Int Urol Nephrol (2015) 47:413–420 DOI 10.1007/s11255-014-0878-8

NEPHROLOGY - ORIGINAL PAPER

Serum neutrophil gelatinase‑associated lipocalin and proinflammatory cytokines in pigs with septic versus non‑septic acute kidney injury Hengjin Wang · Miao Zhang · Huijuan Mao · Zhixiang Cheng · Qingyan Zhang · Chunming Jiang · Chen Sun · Lingyun Sun 

Received: 13 June 2014 / Accepted: 28 October 2014 / Published online: 25 November 2014 © Springer Science+Business Media Dordrecht 2014

Abstract  Background  The aim of this study was to establish a composite pig model with sepsis and ischemic acute kidney injury (AKI) and to investigate the differences in serum neutrophil gelatinase-associated lipocalin (NGAL) and proinflammatory cytokines in septic and non-septic AKI. Methods  Seventeen healthy hybridized pigs (weighed 26.97 ± 2.26 kg) were randomly divided into two groups. Group A (n  = 12) served as the septic AKI model which received cecal ligation and puncture, resulting in abdominal infection plus clamping of renal artery (CRA). Group B (n = 5) received CRA only. Vital signs and the functions of the main organs were observed. Serum NGAL, TNF-α, and IL-6 were measured at 0, 4, 8, 24, and 48 h after surgical admissions.

H. Wang · M. Zhang · Q. Zhang · C. Jiang · C. Sun  Department of Nephrology, Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine (Nanjing Drum Tower Hospital, Nanjing University School of Medicine), Nanjing 210008, China H. Wang · H. Mao  Department of Nephrology, Nanjing Medical University, Nanjing 210029, China Z. Cheng  North Neurosurgical Oncology Laboratory, Department of Neurological Surgery, T4318 Medical Center, Vanderbilt University Medical Center, Nashville, USA L. Sun (*)  Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine (Nanjing Drum Tower Hospital, Nanjing University School of Medicine), Nanjing 210008, China e-mail: [email protected]

Results  Septic AKI model was successfully induced, which manifested as multiple organ dysfunction syndrome, including AKI, liver dysfunction, progressive decline of cardiac function and abnormal pulmonary function. Apparent pathological changes were found in kidney, liver, lung and small intestine of group A. The proinflammatory cytokines in Group A were significantly higher than those in Group B at different time points (P < 0.05). In Group A, serum concentrations of TNF-α reached the peak at 8 h, while IL-6 levels dramatically increased at 24 h. There was a significant difference in serum NGAL between Group A and B at 8 h (P < 0.05). Conclusions  Septic AKI animals have higher serum NGAL compared with non-septic AKI animals. Monitoring the activities of TNF-α, NGAL and IL-6 would make great contributions in discovering sepsis and evaluating the severity of sepsis. Keywords  Acute kidney injury · Sepsis · Neutrophil gelatinase-associated lipocalin · Tumor necrosis factoralpha · Interleukin-6

Introduction Sepsis is a severe and dysregulated inflammatory response to infection and also the most common cause of morbidity and mortality among critically ill patients [1, 2]. Acute kidney injury (AKI) often complicates sepsis, with a linear relationship between the severity of kidney damage and sepsis prognosis [1]. Septic AKI is characterized by a distant pathophysiology and portends a poorer prognosis with lower survival [3–6]. Neutrophil gelatinaseassociated lipocalin (NGAL) is one of the most frequently investigated and most promising biomarkers for the early

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diagnosis of AKI. Meanwhile, cytokines play an important role in the occurrence and development of sepsis (including concomitant AKI), which seriously affects the prognosis of patients. Despite great advances in the understanding of mechanisms involved in septic AKI, progress in diagnosis, treatment and research in septic AKI is limited by few animal models that closely mimic human sepsis and a relative shortage of specific diagnostic tools. Therefore, this study aimed to establish a successful sepsis and AKI model and explore the differences between AKI with and without sepsis in serum NGAL (sNGAL) and proinflammatory cytokines TNF-α and IL-6 in this animal model.

