American Journal of Emergency Medicine 32 (2014) 208–215
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American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem
Original Contribution
Acute kidney injury after cardiac arrest of ventricular fibrillation and asphyxiation swine model☆,☆☆ Chen-Chen Hang, MB, Chun-Sheng Li, MM ⁎, Cai-Jun Wu, MM, Jun Yang, MM Department of Emergency, Beijing Chao-Yang Hospital, Affiliated to Capital Medical University, Beijing 100020, China
a r t i c l e
i n f o
Article history: Received 26 August 2013 Received in revised form 11 October 2013 Accepted 21 October 2013
a b s t r a c t Purposes: The purposes of the study are to investigate the renal function in ventricular fibrillation (VF) and asphyxiation cardiac arrest in a swine model and to estimate the value of novel biomarkers in the acute kidney injury (AKI) after cardiac arrest. Method: Thirty-two healthy inbred Wu-Zhi-Shan miniature piglets were randomized into 2 groups (n = 16 per group). Cardiac arrest was induced by programmed electric stimulation and clamping the endotracheal tube in the VF group and asphyxiation group, respectively. Cardiopulmonary resuscitation was done for return of spontaneous circulation (ROSC). Results: One hundred percent (16/16) ROSC was observed in the VF group, and 50% (8/16) in the asphyxiation group (P b .01). All AKI biomarkers elevated significantly after ROSC. The novel biomarkers changed much earlier than the creatinine. The concentration of novel biomarkers in the asphyxiation group was higher than the VF group. Live animals had an oliguria and developed AKI. Characteristic morphological injuries in renal tissues were observed under light microscope and transmission electron microscope and were more serious in the asphyxiation group. Conclusions: Acute kidney injury at early stage of postresuscitation is common in different causes of cardiac arrest. Asphyxiation has more severe kidney injury and gets worse prognosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Patients who survive in an initial cardiac arrest (CA) have significant morbidity and mortality [1-4]. Postresuscitation morbidity is often attributed to cerebral, myocardial and prolonged, complete, whole-body ischemia-reperfusion injury (IRI) [5], which is an unnatural pathophysiologic stage created by successful cardiopulmonary resuscitation (CPR). Such ischemic injury should have major effects on the kidneys. The acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney's excretory function. It is common in any cohort of critically ill patients, such as the survivors of CA. Post-CA AKI is usually considered related to the post-CA syndrome (PCAS) and might affect the long-term survival. However, it does not attract as much important as the cerebral and myocardial injury do. Serum creatinine (sCr) and urine output are the standard diagnostic indexes of the RIFLE (risk, injury, failure, loss, end stage) criteria, Acute Kidney ☆ Conflicts of interests: None. ☆☆ Sources of fundation: Beijing Natural Science Foundation (No. 81372025); Technology Foundation for the Tutor of Beijing Excellent Doctoral Dissertation (No. 20121002501). ⁎ Corresponding author. Department of Emergency Medicine, Beijing Chao-Yang Hospital, Affiliated to Capital Medical University, Beijing 100020, China. Tel./fax: +86 10 85231051. E-mail addresses: [email protected] (C.-C. Hang), [email protected] (C.-S. Li). 0735-6757/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajem.2013.10.043
Injury Network (the AKIN criteria), and Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guidelines for AKI [6,7]. However, both sCr and urine output have a lack of sensitivity and/or specificity; thus, new biomarkers for earlier and more accurate detection are needed [8,9]. Neutrophil gelatinase–associated lipocalin (NGAL), cystatin C (CysC), kidney injury molecule 1 (KIM-1), and Nasetil-β-glukosaminidase (NAG), which are the clinical utility of certain novel AKI biomarkers, already evaluated in various clinical conditions and were deemed as good as or better than creatinine (Cr) in detecting AKI, particularly in the early stage [10,11]. In this study, we hypothesized that postresuscitation AKI was common in both ventricular fibrillation (VF) CA (VFCA) and asphyxiation CA (ASCA). The purpose of this study was to confirm this hypothesis by carrying out the biomarkers on the characteristics of renal function and structures in swine models of CA. 2. Methods This prospective laboratory study was approved by the Capital Medical University Institutional Animal Care Committee and the Beijing Chao-Yang Hospital Affiliated to the Capital Medical University Animal Care and Use Committee. All animals received treatments in compliance with the National Research Council's 1996 Guide for the Care and Use of Laboratory Animals. Anesthesia was titrated in all surgical interventions to avoid unnecessary suffering. The study was
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performed according to Utstein-style guidelines [12] on 32 healthy inbred Wu-Zhi-Shan miniature piglets of either sex, aged 6 to 8 months, whose average weight was 20 ± 2 kg. Before any procedure, animals were randomized into 2 groups with the use of a sealed envelope indicating the animals assignment to either the VFCA group (n = 16) or ASCA group (n = 16). 2.1. Animal preparation The animals were fasted overnight except for free access to water. Briefly, initial sedation in each animal was achieved with intramuscular ketamine 10 mg/kg, midazolam 0.5 mg/kg, and atropine 0.05 mg/kg, followed by ear vein injection of propofol 1.0 mg/kg. The anesthetized animals were intubated with a 6.5-mm cuffed endotracheal tube via direct laryngoscopy. Animals were mechanically ventilated by a volume-controlled ventilator (Servo 900 c; Siemens, Berlin, Germany) using a tidal volume of 10 mL/kg and a respiratory frequency of 12 per minute with room air (fraction of inspired oxygen, 21%). Propofol 1.0 mg/kg and fentanyl 4 μg/kg were then administered intravenously to reach the desired depth of anesthesia and analgesia. Once this depth was reached, 9 mg/kg per hour of propofol and fentanyl 1 μg/kg per hour were given to maintain the anesthesia level. End-tidal partial pressure of carbon dioxide was monitored with an in-line infrared capnography (CO2SMOplus monitor; Respironics, Inc, Murrysville, PA). The respiratory frequency was adjusted to maintain end-tidal partial pressure of carbon dioxide between 35 and 40 mm Hg. Aortic blood pressure (ABP) was measured with a fluid-filled catheter advanced from the left femoral artery into the thoracic aorta. A Swan-Ganz catheter (7F; Edwards Life Sciences, Irvine, CA) was advanced from the left femoral vein and flow directed into the pulmonary artery to measure cardiac output (CO) by the thermodilution method. A 5F pacing catheter was advanced from the right internal jugular vein into the right ventricle to induce VF for the VFCA group. An 18F urine catheter connecting a urine bag was inserted into the bladder by cystostomy to obtain urine. Electrocardiographic lead II was continuously recorded with a multichannel physiologic recorder (BL-420 F Data Acquisition & Analysis System Chengdu TME Technology Co, Ltd, Sichuan, China). Hemodynamics was monitored by an HP monitor (M1165; Hewlett-Packard Co, Palo Alto, CA). All animals received normal saline solution (10 mL/kg per hour) intraoperatively to replenish fluid losses.
