] and in uremic patients under dialysis [21]. pro-ENK has been established as a reliable surrogate marker for enkephalins [22]. It is stable for at least 48 h in plasma, whereas met- and leu-enkephalin, possess a halflife time in human plasma of less than 15 min [22]. The assay for pro-ENK exhibits ideal accuracy characteristics, indicating that pro-ENK is freely circulating [22]. Recently, plasma pro-ENK was found to be associated with adverse outcome in acute myocardial infarction patients, and a strong association with renal dysfunction was noted [23]. In the present study we assessed the association of plasma pro-ENK in sepsis patients with presence and severity of AKI as well as with short-term mortality.
Materials and methods Study population We enrolled 101 consecutive patients arriving in the ED of our university hospital (Sant’Andrea Hospital, University La Sapienza, Rome, Italy) with the diagnosis of sepsis, severe sepsis or septic shock between December 2011 and April 2012. Sepsis was diagnosed according to the international guidelines of the Surviving Sepsis Campaign [24]. Exclusion criteria were age \18 years and the patient’s inability to give informed consent. All patients gave the informed written consent according to the declaration of Helsinki. The Ethical committee of Sant’Andrea Hospital approved the study. Upon arrival in the ED, each patient underwent recording of anamnestic data, physical examination, electrocardiogram, laboratory tests and radiologic exams. Parameters including temperature, arterial blood pressure, heart rate and respiratory rate were recorded daily, as well as arterial blood gases, daily monitoring of fluid balance and creatinine clearance. RIFLE (risk, injury, failure, loss of function, end-stage renal disease) criteria [25] were applied to determine the acute kidney status of the patients. The patients’ survival status was determined over 7 days post admission. The Acute physiology and chronic health evaluation II (APACHE II) score was calculated as a measure of organ damage severity. Blood samples for measurement of pro-ENK and NGAL were obtained from a peripheral vein or central venous line, collected in ethylenediamine tetraacetic acid (EDTA)
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tubes, centrifuged at 4,000g for 15 min, and plasma was stored at -40 °C until batch measurement. NGAL assay Plasma NGAL levels were measured using an NGAL enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (Kit 036; BioPorto Diagnostics, Gentofte, Denmark). pro-ENK assay An assay for stable pro-ENK (amino acids 119–159 of proenkephalin A) has been previously reported [22] and was modified as recently described [23]. In brief, two mouse monoclonal anti-pro-ENK antibodies were developed by immunization with pro-ENK peptide (amino acids 119–159 of proenkephalin A). One antibody (2 lg) was used to coat polystyrene tubes. The other antibody labelled with methyl-acridinium ester served as the detector antibody. Standards (pro-ENK peptide; amino acids 119–159 of proenkephalin A) and samples (50 ll) were incubated in tubes with the detector antibody (150 ll). After equilibration, tubes were washed and bound chemiluminescence was detected with a luminometer LB952T/16 (Berthold, Bad Wildbad, Germany). The lower detection limit of the assay was 5.5 pmol/l. Intra- and interassay coefficients of variation were, respectively, 6.4 and 9.5 % at 50 pmol/l, and 4.0 and 6.5 % at 150 pmol/l. The normal range for plasma pro-ENK was determined in a general population cohort (Malmo¨ Diet and Cancer Study [26]; n = 4,643): The mean ± standard deviation (SD) was 46.6 ± 14.1 pmol/l, the median [range] was 45 [9–518] pmol/l. The 99th percentile of the normal distribution was 80 pmol/l. Statistical analysis Values were expressed as medians and interquartile ranges (IQR), or counts and percentages, as appropriate. Group comparisons of continuous variables were performed using the non-parametric Kruskal–Wallis test. Biomarker data were log-transformed. Spearman’s correlation coefficient was used to determine the correlation between continuous variables. Multivariate linear regression analysis was performed for pro-ENK (log-transformed) including the variables: age, gender, mean arterial pressure (MAP), concomitant diseases, creatinine clearance, procalcitonin (PCT) and APACHE II score. Logistic regression models were used to evaluate and compare the predictive performance of the biomarkers for predicting kidney disease. Cox proportional hazards regression models were used to analyze the effect of risk
factors on survival. The assumptions of proportional hazard were tested for all variables. For both regression models, the predictive value of each model was assessed by the model likelihood ratio Chi square statistic, and the concordance index (c index or area under the curve, AUC) was given as an effect measure. To demonstrate independence and to test for differences in the predictive value of the biomarkers under investigation, we used the likelihood ratio Chi square test for nested models to assess whether one biomarker adds predictive value to a clinical model with another biomarker and vice versa. Survival curves plotted by the Kaplan–Meier method were used for illustrative purposes. All statistical tests were 2-tailed and a two-sided p value of 0.05 was considered as significant. The statistical analyses were performed using R version 2.5.1 and Statistical Package for the Social Sciences (IBM SPSS) version 22 (SPSS Inc., Chicago, IL, USA).