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Monitor and support of organ function Cardiac and respiratory functions were monitored continuously (Goldway UT4000F). The pigs received intravenous infusions of dopamine 5 μg/kg every minute to maintain blood pressure when they showed signs of cardio-dysfunction. The animals received assisted ventilation when respiratory function was insufficient. After surgery, awake pigs were in harnesses and received sodium chloride–glucose solution (5 % GNS) continuously by intravenous infusion. Additionally, pigs were administered 0.1 g fentanyl by intramuscular injection every 8 h. Indices of observation

Subjects and methods Animals Healthy hybridized pigs, weighing 23.5–27.9 kg, were taken from “the 3rd Suzhong” group in Jiangsu Agricultural Institute. All pigs were accustomed to the experimental surroundings for 17–20 h and fasted before operation. The temperature in the animal room was 20–25 °C. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University Drum tower Hospital. The pigs were randomly divided into two groups. In Group A (n = 12), cecal ligation and puncture (CLP) and clamping of the renal artery (CRA) were performed on the animals. Group B (n = 5) received CRA only. Establishment of pig model with sepsis and AKI The animals received CLP and CRA as described previously [7–10]. Briefly, anesthesia in the pigs was infused with 0.1 % ketamine. The abdomen was opened, and the cecum was identified and ligated with a 4-O silk suture at 8 cm below the ileocecal junction. Patency of the bowel lumen from the ileum to the colon was maintained. The antimesenteric border of the cecum was punctured once and the cecum was then gently compressed to extrude a small amount of cecal content. Then, ischemic kidney injury was induced by clamping the left renal artery for 60 min immediately after right-side nephrectomy. The cecum was returned to the abdomen, which was closed in two layers with 4-O silk sutures. The study was approved by the Animal Care and Use committee at Nanjing University Drum tower Hospital and conformed with the “Guide for the Care and Use of Laboratory Animals”.

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Symptom and vital signs: Heart rate (HR), mean artery press (MAP), respiratory frequency and temperature were recorded every 30 min. Organs function: Blood biochemical markers including electrolytes, creatinine (Cr), urea nitrogen (BUN), alanine aminotransferase and arterial blood gas (ABG) were recorded or measured at 0 (operation beginning), 4, 8, 16, 24 and 48 h (ABG at 0, 4, 8, 24 and 48 h) following surgery operation. Cytokine assays: Serum samples were collected at 0, 4, 8, 24 and 48 h and stored at −80 °C for later cytokine assay. The concentrations of IL-6 and TNF-α were measured by ELISA as recommended by the manufacturers (Biosource Co., Camarillo, CA, and R&D Systems Co., Minneapolis, MN, Specific swine IL-6 and TNF-α antibody). Sampling and quantification of NGAL: Serum samples for quantification of NGAL were obtained from each animal, immediately centrifuged at 3,000g for 10 min, divided into aliquots and stored at −80 °C. NGAL was measured using a commercially available ELISA kit (BioPorto Diagnostics, Gentofte, Denmark) according to manufacturer’s instruction. Survival times of all the pigs were recorded. Study definitions AKI was defined according to the RIFLE criteria [11]. Sepsis was defined according to consensus guidelines [12]. Shock was defined as a MAP < 60 mmHg and/or need for vasoactive support. Pathological examination The lung, kidney, liver and small intestine of pigs with sepsis and AKI were obtained for histological examination after 6 days. The tissues were fixed in the 10 % neutral formalin, stained by hematoxylin–eosin staining/periodic acid–Schiff staining, and evaluated by light microscope (OLYMPUS BX41 microscope, Nikon COOLPIX 4500 digital camera).

Int Urol Nephrol (2015) 47:413–420 Table 1  Dynamic changes of MAP, WBC and platelets in pigs with septic and non-septic AKI

415 Parameter

Time (h)

MAP (mmHg)

WBC (×109/ml)

Platelets (×109/ml)

Septic AKI

0  4  8  16  24  48  0  4  8  16  24  48 

159.03 ± 17.92 154.69 ± 20.37 148.57 ± 15.04 141.94 ± 18.61* 136.01 ± 22.83* 120.14 ± 10.31** 157.96 ± 19.03 155.01 ± 18.94 149.83 ± 20.15 147.18 ± 21.33 144.67 ± 19.23 137.51 ± 15.46*