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Table 1 Baseline and preparatory phase characteristic in VFCA and ASCA group (mean ± SD)
Male/female Weights (kg) Propofol (mg) Fentanyl (μg) Preparation time (min)
VFCA group (n = 16)
ASCA group (n = 16)
P
9/7 20.3 ± 0.7 120.7 ± 2.4 72.1 ± 1.7 65.6 ± 2.5
10/6 20.1 ± 0.8 120.6 ± 2.1 71.5 ± 2.1 64.1 ± 2.5
1.000 .563 .878 .340 .117
Cardiopulmonary resuscitation was resumed for another 2 minutes after defibrillation attempt. The sequence continued until return of spontaneous circulation (ROSC) or death. ROSC was defined as the maintenance of a systolic blood pressure of at least 50 mm Hg for at least 10 consecutive minutes. The animal death was defined as the resuscitation maintenance of 30 minutes but the ROSC still not achieved. The animals with ROSC received intensive care for 6 hours, and mechanical ventilation with supplemental oxygen continued throughout the immediate postresuscitation period. Six hours after ROSC, all catheters were removed by a surgical procedure [15]. Then the animals underwent euthanasia with propofol 60 mg and were killed with 20 mL of 10 mol/L potassium chloride solution intravenously. If the animals could not live to the prospective end point, the living time was recorded. 2.3. Measurements Hemodynamics, such as heart rate (HR), ABP and CO, was measured continuously. Urine output was recorded per hour. Blood and urine samples were collected for laboratory testing at baseline, immediately at ROSC (ROSC 0), 1 hour after ROSC (ROSC 1 h), 2 hours after ROSC (ROSC 2 h), 3 hours after ROSC (ROSC 3 h), 4 hours after ROSC (ROSC 4 h), and 6 hours after ROSC (ROSC 6 h). Blood was used for measurement of Cr (sCr), serum CysC (sCysC), and serum NGAL (sNGAL), whereas urine was used for the determination of CysC (uCysC), NGAL (uNGAL), KIM-1 (uKIM-1), and NAG (uNAG). Blood and urine samples were centrifuged (3000 rpm) for 20 minutes; then
2.2. Experimental protocol After surgery, the animals were allowed to equilibrate for 1 hour to achieve a stable resting level, and the baseline data were collected. In the VFCA group, VF was induced by a programmed electrical stimulation instrument (GY-600A; Kaifeng Huanan Instrument Co, Kaifeng, Henan, China) with mode S1S2 (300/200 milliseconds), 40 V, 8:1 proportion, and − 10-millisecond step length. VF was verified by the presence of a characteristic electrocardiographic waveform and an immediate drop in ABP. In the ASCA group, animals were paralyzed with 0.2 mg/kg cisatracurium to avoid gasping, and then CA was induced by clamping the endotracheal tube. The piglets were asphyxiated until simulated pulselessness, defined by an aortic systolic pressure less than 30 mm Hg [13]. The electrocardiograph waveform and the asphyxiation induction time were recorded. After 8 minutes of untreated CA [14], mechanical ventilation was resumed with 100% oxygen, and CPR was performed manually. Manual chest compressions were rapidly initiated at a rate of 100 per minute with equal compression-relaxation duration. The compression depth was approximately equivalent to one-third of anteroposterior diameter of the thorax. After 2 minutes of CPR, epinephrine (0.02 mg/kg) was injected into the right atrium, and then CPR was performed manually for 2 minutes. After 4 minutes of CPR, defibrillation was attempted using 4 J/kg (biphasic waveforms shock) for the first attempt.
Fig. 1. Survival function of animals with ROSC in VFCA group (n = 16) and ASCA group (n = 8).
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serum and urine supernatants were stored at − 80°C until measurements, which were performed by an investigator blinded to the study design. Commercially available enzyme-linked immunosorbent assays were used for swine serum and urine AKI biomarkers measurements, according to the manufacturer's instructions (Swine Cr/BUN/CysC/NGAL/KIM-1/NAG; Beijing Xin Fangcheng Biotechnology Company, Ltd, Beijing, China). Assays were run in duplicate. All intra-assay coefficients of variation were less than 10%. After the animals underwent euthanasia, the kidney was excised; a part of tissue samples of lower pole kidney bilaterally was taken rapidly and preserved in 10% formaldehyde or 4% paraformaldehyde to observe pathologic changes under a light microscope (LM) or a transmission electron microscope (TEM), respectively.
test was used to determine differences over time within groups, as appropriate; the Bonferroni t test, for multiple comparisons. Pearson correlation coefficient was calculated to determine the correlations of biomarkers between the serum and urine. Log-rank test was used for survival analysis. Fisher exact test was used for the rate of ROSC analysis. A 2-sided P b .05 was considered statistically significant. 3. Results 3.1. Characteristics of animals The animal profile (sex and weight), time of preparatory phase, and the extra doses of propofol and fentanyl administered during the preparatory phase did not differ significantly in the 2 groups (Table 1).
2.4. Statistical analysis 3.2. Rate of ROSC and survivals Statistical analysis was performed with SPSS 19.0 software (SPSS, Inc, Chicago, IL). Values were shown as mean ± SD. Continuous variables between groups were compared using the Student t test. One-way repeated-measures analysis of variance (ANOVA) or paired t
All animals in both groups had CA. The duration between clamping the tube and CA in the ASCA group was between 13 and 20 minutes (16.8 ± 1.3 minutes). Return of spontaneous circulation was observed
Fig. 2. Invasive hemodynamic values in VFCA and ASCA group at baseline and throughout ROSC 6 h. A, Heart rate. B, Mean arterial pressure. C, Cardiac output. The values are presented as mean (SD). ⁎P b .05 and ⁎⁎P b .01 vs baseline (1-way repeated-measures ANOVA). #P b .05 and ##P b .01 vs VFCA group (Student t test).