Results pro-ENK and kidney disease Patient characteristics are presented in Table 1. pro-ENK was elevated in patients with severe sepsis or shock compared to patients with sepsis (median 205 [87–485] vs. 73 [47–123] pmol/l, p \ 0.001); 47 % of all patients were above the upper 99th percentile of the normal range (80 pmol/l). pro-ENK levels were inversely correlated to creatinine clearance (r = -0.72, p \ 0.0001) (Fig. 1). At multivariate linear regression analysis, creatinine clearance was the strongest determinant for pro-ENK (partial r = 0.52, followed by r = 0.16 for concomitant cardiovascular disease and r = 0.13 for MAP; all other variables including age and gender were non-significant). Overall, 49 (49 %) patients showed signs of AKI. The concentration of pro-ENK was associated to severity of acute renal dysfunction: pro-ENK increased with severity of RIFLE stages (global p \ 0.0001) (Fig. 2): median [IQR] concentration (pmol/l) of pro-ENK for patients without AKI (n = 52) was 71 [41–97], comparable to that for patients at risk for AKI (n = 15): 72 [51–120]. Patients with kidney injury (n = 8) showed a median pro-ENK of 200 [104–259] pmol/l, those with failure (n = 24) had a median of 230 [104–670], and those with loss of kidney function 947 [273–811] pmol/l. Post-hoc comparisons demonstrated significant differences between stages of failure and no AKI, as well as between failure and patients at risk (both p \ 0.05). For NGAL, the separation between RIFLE classes was comparable to that for pro-ENK (Fig. 2): median [IQR] concentration was 0.5 [0.3–0.8], 0.4 [0.3–0.8], 0.8
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J Nephrol Table 1 Patient characteristics Variable
All patients (n = 101)
Demographics Gender, male (%)
61 (60 %)
Age (years), median [IQR]
78 [72–83]
Concomitant diseases Cardiovascular, n (%)
26 (26 %)
Hypertensive, n (%)
47 (47 %)
Diabetes, n (%) Hepatic cancer, n (%)
35 (35 %) 1 (1 %)
Lung cancer, n (%)
8 (8 %)
Gastric cancer, n (%)
2 (2 %)
Colon cancer, n (%)
2 (2 %)
Antihypertensive drugs ARBs, n (%)
18 (18 %)
ACE inhibitors, n (%)
29 (29 %)
Routine laboratory tests Creatinine clearance (ml/min), median [IQR]
48 [23–77]
PCT (ng/ml), median [IQR]
2.8 [0.6–10.7]
Blood cultures Performed, n (%)
35 (35 %)
Positive, n (% of performed)
25 (71 %)
Gram- bacteria, n (% of all positives)
11 (44 %)
Gram? bacteria, n (% of all positives) Site of infection
14 (56 %)
Skin, n (%)
2 (2 %)
Lung, n (%)
46 (46 %)
Abdomen, n (%)
10 (10 %)
Urinary tract, n (%)
33 (33 %)
Other APACHE II score (points), median [IQR]
16 [13–21]
Final diagnosis, severe sepsis or shock
29 (29 %)
Days hospitalized, median [IQR]
5 [2–7]
Deaths within 7 days after admission
28 (28 %)
IQR interquartile range, ARBs angiotensin receptor blockers, ACE angiotensin converting enzyme, PCT procalcitonin, APACHE II acute physiology and chronic health evaluation II
[0.6–1.4], 1.3 [0.6–3.2] and 2.7 [2.5–2.8] lg/ml for patients without AKI, at risk, with kidney injury, failure and loss, respectively (global p \ 0.0001). Post-hoc comparisons showed significant differences between the stage of failure and no AKI, as well as between failure and patients at risk (both, p \ 0.05). The majority of patients without AKI or at risk, i.e. 40 out of 67 (60 %) for both groups, had pro-ENK concentrations below the 99th percentile of the normal range (80 pmol/l), despite septic inflammation. For patients with AKI (injury, failure or loss), 27 out of 34 patients (79 %) were above the normal range. In contrast, for NGAL, all
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Fig. 1 Correlation of creatinine clearance and pro-ENK. Creatinine clearance and plasma pro-ENK concentrations were determined for all patients on admission. The Spearman correlation coefficient was r = -0.72, p \ 0.0001. pro-ENK proenkephalin