12.01 (7.38) 11.97 (8.25) 18.03 (7.12)* 19.27(8.01)* 20.49 (6.95)** 25.14 (10.27)** 11.93 (6.95) 12.07 (9.13) 13.48 (8.32) 13.15 (9.24) 14.06 (7.83) 15.02 (9.18)*,‡

671.33 ± 67.50 612.98 ± 78.33 537.81 ± 59.74* 514.26 ± 63.17* 489.17 ± 90.36** 307.46 ± 109.53** 654.73 ± 78.52 627.41 ± 80.36 609.33 ± 67.41 597.08 ± 73.59 581.52 ± 83.46* 559.61 ± 97.36*,†

Non-septic AKI

AKI acute kidney injury, MAP mean artery press, WBC white blood cells In the same group, compared with 0 h, * P < 0.05, ** P < 0.01, while compared between groups, † P < 0.05, ‡  P < 0.01

Parameter

Time (h)

Temperature (°C) (mean ± SD)

Heart rate (beats/min) (mean ± SD)

Respiratory rate (/ min) (mean ± SD)

Septic AKI

0  4  8  16  24  48  0  4  8 

38.71 ± 0.39 39.04 ± 0.51 39.16 ± 0.72 40.53 ± 0.94 41.40 ± 1.02* 40.97 ± 0.89* 38.80 ± 0.45 39.11 ± 0.68 39.24 ± 0.59

84.93 ± 10.40 93.68 ± 9.47 104.07 ± 8.16* 107.11 ± 9.37* 114.38 ± 10.72** 110.52 ± 10.14* 85.13 ± 9.84 94.25 ± 8.96 99.41 ± 10.06

16  24 

39.83 ± 0.76 * 39.41 ± 0.81*

101.53 ± 10.27*,† 98.54 ± 9.33*,†

13.85 ± 0.64 18.01 ± 1.37 21.03 ± 2.96* 24.51 ± 3.04* 26.97 ± 3.58** 25.03 ± 2.85** 14.02 ± 0.71 18.62 ± 1.09 20.41 ± 1.37 21.62 ± 1.58*,‡ 19.83 ± 1.16*,†

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38.97 ± 0.65

Non-septic AKI

Statistical analysis Analysis was performed with SPSS software 17.0. Normally distributed variables are reported as mean with standard deviations (SD) and compared with Student’s t test or repeated measures analysis of variance (r-ANOVA), as appropriate. Non-normally distributed data are reported as medians with inter-quartile range (IQR) and compared with Mann–Whitney U test. Categorical data were reported as proportions and compared using Fisher’s exact test. Kaplan–Meier analysis and logrank test was used to assess the survival time of the study animals. A P value <0.05 was considered statistically significant.

92.45 ± 9.61†

17.19 ± 0.82†

8 h after surgery, and the respiratory rate increased from (13.85  ± 0.64)/min to (21.03 ± 2.96)/min. Changes of MAP, white blood cells (WBC) and platelets are shown in Table 1. At 16 and 24 h, there were significant differences of HR and respiratory rate between two groups (P < 0.05). At 48 h, there were significant difference of WBC, platelets, HR and respiratory rate between two groups (P < 0.05). As shown in Table 2, in septic AKI group, compared with baseline, there is a significant increase in PaCO2 and a decrease in pH and PaO2 (P < 0.05). At 48 h, there was significant difference of PaO2 between two groups (P < 0.05). Changes in functions of liver and kidney

Results Variations of animal symptoms, physical signs, blood routine and blood gas analysis Rectal temperature reached 39.1–41.4 °C after 8–24 h, 1 °C higher than baseline. The HR increased from preoperative baseline (84.93 ± 10.04) to (104.07 ± 8.16) bpm

As shown in Figs. 1 and 2, both groups (septic and nonseptic AKI) have significantly higher renal function marker Cr and BUN 24 h after surgery. However, the levels of Cr and BUN were similar between septic and non-septic AKI groups (P > 0.05). The liver function markers were only elevated in septic AKI animals but exhibited no obvious change in non-septic AKI group during the observation time. Mean blood potassium of septic AKI animals

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ascended to (6.13 ± 0.74) mmol/L 48 h after surgery, which was significantly higher compared with the preoperative value (4.06 ± 0.49) mmol/L.

organs and tissues were discovered in the non-septic AKI group, except kidney. Changes of cytokines levels