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in 100% (16/16) of animals of the VFCA group and only 50% (8/16) of animals of the ASCA group (P b .01). The duration of CPR in the VFCA group was shorter than in the ASCA group (4.8 ± 1.2 vs 8.3 ± 1.4 minutes; P b .01). Average survival time in the VFCA group (n = 16) was longer than that in the ASCA group (n = 16) (5.90 ± 0.09 vs 2.68 ± 0.70 hours; P b .01; Fig. 1). 3.3. Hemodynamics Heart rate, mean arterial pressure (MAP), and CO at baseline did not differ significantly between the 2 groups (P N .05; Fig. 2). Heart rate and MAP increased significantly, whereas CO decreased significantly, after ROSC in both groups (Fig. 2). Heart rate, MAP, and CO were higher in the VFCA group than in the ASCA group at all time points after ROSC (Fig. 2). 3.4. Urine outputs The urine output at baseline was between 6 and 13 mL/kg per hour; no significant difference was found between the VFCA group and ASCA group (7.85 ± 2.05 vs 7.27 ± 2.75 mL/kg per hour; P = .609). Among the live animals, urine outputs of 17 animals were less than 0.5 mL/kg per hour for 6 hours, 12 animals in the VFCA group, and 5 animals in the ASCA group (Table 2). 3.5. Serum and urine AKI biomarkers Serum and urine AKI biomarkers at baseline did not differ significantly between the 2 groups (P N .05; Table 3, Fig. 3). Neutrophil gelatinase–associated lipocalin was the most sensitive one and increased at ROSC 1 h in both serum and urine. Cystatin C increased significantly at ROSC 2 h in serum and at ROSC 3 h in urine. Urine KIM1 and uNAG were less sensitive than NGAL and CysC and increased at ROSC 4 h. Serum Cr lack sensitivity compared with the novel biomarkers. Serum NGAL and uNGAL were higher in the ASCA group than those in the VFCA group after ROSC 1 h. Serum CysC after ROSC 2 h and uCysC, uKIM-1, and uNAG after ROSC 3 h were higher in the ASCA group than those in the VFCA group too (Table 3; Fig. 3).
Table 2 Urine output in VFCA and ASCA group at baseline and throughout ROSC 6 h Group Number Urine output (mL/kg per h) ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 5 h ROSC 6 h VFCA
ASCA
a
1a 2a 3 4a 5a 6 7a 8a 9a 10a 11a 12 13a 14a 15a 16 1a 2a 3 4a 5a 6a 7 8
0.48 0.46 1.20 0.49 0.45 1.20 0.40 0.40 0.49 0.48 0.45 1.00 0.45 0.40 0.45 0.40 0.45 0.45 1.00 0.4 0.45 0.4 0.40 0.35
0.45 0.45 1.10 0.48 0.45 1.25 0.45 0.40 0.45 0.45 0.40 0.80 0.48 0.35 0.48 0.35 0.42 0.48 0.80 0.30 0.48 0.45 0.45 0.25
0.45 0.38 1.10 0.45 0.40 1.20 0.42 0.35 0.48 0.40 0.40 0.90 0.40 0.45 0.49 0.30 0.48 0.40 0.50 0.35 0.40 0.38 0.35
0.44 0.40 1.00 0.48 0.40 1.15 0.48 0.30 0.45 0.45 0.35 0.70 0.35 0.35 0.42 0.30 0.40 0.45 0.75 0.36 0.45 0.42 0.30
0.43 0.42 0.95 0.45 0.30 1.00 0.45 0.25 0.45 0.45 0.40 1.00 0.40 0.36 0.48
0.45 0.44 1.00 0.48 0.36 1.20 0.40 0.25 0.48 0.45 0.38 0.80 0.42 0.35 0.46
0.40 0.42 0.6 0.28 0.42 0.45
0.34 0.45 0.75 0.25 0.48 0.44
Urine output were less than 0.5 mL/kg per hour for 6 hours.
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Table 3 Serum and urine AKI biomarkers in VFCA and ASCA group at baseline and throughout ROSC 6 h (mean ± SD) Biomarker sCr (μmol/L) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h sCysC (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uCysC (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h sNGAL (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uNGAL (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uKIM-1 (pg/ mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uNAG (U/L) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h
VFCA group (n = 16)
ASCA group (n = 8)
P
62.38 61.35 62.70 63.29 62.84 66.92 66.90
62.75 62.83 63.13 62.89 67.92 66.90 88.99
.877 .500 .857 .867 .081 .996 b.0001b
± 6.03 ±4.52 ±5.50 ±4.90 ± 6.83 ± 5.72 ± 6.21 (n = 15)
± 4.42 ± 5.84 ± 5.19 ± 6.49 ± 3.78 (n = 7) ± 6.11 (n = 7) ±8.46a (n = 6)
511.86 ± 10.10 511.46 ± 10.71 514.07 ±11.48 517.37 ± 13.73 906.48 ± 14.81d 1011.87 ± 23.65d 1177.46 ± 65.19d (n = 15)
512.65 ± 9.58 517.52 ± 10.50 513.50 ± 9.83 565.96 ± 41.45 1021.89 ±49.82d (n = 7) 1174.37 ± 64.48d (n = 7) 1303.91 ± 87.47d (n = 6)
.856 .203 .906 .013c b.0001b b.0001b .794
207.66 ± 3.60 210.05 ± 4.51 212.66 ± 6.27 216.40 ± 6.57a 217.69 ±6.16d 407.79 ± 8.30d 508.66 ± 11.57 d (n = 15)
206.95 208.60 214.58 218.81 295.99 506.64 635.39
.693 .458 .521 .484 b.0001b b.0001b b.0001b
2.18 ± 2.21 ± 3.78 ± 4.14 ± 4.45 ± 5.03 ± 6.78 ±
0.11 0.12 0.09d 0.12d 0.18d 0.15d 0.17d (n = 15)
2.19 2.22 4.42 4.75 5.46 6.67 7.85
± 0.12 ± 0.13 ± 0.24d ± 0.32d ± 0.25d (n = 7) ± 0.30d (n = 7) ± 0.34d (n = 6)
.922 .866 b.0001b b.0001b b.0001b b.0001b b.0001b
0.58 ± 0.60 ± 1.32 ± 1.53 ± 1.83 ± 2.82 ± 4.24 ±
0.04 0.03 0.06d 0.07d 0.06d 0.08d 0.17d (n = 15)
0.56 0.60 2.20 2.66 3.68 4.92 6.21
± 0.03 ± 0.04 ± 0.15d ± 0.14d ± 0.17d (n = 7) ± 0.27d (n = 7) ± 0.19d (n = 6)
.160 .705 b.0001b b.0001b b.0001b b.0001b b.0001b
± 4.92 ± 4.30 ± 7.80 ± 9.93 ± 16.73d (n = 7) ± 15.11d (n = 7) ± 16.40d (n = 6)
70.36 ± 4.09 71.33 ± 3.63 71.59 ±3.50 71.48 ± 3.21 73.15 ± 1.81 115.21 ± 4.44d 119.53 ± 5.85d (n = 15)
68.27 ± 3.70 70.61 ± 2.62 72.09 ± 1.57 71.93 ± 1.74 83.14 ± 6.44 (n = 7) 139.40 ± 3.98d (n = 7) 143.58 ± 4.03d (n = 6)
.238 .623 .707 .719 .006b b.0001b b.0001b
162.00 ± 4.87 163.90 ± 4.56 164.19 ± 4.35 166.80 ± 3.30 166.95 ± 3.65 280.95 ± 8.60d 306.55 ± 10.99d (n = 15)
159.81 161.13 162.34 166.37 248.11 305.04 330.35
.343 .183 .403 .827 b.0001b b.0001b .001b
± 5.86 ± 4.84 ±6.17 ± 6.24 ± 16.38d (n = 7) ± 9.66d (n = 7) ± 18.09d (n = 6)
a Significant differences compared with baseline: P b .05 (1-way repeated-measures ANOVA). b Significant differences between the VFCA group and the ASCA group: P b .01 (Student t test). c Significant differences between the VFCA group and the ASCA group: P b .05 (Student t test). d Significant differences compared with baseline: P b .01 (1-way repeated-measures ANOVA).