but three patients (two without AKI and one at risk) had concentrations above the upper normal limit (0.1 lg/ml); the mean NGAL concentration in plasma from healthy donors has been reported as 0.063 lg/ml (range 0.037–0.106) with the NGAL Rapid ELISA Kit (Kit 036; BioPorto Diagnostics, Hellerup, Denmark) (for more detail, a brochure can be downloaded at http://www.bio porto.com/products/bioporto_diagnostics/ngal_elisa_kits/ ngal_rapid_elisa_kit_ruo) [27]. The diagnostic accuracy of pro-ENK for detecting kidney disease within the investigated population (no AKI or at risk vs. all other RIFLE categories) was comparable to that of NGAL (pro-ENK: v2 34.9, p \ 0.001, AUC 0.815 vs. NGAL: v2 31.3, p \ 0.001, AUC 0.800). The predictive information of pro-ENK was independent from that of NGAL: the combination of both biomarkers provided additional predictive information for detecting kidney dysfunction (added v2 10.0, p = 0.0016 for the additive effect of pro-ENK to NGAL). pro-ENK and short-term outcome Within 7 days post admission, 28 (28 %) patients died. Both admission pro-ENK and NGAL were elevated in those early deaths, while PCT and creatinine clearance was not (Table 2). Despite the high correlation between proENK and creatinine clearance, pro-ENK was a better predictor of mortality within 7 days (pro-ENK: v2 13.4, p \ 0.001, AUC 0.69 vs. creatinine clearance: v2 4, p = 0.045, AUC 0.61). pro-ENK was independent from creatinine clearance and provided superior prognostic information (added v2 11.2, p \ 0.001). NGAL performed
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Fig. 2 Concentration of pro-ENK (a) and NGAL (b) in patients according to the RIFLE criteria. Plasma levels of both markers were determined for all patients on admission and are shown here for patient subgroups according to presence and severity of AKI using the RIFLE criteria (box 25th–75th percentile, whiskers 10th–90th
percentile). The upper normal limits are 80 pmol/l (pro-ENK) and 0.1 lg/ml (NGAL). pro-ENK proenkephalin, NGAL neutrophil gelatinase-associated lipocalin, RIFLE risk, injury, failure, loss of function, end-stage renal disease, AKI acute kidney injury
Table 2 Selected variables by all-cause mortality within 7 days after admission Variable median [IQR]
All (n = 101)
No. of deaths within 7 days (n = 28)
7-day survivors (n = 73)
PCT (ng/ml) pro-ENK (pmol/l)
p value
2.8 [0.6–10.7]
4.1 [1.3–13.0]
2.2 [0.6–9.0]
0.102
87 [50–205]
209 [77–499]
75 [47–124]
\0.001 \0.001
NGAL (lg/ml)
0.6 [0.4–1.2]
1.3 [0.5–2.1]
0.6 [0.3–0.8]
Creatinine clearance (ml/min)
48 [23–77]
33 [15–69]
56 [29–81]
0.071
APACHE II score (points)
16 [13–21]
23 [18–27]
14 [12–18]
\0.001
Values are median and inter-quartile range (IQR); p value from non-parametric Kruskal–Wallis test PCT procalcitonin, pro-ENK proenkephalin, NGAL neutrophil gelatinase-associated lipocalin, APACHE II acute physiology and chronic health evaluation II
comparably to pro-ENK (v2 14.2, p \ 0.001, AUC 0.69), while the APACHE II score was by far the strongest predictor in our population (v2 21.3, p \ 0.001, AUC 0.75). PCT was not predictive of mortality within 7 days (v2 2.1, p = 0.144, AUC 0.60). Serial measurement of pro-ENK further improved its predictive performance: the c index (AUC) increased from 0.69 for pro-ENK measured at baseline to 0.90 for the 49 patients still alive and hospitalized at day 4 post admission. To illustrate the dynamic and added value of serial proENK measurement, we applied a cutoff at [100 pmol/l to define patients at risk. This cut-point was at the 75th percentile of patients without AKI, and is slightly above the upper normal range. It also appears to offer the best distinction between groups. At baseline, 43 patients were at risk. Their 7-day survival rate was 56 % (Fig. 3, left). Of those, 5 (12 %) dropped below our risk cutoff as early as one day post admission, with an observed survival rate of 100 % (Fig. 3, right).