Pathological changes of organs Lung, liver, kidney and gastrointestinal tract have different degrees of pathological changes in the septic AKI group, as shown in Fig. 3. No significant pathological changes in

Table 2  Dynamic changes of artery blood gas in pigs with septic and non-septic AKI Parameter

Time pH (h)

Septic AKI

0 4 8 24 48 Non-septic 0 AKI 4 8 24 48

7.42 ± 0.04 7.43 ± 0.07 7.41 ± 0.05 7.38 ± 0.05 7.33 ± 0.06* 7.42 ± 0.08 7.40 ± 0.05 7.41 ± 0.07 7.39 ± 0.05

PaCO2 (mmHg) PaO2 (mmHg) 38.27 ± 3.01 39.13 ± 3.19 41.87 ± 4.06 43.92 ± 2.87 49.17 ± 4.93* 41.87 ± 1.79 40.15 ± 2.31 42.11 ± 3.72 44.02 ± 4.03

94.32 ± 5.33 93.07 ± 4.26 90.35 ± 5.81 88.27 ± 6.91* 69.11 ± 7.25** 93.68 ± 5.04 92.57 ± 6.10 89.81 ± 5.49 88.19 ± 3.57

7.34 ± 0.09* 46.83 ± 5.18*

79.46 ± 4.02 *†

AKI acute kidney injury In the same group, compared with 0 h, * P < 0.05, ** P < 0.01, while compared between groups, † P < 0.05 Fig. 1  Changes in serum Cr and BUN levels in the two groups. Compared with 0 h, *P < 0.05, **P < 0.01

Fig. 2  Changes in levels of serum alanine aminotransferase and aspartate aminotransferase in the two groups. Compared with 0 h, *P < 0.05, **P < 0.01; between septic AKI groups and non-septic AKI groups, △ P < 0.05

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No significant differences were found in serum levels of TNFα and IL-6 at 0 h (P > 0.05) between the two groups. In septic AKI group, the level of serum TNF-α increased after surgery and peaked at 8 h (621.5 ± 207.3 pg/ml), which was significantly higher than that at 0 h (74.1 ± 10.3 pg/ml) (P < 0.01) and that in non-septic AKI group at 8 h (194.8 ± 57.1 pg/ ml) (P < 0.01). The level of IL-6 elevated markedly at 24 h in septic AKI group, significantly higher than that of baseline. There was no obvious change regarding both TNF-α and IL-6 in non-septic AKI animals (Figs. 4 and 5). Changes of serum NGAL levels Serum NGAL levels were similar between the two groups (69.5 ± 15.2 vs. 76.1 ± 14.1 ng/ml, P > 0.05) at 0 h, and serum NGAL concentrations peaked at 8 h after surgery in both groups but was higher in septic AKI than in nonseptic AKI animals (463.1 ± 75.3 vs. 317.4 ± 58.5 ng/ml, P < 0.05) (Fig. 6). A sNGAL ≥ 291.6 ng/ml is associated with a sensitivity 72.7 % and specificity 66.7 % for the diagnosis of septic versus non-septic AKI (AuROC 0.79; 95 % CI, 0.65–0.93) at 8 h, which is shown in Fig. 7. Peak sNGAL is correlated with white blood cell count for septic (R = 0.41, P = 0.04) but not non-septic animals (R = 0.33, P = 0.12).

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Fig. 3  Histological variation of organs’ damage in pig models with septic AKI (“C” for periodic acid–Schiff staining, the others for hematoxylin-eosin staining, all ×400). a The septal capillaries revealed generalized dilation and congestion. Interstitium in septic tissue was infiltrated by inflammatory cells. Alveolar septa widening and alveolar edema and bleeding were seen locally. b Sinus hepaticus are widespread expanding and congestive. Animals noted granular degeneration in some hepatocytes and piecemeal hepatocellular

necrosis at local region of the liver. Neutrophil infiltrates were noted at the interface of some portal tracts. c Renal tubular epithelial cells are cloudy swollen, and hyaline cast is found in many tubules. The small blood vessels in mesenchymal are expanded and congested, and congestion and damage of ischemic are both presented in glomerular capillary plexus. d Degeneration and necrosis occurred in the small intestinal epithelial cells. Marked edema and massive inflammatory cells infiltration were presented in subepithelial lamina

Fig. 4  Changes in serum TNF-α levels in the two groups. **P < 0.01 (septic vs. non-septic AKI group)

Fig. 5  Changes in serum IL-6 levels in the two groups. **P < 0.01 (septic vs. non-septic AKI group)

Survival time and mortality

Discussion

Mortality within 6 days of the septic AKI group was 100 %, and the average survival time was 72.15 ± 16.64 h. Two died in the non-septic AKI group, so there was a significant difference (P = 0.007) between the two groups (Fig. 8).