3.6. Correlations of NGAL and CysC between serum and urine High correlations between serum and urine were found in both NGAL and CysC. The correlation of NGAL (r = 0.956; P b .01) was better than CysC (r = 0.788; P b .01).
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Fig. 3. Serum and urine AKI biomarkers in VFCA and ASCA group at baseline and throughout ROSC 6 h. A, Serum CysC. B, Urine CysC. C, Serum NGAL. D, Urine NGAL. E, Urine KIM-1. F, Urine NAG. G, Serum Cr. The values are presented as mean (SD). ⁎P b .05 and ⁎⁎P b .01 vs baseline (1-way repeated-measures ANOVA). #P b .05 and ##P b .01 vs VFCA group (Student t test).
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3.7. Renal histopathology Under the LM, interstitial edema, renal tubular necrosis, glomerular capillaries angiectasis, mesangial proliferation, and inflammatory cells infiltration could be seen in both groups (Fig. 4A and B). Compared with the VFCA group, interstitial edema and acute renal tubular necrosis were more serious. Glomerular capillaries were highly dilated, with more inflammatory cells degenerating in the ASCA group. Moreover, irregular brush border of the renal tubular epithelial cells with more serious
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erosion was found in the ASCA group (Fig. 4B). Under the TEM, there were characteristic morphological changes of apoptosis in both groups, but the changes were more serious in the ASCA group than those in the VFCA group. In the VFCA group, nuclei were in different sizes and shapes, and chromatin was agglutinated. Abundant mitochondria were observed with crista swelling (Fig. 4C and E). However, in the ASCA group, nuclei deformation, chromatin pyknosis, and heterochromatin marginalized were extensive; the mitochondria vacuolization and autophagy could be observed (Fig. 4D and F).
Fig. 4. Renal ultramicrostructure of VFCA (A, C, and E) and ASCA (B, D, and F) groups at 6 hours after ROSC. A, Interstitial edema (①) and mesangial inflammatory cells infiltration (②). B, More severe than A, with serious brush border of the renal tubular epithelial cells erosion and necrosis (③) and glomerular capillaries angiectasis (④). C, Abundant mitochondria (⑤), nuclei in different sizes and shapes (⑥), chromatin agglutinated. D, Nuclei deformation (⑦), chromatin pyknosis, and heterochromatin marginalized. E, Abundant mitochondria (⑧) with crista damage accidentally. F, Mitochondria vacuolization and autophagy (⑨).
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4. Discussion The high mortality of patients who initially achieve ROSC after CA can be attributed to PCAS that involves multiple organs, and epidemiological data suggested that the mortality of PCAS was nearly 70% [5]. Because there is no doubt about the importance of heart and brain, post-CA myocardial dysfunction and brain injury as well as the systemic IRI are always paid close attention. However, the kidney is ignored, although the injured kidneys may play a key role in the systemic derangement present during multiorgan failure. Post-CA AKI is so common in the survivors of CA with a reported incidence of 23.2% [16], but there is limited information on the epidemiology. Incomplete recovery of renal function from AKI may cause excessive long-term morbidity and mortality and may have a higher risk of chronic kidney disease [17-19]. Many survivors need renal replacement therapy during the advanced life support phase or for lifelong time. So it is worth paying more attention to post-CA AKI. In this study, we measured 4 previously identified candidate biomarkers combined with the Cr to predict the AKI in 2 CA models and found that all the survival animals in both groups got abnormalities in AKI biomarkers. Serum NGAL and uNGAL increased much earlier than other biomarkers. Cystatin C increased within ROSC 2 to 3 hours in serum and urine. Urine KIM-1 increased at ROSC 4 h as well as NAG. Creatinine remained stable until ROSC 6 h. Seventeen live animals had an oliguria and developed to RIFLE injury or KDIGO/AKIN Stage 1. Combining with the kidneys' histopathology evidence, both indicated that animals with different causes of CA did suffer AKI, due to glomerular dysfunction and tubular injury. The mechanism of post-CA AKI is thought to be associated with IRI. Milder ischemia can lead to pre-renal dysfunction, which is relatively slight in all AKIs and may be reversed. When ischemia is more severe and renal tubular injury evolves to tubular necrosis [20], the prognosis of AKI will be disappointing. After ischemia, the whole-body reperfusion will further aggravate the kidney injury. Moreover, it has recently been demonstrated that AKI may not just be a consequence of CA but of time without spontaneous circulation. The underlying cause of CA, PCAS, and cardiogenic shock may strongly influence the prognosis of AKI [21]. In addition, we could also get knowledge that novel biomarkers had a higher sensitivity in diagnosis of AKI. As the international criterion standard for identification and classification of AKI was still dependent on Cr and urine output measurements, which were not specific for renal tubular lesions, the incidence of post-CA AKI might be higher than previously anticipated. In our study, all of the novel AKI biomarkers indeed increased, which suggested that all survival animals had AKI more or less. Some animals had AKI so early and so mild that they had normal Cr. Furthermore, AKI might happen much earlier than expected because the NGAL increased as early as ROSC 1 h and the other novel biomarkers increased before ROSC 6 h. Because the study of the CA animal models is mainly on VF, there is still confusion whether the post-CA kidney function was the same in asphyxiation. In our study, we compared the biomarkers in the 2 different models and found that the concentration of sNGAL and uNGAL in the ASCA group was higher than those in the VFCA group after ROSC 1 h. The similar results were found in sCysC after ROSC 2 h and in uCysC, uKIM-1, and uNAG after ROSC 3 h. These differences might suggest that the animals in the ASCA group had more serious kidney injury, especially tubular necrosis, than the animals in the VFCA group. This conclusion was verified by the histopathologic evidence of LM and TEM. The mechanism of the difference post-CA kidney injury is yet unknown but is thought to be related to either IRI or anoxia injury. In both groups, the kidney had IRI after ROSC. Oxygen debt, generalized activation of immunologic and coagulation pathways, adrenal insufficiency, and