Discussion In the present study we demonstrate that plasma pro-ENK in sepsis patients is independently associated with the presence and severity of AKI as well as with short-term mortality. As opposed to NGAL, plasma pro-ENK in septic patients without kidney dysfunction was essentially in the normal range. Approximately 2 million people worldwide die each year due to AKI, and hospitalized patients with AKI have a post-discharge mortality rate twice as high as those without AKI [5]. Moreover, the increased risk of death rises incrementally with the severity of AKI [11]. Recent data indicate that even transient perturbations in kidney function in hospitalized patients increase the risk of death [28]. Therefore it is important to diagnose AKI early, and to apply prompt and specific treatment strategies in order to reduce the risk. As a consequence, there has been a growing interest in the discovery of new biomarkers for
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Fig. 3 Illustration of added value of serial measurement of pro-ENK using a cutoff at 100 pmol/l. On the left, Kaplan–Meier plot showing survival rate of patients with plasma pro-ENK concentration \100 vs. [100 pmol/l on admission. On the right, Kaplan–Meier plot showing
survival rate of a subgroup of patients with plasma pro-ENK concentration on admission \100 pmol/l and which, on the next day, remained below vs. rose above 100 pmol/l
early diagnosis of AKI. Recently pro-ENK, a stable surrogate marker for endogenous enkephalins, has emerged as a novel candidate biomarker strongly associated with kidney dysfunction in acute myocardial infarction [22, 23]. Endogenous opioids mediate some of the circulatory and renal excretory responses to plasma volume expansion, and the delta opioid receptor evokes changes in renal excretory function [29]. Opioids have a strong influence on renal function. One mechanism is the accumulation of oxidative stress factors in the kidney during pathological processes like sepsis, which in turn increase pro-ENK expression [30]. During sepsis, an internal redistribution of blood flow occurs to the cortex and away from the medulla, which leads to a massive collapse of the renal and tubular cells, leading to the loss of kidney function [31]. Another possible mechanism is the interplay of opioids and pain proprioception with pathological processes that take place in the kidney [32]. These integrated neural and hormonal mechanisms within the central nervous system and kidneys may culminate in alterations in urine output and urine sodium excretion [33]. It is well known that clearance of creatinine has limitations in detecting early changes in renal function, including its dependency on factors independent of AKI, including muscle mass, liver function, fever, age, sex, race, protein intake and hydration status [25, 34]. Our data suggest that pro-ENK is more useful as an early biomarker of AKI than sCr upon entrance to the ED. Analysis in a general population cohort has revealed measures of kidney function as by far the strongest determinants of plasma
pro-ENK (Melander et al., unpublished). Levels of proENK were essentially not influenced by underlying comorbidities such as diabetes. Even though the present study includes too few patients to draw definite conclusions, we could not find any indication of comorbidities impairing pro-ENK’s capacity to diagnose AKI. We compared pro-ENK to NGAL on the basis of RIFLE stages and demonstrated that for patients without AKI proENK levels were essentially within the normal range, despite the presence of inflammation, while pro-ENK concentration was increased in patients with AKI. On the contrary, NGAL was elevated above the normal range for almost all patients without AKI, confirming that NGAL is influenced by inflammation. This is supportive of our recent observations that plasma NGAL could be higher in the occurrence of AKI in the ED in patients with sepsis compared to those free from infections [17]. Thus, we conclude that pro-ENK is more specific than NGAL for assessing AKI in septic patients. In our study, we did not collect urine samples, so that a comparison of plasma pro-ENK and NGAL with other potential AKI biomarkers to be measured in urine was not possible. Such comparison should be considered for further studies in a larger cohort of patients. Since AKI aggravates the prognosis of sepsis patients, it is notable that baseline pro-ENK is superior to creatinine clearance and PCT in predicting death. In addition, proENK proved a dynamic marker: the 24-h reassessment significantly improved risk stratification. In conclusion, in patients admitted for sepsis, plasma pro-ENK is an early biomarker useful for promptly
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detecting AKI and, in contrast to NGAL, it is not influenced by inflammation. Thus, the use of pro-ENK in septic patients could allow physicians to immediately assess the presence and severity of AKI. This would enable adequate treatment to be started promptly to prevent ongoing kidney failure, or the search for and removal of any offending agent. Moreover, pro-ENK is highly predictive of shortterm mortality and could enable an early identification of patients at risk of death. Acknowledgments We would like to thank Janine Sachse and Julia Weber for their excellent technical assistance. Conflict of interest University La Sapienza Rome, Sant’Andrea Hospital, received a research grant from Sphingotec GmbH for this study. Joachim Struck, Oliver Hartmann, Miriam Rehfeldt and Andreas Bergmann are employed by Sphingotec GmbH, and Andreas Bergmann holds shares in Sphingotec GmbH. Sphingotec GmbH holds patent rights related to the pro-ENK assay. Alan S. Maisel is a consultant for Sphingotec GmbH.
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