Sepsis (including concomitant AKI) remains the leading cause of death in critically ill patients [13, 14]. Establishing animal model of sepsis with AKI is the foundation to research the pathogenesis and clinical preventive treatment.

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Fig. 6  Changes in serum NGAL levels in the two groups. *P < 0.05 (septic vs. non-septic group)

Fig. 8  Cumulative survival rate curves of the two groups. Survival time was significantly greater in the non-septic AKI animals than in the septic AKI ones (P = 0.007)

Fig. 7  Area under receiver operating characteristic curve for diagnosis of septic versus non-septic AKI for peak serum NGAL. A sNGAL ≥ 291.6 ng/ml was associated with a sensitivity 72.7 % and specificity 66.7 % for the diagnosis of septic versus non-septic AKI (AuROC 0.79; 95 % CI, 0.65–0.93) at 8 h

Thus, we compared a variety of animal models of sepsis and then selected the CLP animal models, because this was similar to natural spontaneous occurrence. Recently, some scholars have also proposed that abdominal infection is most commonly used to induce sepsis model [15]. In this study, we induced sepsis successfully by CLP. The results showed that CLP plus CRA model injured the function and morphology of major organs seriously as follows: (1) Temperature >39 °C; (2) HR >90 bpm; (3) WBC twice more than baselines; (4) MAP ≤70 % baseline; (5) PLT ≤50 % baseline; (6) significantly higher alanine aminotransferase values; (7) increased Cr and BUN; and (8) abdominal flatulence. The above markers and the progress of the disease were consistent with the diagnostic criteria of sepsis/AKI. We not only successfully replicated a new compound pig

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model of sepsis and AKI in this study, but also provided the basis for pathomorphism. This model has the following advantages and innovations: (1) pigs, used as experimental animals, are very easy to obtain, and moreover, the anatomy and physiological function of their kidney, cardiovascular and digestive system are similar with humans; (2) compounded injury factors, including experimental surgical trauma, abdominal infection caused by intestinal contents and intestinal barrier dysfunction induced by cutting off the blood supply, are applied to the animals at the same time; (3) intestinal tract is the expander of the inflammation medium, as well as the start position and target organ of sepsis. Therefore, a serious trauma to the sensitive intestinal organs could successfully induce sepsis/MODS; (4) because infection is the most common and important initial factor of sepsis, severe abdominal infection, as we used, to induce sepsis is an classic model of clinical pathophysiology [16]; (5) the survival time of the animal models is longer, and the incidence of sepsis is higher. The model, which is simple and easy to copy, simulates better the clinical features of sepsis complicated with AKI including organ function and morphology damage, provides an experimental basis for presentation of its pathogenesis and establishes a reliable and practical model for its prevention and treatment. This study found that serum TNF-α and IL-6 increased in septic AKI animals, which was consistent with other reports [17, 18]. It is well known that cytokines play an important role in the pathogenesis of sepsis and septic AKI. Sepsis is mainly due to organ damage caused by uncontrolled inflammatory reaction, and a variety of