necrosis are all important pathophysiologic mechanisms that may contribute to microcirculatory reperfusion disorders and kidney injury [22]. It is obvious that the ASCA group had a severe anoxia besides the IRI. As known, kidney is sensitive to anoxia, especially the tubular. When asphyxiation occurs, the whole body suffers hypoxemia, hypercarbia, and acidemia, which may led to a severe and irreversible acute tubular necrosis. Furthermore, in the ASCA group, MAP and CO decreased significantly after resuscitation compared with the VFCA group, which might result in worse renal reperfusion after ROSC. All those would exacerbate AKI. Collectively, the results of ecsomatics and histology demonstrated that both VFCA and ASCA could result in a severe kidney injury, but the injury was more serious in ASCA. 4.1. Limitations The number of animals achieved ROSC in the ASCA group is relatively small, which may influence the outcome. The primary end point of the experiment was set at 6 hours after ROSC, so we could not get knowledge of the tendency of the AKI biomarkers after then, which is thought to be important. The experiment was conducted on apparently healthy animals, whereas most individuals who had CA have underlying pathologic findings. 5. Conclusions Our findings suggest that post-CA AKI that occurs at an early stage of postresuscitation is common in both VFCA and ASCA, but ASCA has more severe kidney injury and worse prognosis. The novel AKI biomarkers in serum and urine, such as NGAL, CysC, KIM-1, and NAG, are of significant importance as early predictors of post-CA AKI. Acknowledgments The authors thank Yi Zhang, Qin Yin, Zhi-Jun Guo, Shuo Wang, and Qian Zhang for excellent technical assistance. References [1] Nichol G, Thomas E, Callaway CW, et al. Regional variation in out-of-hospital cardiac arrest incidence and outcome. JAMA 2008;300:1423–31. [2] Sandroni C, Nolan J, Cavallaro F, et al. In-hospital cardiac arrest: incidence, prognosis and possible measures to improve survival. Intensive Care Med 2007;33:237–45. [3] Deasy C, Bray JE, Smith K, et al. Out-of-hospital cardiac arrests in the older age groups in Melbourne, Australia. Resuscitation 2011;82:398–403. [4] Deasy C, Bray JE, Smith K, et al. Out-of hospital cardiac arrests in young adults in Melbourne, Australia. Resuscitation 2011;82:830–4. [5] Neumar RW, Nolan JP, Adrie C, et al. Post-cardiac arrest syndrome: epidemiology, pathophysiology, treatment, and prognostication. A consensus statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian and New Zealand Council on Resuscitation, European Resuscitation Council, Heart and Stroke Foundation of Canada, Inter American Heart Foundation, Resuscitation Council of Asia, and the Resuscitation Council of Southern Africa), the American Heart Association Emergency Cardiovascular Care Committee, the Council on Cardiovascular Surgery and Anesthesia, the Council on Cardiopulmonary, Perioperative, and Critical Care, the Council on Clinical Cardiology, and the Stroke Council. Circulation 2008; 118(23):2452–83. [6] Bellomo R, Kellum JA, Ronco C. Acute kidney injury. Lancet 2012;380:756–66. [7] KDIGO. Clinical practice guideline for acute kidney injury. Kidney Int Suppl 2012;2:1 e138. [8] Uchino S, Kellum JA, Bellomo R, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005;294:813–8. [9] Hewitt SM, Dear J, Star RA. Discovery of protein biomarkers for renal diseases. J Am Soc Nephrol 2004;15:1677–89. [10] Sharma RK. Biomarkers of acute kidney injury. Clin Queries: Nephrol 2012;0101: 13–7. [11] Adiyanti SS, Loho T. Acute kidney injury (AKI) biomarker. Acta Med IndonesIndones J Intern Med 2012;44(3):246–55. [12] Idris AH, Becker LB, Ornato JP, et al. Utstein-style guidelines for uniform reporting of laboratory CPR research. A statement for healthcare professionals from a task force of the American Heart Association, the American College of Emergency Physicians, the American College of Cardiology, the European
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[17] Schiffl H. Renal recovery from acute tubular necrosis requiring renal replacement therapy: a prospective study in critically ill patients. Nephrol Dial Transplant 2006;21(5):1248–52. [18] Coca SG, Yusuf B, Shlipak MG, et al. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis 2009;53:961–73. [19] Wald R, Quinn RR, Luo J, Li P, Scales DC, Mamdani MM, Ray JG. Chronic dialysis and death among survivors of acute kidney injury requiring dialysis. JAMA 2009;302: 1179–85. [20] Kumar Jitendra. Pathophysiology of ischemic acute tubular necrosis. Clin Queries: Nephrol 2012;0101:18–26. [21] Chua HR, Glassford N, Bellono R. Acute kidney injury after cardiac arrest. Resuscitation 2012;83:721–7. [22] Adrie C, Monchi M, Laurent I, et al. Coagulopathy after successful cardiopulmonary resuscitation following cardiac arrest: implication of the protein C anticoagulant pathway. J Am Coll Cardiol 2005;46:21–8.
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Original Contribution
Acute kidney injury after cardiac arrest of ventricular fibrillation and asphyxiation swine model☆,☆☆ Chen-Chen Hang, MB, Chun-Sheng Li, MM ⁎, Cai-Jun Wu, MM, Jun Yang, MM Department of Emergency, Beijing Chao-Yang Hospital, Affiliated to Capital Medical University, Beijing 100020, China
a r t i c l e
i n f o
Article history: Received 26 August 2013 Received in revised form 11 October 2013 Accepted 21 October 2013
a b s t r a c t Purposes: The purposes of the study are to investigate the renal function in ventricular fibrillation (VF) and asphyxiation cardiac arrest in a swine model and to estimate the value of novel biomarkers in the acute kidney injury (AKI) after cardiac arrest. Method: Thirty-two healthy inbred Wu-Zhi-Shan miniature piglets were randomized into 2 groups (n = 16 per group). Cardiac arrest was induced by programmed electric stimulation and clamping the endotracheal tube in the VF group and asphyxiation group, respectively. Cardiopulmonary resuscitation was done for return of spontaneous circulation (ROSC). Results: One hundred percent (16/16) ROSC was observed in the VF group, and 50% (8/16) in the asphyxiation group (P b .01). All AKI biomarkers elevated significantly after ROSC. The novel biomarkers changed much earlier than the creatinine. The concentration of novel biomarkers in the asphyxiation group was higher than the VF group. Live animals had an oliguria and developed AKI. Characteristic morphological injuries in renal tissues were observed under light microscope and transmission electron microscope and were more serious in the asphyxiation group. Conclusions: Acute kidney injury at early stage of postresuscitation is common in different causes of cardiac arrest. Asphyxiation has more severe kidney injury and gets worse prognosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Patients who survive in an initial cardiac arrest (CA) have significant morbidity and mortality [1-4]. Postresuscitation morbidity is often attributed to cerebral, myocardial and prolonged, complete, whole-body ischemia-reperfusion injury (IRI) [5], which is an unnatural pathophysiologic stage created by successful cardiopulmonary resuscitation (CPR). Such ischemic injury should have major effects on the kidneys. The acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney's excretory function. It is common in any cohort of critically ill patients, such as the survivors of CA. Post-CA AKI is usually considered related to the post-CA syndrome (PCAS) and might affect the long-term survival. However, it does not attract as much important as the cerebral and myocardial injury do. Serum creatinine (sCr) and urine output are the standard diagnostic indexes of the RIFLE (risk, injury, failure, loss, end stage) criteria, Acute Kidney ☆ Conflicts of interests: None. ☆☆ Sources of fundation: Beijing Natural Science Foundation (No. 81372025); Technology Foundation for the Tutor of Beijing Excellent Doctoral Dissertation (No. 20121002501). ⁎ Corresponding author. Department of Emergency Medicine, Beijing Chao-Yang Hospital, Affiliated to Capital Medical University, Beijing 100020, China. Tel./fax: +86 10 85231051. E-mail addresses: [email protected] (C.-C. Hang), [email protected] (C.-S. Li). 0735-6757/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajem.2013.10.043
Injury Network (the AKIN criteria), and Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guidelines for AKI [6,7]. However, both sCr and urine output have a lack of sensitivity and/or specificity; thus, new biomarkers for earlier and more accurate detection are needed [8,9]. Neutrophil gelatinase–associated lipocalin (NGAL), cystatin C (CysC), kidney injury molecule 1 (KIM-1), and Nasetil-β-glukosaminidase (NAG), which are the clinical utility of certain novel AKI biomarkers, already evaluated in various clinical conditions and were deemed as good as or better than creatinine (Cr) in detecting AKI, particularly in the early stage [10,11]. In this study, we hypothesized that postresuscitation AKI was common in both ventricular fibrillation (VF) CA (VFCA) and asphyxiation CA (ASCA). The purpose of this study was to confirm this hypothesis by carrying out the biomarkers on the characteristics of renal function and structures in swine models of CA. 2. Methods This prospective laboratory study was approved by the Capital Medical University Institutional Animal Care Committee and the Beijing Chao-Yang Hospital Affiliated to the Capital Medical University Animal Care and Use Committee. All animals received treatments in compliance with the National Research Council's 1996 Guide for the Care and Use of Laboratory Animals. Anesthesia was titrated in all surgical interventions to avoid unnecessary suffering. The study was
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performed according to Utstein-style guidelines [12] on 32 healthy inbred Wu-Zhi-Shan miniature piglets of either sex, aged 6 to 8 months, whose average weight was 20 ± 2 kg. Before any procedure, animals were randomized into 2 groups with the use of a sealed envelope indicating the animals assignment to either the VFCA group (n = 16) or ASCA group (n = 16). 2.1. Animal preparation The animals were fasted overnight except for free access to water. Briefly, initial sedation in each animal was achieved with intramuscular ketamine 10 mg/kg, midazolam 0.5 mg/kg, and atropine 0.05 mg/kg, followed by ear vein injection of propofol 1.0 mg/kg. The anesthetized animals were intubated with a 6.5-mm cuffed endotracheal tube via direct laryngoscopy. Animals were mechanically ventilated by a volume-controlled ventilator (Servo 900 c; Siemens, Berlin, Germany) using a tidal volume of 10 mL/kg and a respiratory frequency of 12 per minute with room air (fraction of inspired oxygen, 21%). Propofol 1.0 mg/kg and fentanyl 4 μg/kg were then administered intravenously to reach the desired depth of anesthesia and analgesia. Once this depth was reached, 9 mg/kg per hour of propofol and fentanyl 1 μg/kg per hour were given to maintain the anesthesia level. End-tidal partial pressure of carbon dioxide was monitored with an in-line infrared capnography (CO2SMOplus monitor; Respironics, Inc, Murrysville, PA). The respiratory frequency was adjusted to maintain end-tidal partial pressure of carbon dioxide between 35 and 40 mm Hg. Aortic blood pressure (ABP) was measured with a fluid-filled catheter advanced from the left femoral artery into the thoracic aorta. A Swan-Ganz catheter (7F; Edwards Life Sciences, Irvine, CA) was advanced from the left femoral vein and flow directed into the pulmonary artery to measure cardiac output (CO) by the thermodilution method. A 5F pacing catheter was advanced from the right internal jugular vein into the right ventricle to induce VF for the VFCA group. An 18F urine catheter connecting a urine bag was inserted into the bladder by cystostomy to obtain urine. Electrocardiographic lead II was continuously recorded with a multichannel physiologic recorder (BL-420 F Data Acquisition & Analysis System Chengdu TME Technology Co, Ltd, Sichuan, China). Hemodynamics was monitored by an HP monitor (M1165; Hewlett-Packard Co, Palo Alto, CA). All animals received normal saline solution (10 mL/kg per hour) intraoperatively to replenish fluid losses.