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inflammatory mediators involved is the key to its pathogenesis. TNF-α and IL-6 are considered as the most important inflammatory cytokines [19, 20]. TNF-α is subject to risk factors in the role of the body after the first cytokines produced, starting material for the inflammatory response, with a wide range of biological effects. TNF-α acts on a variety of cells, stimulates TNF-IL-1-IL-6 cytokine cascade reactions or “waterfall effect”, increases the permeability of capillary endothelial cells, and leads to ischemia and thrombosis. It can also activate inflammatory cells, increase the expression of adhesion molecules and oxygen free radicals, and thereby cause congestion microcirculation, increase the development of microthrombi, resulting in tissue ischemia, hypoxia, cell apoptosis and the occurrence and development of SIRS and MODS. The results showed that serum concentrations of TNF-α in septic AKI group peaked at 8 h after surgery, which was significantly higher than the non-septic AKI group. TNF-α levels increased rapidly after trauma or infection, suggesting that TNF-α activation of cytokine network may be the early biomarkers. It is thought that they are among the first inflammatory mediators that could induce sepsis. We theorize that measurements of TNF-α offer clinical utility to facilitate earlier prediction of sepsis in a subset of patients at risk of infection. The results also showed that the concentrations of serum IL-6 in septic AKI group increased gradually after surgery. IL-6 is mainly composed of mononuclear macrophages, fibroblasts and other important biological activities produced by the cytokines, which can be induced by TNF and may enhance the harmful effects of TNF, which induce T, B lymphocyte differentiation and stimulate liver cells to synthesize acute-phase proteins, catalysis and amplification of the inflammatory response and toxicity, resulting in tissue damage. Some studies have shown that serum IL-6 levels and the severity of infection in patients with inflammatory response and the degree of positive correlation can be used as discriminant sensitive index of disease severity [21, 22]. Poor prognosis is considered if the serum or plasma IL-6 continued to rise, which is an important indicator of ongoing inflammation [23]. In summary, the dynamic monitoring of inflammatory cytokines TNF-α and IL-6 levels, contributed to an early detection and assessment of the severity of sepsis/ MODS with ischemic AKI. In the latest investigation, increased serum and urine NGAL levels were associated with AKI [24–26]. However, whether serum NGAL concentration of septic AKI animals is different to those of non-septic AKI is rarely reported. In this study, we found septic AKI was associated with higher peak serum NGAL compared with non-septic AKI. NGAL, a small 25-kD peptide, originally discovered as an antibacterial factor of natural immunity and an acute-phase protein [27]. Several studies highlight its role as an organ

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injury and disease activity biomarker [28]. Indeed, Wheeler et al. [29] found higher values of NGAL in children with septic shock compared with those with SIRS or healthy controls. NGAL is constitutively expressed in several tissues including lung, liver, kidney and the gastrointestinal tract [30]. Likewise, NGAL expression has been found to be increased during acute inflammatory and/or infectious processes [31–33]. These observations suggest that NGAL may represent an emerging global biomarker for inflammation, tissue injury, illness severity and organ failure and correlate with survival in sepsis. In summary, the levels of serum NGAL were higher in septic AKI compared with non-septic AKI animals. The possible reason was that septic AKI animals had a higher burden of illness and were sicker which were compared with non-septic AKI ones. These observations suggested that there were differential biomarker patterns in septic AKI that might have clinical relevance and prognostic importance. However, there were limitations in this study. First, due to the lack of urine sample in animals with AKI, the measurements of urine NGAL were not complete in each time point. Moreover, it was suggested that early diagnosis of AKI by the concentration of serum NGAL would be more beneficial, because it could not only reduce the difficulty of sampling as a result of oliguria or anuria, but also avoid the interference from the use of diuretics in the clinical work. Meanwhile, serum NGAL was an accurate biomarker for prediction of AKI in patients [34]. So we did not report the values of urine NGAL. Second, without the floating catheter implantation or PiCCO monitoring, it was lost that the data regarding renal blood flow, cardiac output and systemic vascular resistance in the monitoring of septic AKI. PiCCO monitoring was performed in the next investigations. Third, prospectively, this study was small and potentially prone to type I error, and it has limited statistical power. Therefore, further research is warranted. Conclusion Septic AKI animals have higher serum NGAL compared with non-septic AKI animals. Monitoring the activities of TNF-α, NGAL and IL-6 would make great contributions in discovering sepsis and evaluating the severity of sepsis. Acknowledgments  We thank professor Xiaoyun Wang for her guidance. This study was supported by Grants from the National Natural Science Foundation of China (No. 30170433), the Key project of Nanjing medical science and technology development foundation (ZKX13021, YKK10077, Youth health personnel), the Foundation of Nanjing Science and Technology Development Program (201104002) and Jiangsu “six talent peaks” high-level talent project (WSN-048). Conflict of interest None.

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