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Table 1 Baseline and preparatory phase characteristic in VFCA and ASCA group (mean ± SD)
Male/female Weights (kg) Propofol (mg) Fentanyl (μg) Preparation time (min)
VFCA group (n = 16)
ASCA group (n = 16)
P
9/7 20.3 ± 0.7 120.7 ± 2.4 72.1 ± 1.7 65.6 ± 2.5
10/6 20.1 ± 0.8 120.6 ± 2.1 71.5 ± 2.1 64.1 ± 2.5
1.000 .563 .878 .340 .117
Cardiopulmonary resuscitation was resumed for another 2 minutes after defibrillation attempt. The sequence continued until return of spontaneous circulation (ROSC) or death. ROSC was defined as the maintenance of a systolic blood pressure of at least 50 mm Hg for at least 10 consecutive minutes. The animal death was defined as the resuscitation maintenance of 30 minutes but the ROSC still not achieved. The animals with ROSC received intensive care for 6 hours, and mechanical ventilation with supplemental oxygen continued throughout the immediate postresuscitation period. Six hours after ROSC, all catheters were removed by a surgical procedure [15]. Then the animals underwent euthanasia with propofol 60 mg and were killed with 20 mL of 10 mol/L potassium chloride solution intravenously. If the animals could not live to the prospective end point, the living time was recorded. 2.3. Measurements Hemodynamics, such as heart rate (HR), ABP and CO, was measured continuously. Urine output was recorded per hour. Blood and urine samples were collected for laboratory testing at baseline, immediately at ROSC (ROSC 0), 1 hour after ROSC (ROSC 1 h), 2 hours after ROSC (ROSC 2 h), 3 hours after ROSC (ROSC 3 h), 4 hours after ROSC (ROSC 4 h), and 6 hours after ROSC (ROSC 6 h). Blood was used for measurement of Cr (sCr), serum CysC (sCysC), and serum NGAL (sNGAL), whereas urine was used for the determination of CysC (uCysC), NGAL (uNGAL), KIM-1 (uKIM-1), and NAG (uNAG). Blood and urine samples were centrifuged (3000 rpm) for 20 minutes; then
2.2. Experimental protocol After surgery, the animals were allowed to equilibrate for 1 hour to achieve a stable resting level, and the baseline data were collected. In the VFCA group, VF was induced by a programmed electrical stimulation instrument (GY-600A; Kaifeng Huanan Instrument Co, Kaifeng, Henan, China) with mode S1S2 (300/200 milliseconds), 40 V, 8:1 proportion, and − 10-millisecond step length. VF was verified by the presence of a characteristic electrocardiographic waveform and an immediate drop in ABP. In the ASCA group, animals were paralyzed with 0.2 mg/kg cisatracurium to avoid gasping, and then CA was induced by clamping the endotracheal tube. The piglets were asphyxiated until simulated pulselessness, defined by an aortic systolic pressure less than 30 mm Hg [13]. The electrocardiograph waveform and the asphyxiation induction time were recorded. After 8 minutes of untreated CA [14], mechanical ventilation was resumed with 100% oxygen, and CPR was performed manually. Manual chest compressions were rapidly initiated at a rate of 100 per minute with equal compression-relaxation duration. The compression depth was approximately equivalent to one-third of anteroposterior diameter of the thorax. After 2 minutes of CPR, epinephrine (0.02 mg/kg) was injected into the right atrium, and then CPR was performed manually for 2 minutes. After 4 minutes of CPR, defibrillation was attempted using 4 J/kg (biphasic waveforms shock) for the first attempt.
Fig. 1. Survival function of animals with ROSC in VFCA group (n = 16) and ASCA group (n = 8).
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serum and urine supernatants were stored at − 80°C until measurements, which were performed by an investigator blinded to the study design. Commercially available enzyme-linked immunosorbent assays were used for swine serum and urine AKI biomarkers measurements, according to the manufacturer's instructions (Swine Cr/BUN/CysC/NGAL/KIM-1/NAG; Beijing Xin Fangcheng Biotechnology Company, Ltd, Beijing, China). Assays were run in duplicate. All intra-assay coefficients of variation were less than 10%. After the animals underwent euthanasia, the kidney was excised; a part of tissue samples of lower pole kidney bilaterally was taken rapidly and preserved in 10% formaldehyde or 4% paraformaldehyde to observe pathologic changes under a light microscope (LM) or a transmission electron microscope (TEM), respectively.
test was used to determine differences over time within groups, as appropriate; the Bonferroni t test, for multiple comparisons. Pearson correlation coefficient was calculated to determine the correlations of biomarkers between the serum and urine. Log-rank test was used for survival analysis. Fisher exact test was used for the rate of ROSC analysis. A 2-sided P b .05 was considered statistically significant. 3. Results 3.1. Characteristics of animals The animal profile (sex and weight), time of preparatory phase, and the extra doses of propofol and fentanyl administered during the preparatory phase did not differ significantly in the 2 groups (Table 1).
2.4. Statistical analysis 3.2. Rate of ROSC and survivals Statistical analysis was performed with SPSS 19.0 software (SPSS, Inc, Chicago, IL). Values were shown as mean ± SD. Continuous variables between groups were compared using the Student t test. One-way repeated-measures analysis of variance (ANOVA) or paired t
All animals in both groups had CA. The duration between clamping the tube and CA in the ASCA group was between 13 and 20 minutes (16.8 ± 1.3 minutes). Return of spontaneous circulation was observed
Fig. 2. Invasive hemodynamic values in VFCA and ASCA group at baseline and throughout ROSC 6 h. A, Heart rate. B, Mean arterial pressure. C, Cardiac output. The values are presented as mean (SD). ⁎P b .05 and ⁎⁎P b .01 vs baseline (1-way repeated-measures ANOVA). #P b .05 and ##P b .01 vs VFCA group (Student t test).
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in 100% (16/16) of animals of the VFCA group and only 50% (8/16) of animals of the ASCA group (P b .01). The duration of CPR in the VFCA group was shorter than in the ASCA group (4.8 ± 1.2 vs 8.3 ± 1.4 minutes; P b .01). Average survival time in the VFCA group (n = 16) was longer than that in the ASCA group (n = 16) (5.90 ± 0.09 vs 2.68 ± 0.70 hours; P b .01; Fig. 1). 3.3. Hemodynamics Heart rate, mean arterial pressure (MAP), and CO at baseline did not differ significantly between the 2 groups (P N .05; Fig. 2). Heart rate and MAP increased significantly, whereas CO decreased significantly, after ROSC in both groups (Fig. 2). Heart rate, MAP, and CO were higher in the VFCA group than in the ASCA group at all time points after ROSC (Fig. 2). 3.4. Urine outputs The urine output at baseline was between 6 and 13 mL/kg per hour; no significant difference was found between the VFCA group and ASCA group (7.85 ± 2.05 vs 7.27 ± 2.75 mL/kg per hour; P = .609). Among the live animals, urine outputs of 17 animals were less than 0.5 mL/kg per hour for 6 hours, 12 animals in the VFCA group, and 5 animals in the ASCA group (Table 2). 3.5. Serum and urine AKI biomarkers Serum and urine AKI biomarkers at baseline did not differ significantly between the 2 groups (P N .05; Table 3, Fig. 3). Neutrophil gelatinase–associated lipocalin was the most sensitive one and increased at ROSC 1 h in both serum and urine. Cystatin C increased significantly at ROSC 2 h in serum and at ROSC 3 h in urine. Urine KIM1 and uNAG were less sensitive than NGAL and CysC and increased at ROSC 4 h. Serum Cr lack sensitivity compared with the novel biomarkers. Serum NGAL and uNGAL were higher in the ASCA group than those in the VFCA group after ROSC 1 h. Serum CysC after ROSC 2 h and uCysC, uKIM-1, and uNAG after ROSC 3 h were higher in the ASCA group than those in the VFCA group too (Table 3; Fig. 3).
Table 2 Urine output in VFCA and ASCA group at baseline and throughout ROSC 6 h Group Number Urine output (mL/kg per h) ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 5 h ROSC 6 h VFCA
ASCA
a
1a 2a 3 4a 5a 6 7a 8a 9a 10a 11a 12 13a 14a 15a 16 1a 2a 3 4a 5a 6a 7 8
0.48 0.46 1.20 0.49 0.45 1.20 0.40 0.40 0.49 0.48 0.45 1.00 0.45 0.40 0.45 0.40 0.45 0.45 1.00 0.4 0.45 0.4 0.40 0.35
0.45 0.45 1.10 0.48 0.45 1.25 0.45 0.40 0.45 0.45 0.40 0.80 0.48 0.35 0.48 0.35 0.42 0.48 0.80 0.30 0.48 0.45 0.45 0.25
0.45 0.38 1.10 0.45 0.40 1.20 0.42 0.35 0.48 0.40 0.40 0.90 0.40 0.45 0.49 0.30 0.48 0.40 0.50 0.35 0.40 0.38 0.35
0.44 0.40 1.00 0.48 0.40 1.15 0.48 0.30 0.45 0.45 0.35 0.70 0.35 0.35 0.42 0.30 0.40 0.45 0.75 0.36 0.45 0.42 0.30
0.43 0.42 0.95 0.45 0.30 1.00 0.45 0.25 0.45 0.45 0.40 1.00 0.40 0.36 0.48
0.45 0.44 1.00 0.48 0.36 1.20 0.40 0.25 0.48 0.45 0.38 0.80 0.42 0.35 0.46
0.40 0.42 0.6 0.28 0.42 0.45
0.34 0.45 0.75 0.25 0.48 0.44
Urine output were less than 0.5 mL/kg per hour for 6 hours.
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Table 3 Serum and urine AKI biomarkers in VFCA and ASCA group at baseline and throughout ROSC 6 h (mean ± SD) Biomarker sCr (μmol/L) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h sCysC (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uCysC (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h sNGAL (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uNGAL (ng/mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uKIM-1 (pg/ mL) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h uNAG (U/L) Baseline ROSC 0 ROSC 1 h ROSC 2 h ROSC 3 h ROSC 4 h ROSC 6 h
VFCA group (n = 16)
ASCA group (n = 8)
P
62.38 61.35 62.70 63.29 62.84 66.92 66.90
62.75 62.83 63.13 62.89 67.92 66.90 88.99
.877 .500 .857 .867 .081 .996 b.0001b
± 6.03 ±4.52 ±5.50 ±4.90 ± 6.83 ± 5.72 ± 6.21 (n = 15)
± 4.42 ± 5.84 ± 5.19 ± 6.49 ± 3.78 (n = 7) ± 6.11 (n = 7) ±8.46a (n = 6)
511.86 ± 10.10 511.46 ± 10.71 514.07 ±11.48 517.37 ± 13.73 906.48 ± 14.81d 1011.87 ± 23.65d 1177.46 ± 65.19d (n = 15)
512.65 ± 9.58 517.52 ± 10.50 513.50 ± 9.83 565.96 ± 41.45 1021.89 ±49.82d (n = 7) 1174.37 ± 64.48d (n = 7) 1303.91 ± 87.47d (n = 6)
.856 .203 .906 .013c b.0001b b.0001b .794
207.66 ± 3.60 210.05 ± 4.51 212.66 ± 6.27 216.40 ± 6.57a 217.69 ±6.16d 407.79 ± 8.30d 508.66 ± 11.57 d (n = 15)
206.95 208.60 214.58 218.81 295.99 506.64 635.39
.693 .458 .521 .484 b.0001b b.0001b b.0001b
2.18 ± 2.21 ± 3.78 ± 4.14 ± 4.45 ± 5.03 ± 6.78 ±
0.11 0.12 0.09d 0.12d 0.18d 0.15d 0.17d (n = 15)
2.19 2.22 4.42 4.75 5.46 6.67 7.85
± 0.12 ± 0.13 ± 0.24d ± 0.32d ± 0.25d (n = 7) ± 0.30d (n = 7) ± 0.34d (n = 6)
.922 .866 b.0001b b.0001b b.0001b b.0001b b.0001b
0.58 ± 0.60 ± 1.32 ± 1.53 ± 1.83 ± 2.82 ± 4.24 ±
0.04 0.03 0.06d 0.07d 0.06d 0.08d 0.17d (n = 15)
0.56 0.60 2.20 2.66 3.68 4.92 6.21
± 0.03 ± 0.04 ± 0.15d ± 0.14d ± 0.17d (n = 7) ± 0.27d (n = 7) ± 0.19d (n = 6)
.160 .705 b.0001b b.0001b b.0001b b.0001b b.0001b
± 4.92 ± 4.30 ± 7.80 ± 9.93 ± 16.73d (n = 7) ± 15.11d (n = 7) ± 16.40d (n = 6)
70.36 ± 4.09 71.33 ± 3.63 71.59 ±3.50 71.48 ± 3.21 73.15 ± 1.81 115.21 ± 4.44d 119.53 ± 5.85d (n = 15)
68.27 ± 3.70 70.61 ± 2.62 72.09 ± 1.57 71.93 ± 1.74 83.14 ± 6.44 (n = 7) 139.40 ± 3.98d (n = 7) 143.58 ± 4.03d (n = 6)
.238 .623 .707 .719 .006b b.0001b b.0001b
162.00 ± 4.87 163.90 ± 4.56 164.19 ± 4.35 166.80 ± 3.30 166.95 ± 3.65 280.95 ± 8.60d 306.55 ± 10.99d (n = 15)
159.81 161.13 162.34 166.37 248.11 305.04 330.35
.343 .183 .403 .827 b.0001b b.0001b .001b
± 5.86 ± 4.84 ±6.17 ± 6.24 ± 16.38d (n = 7) ± 9.66d (n = 7) ± 18.09d (n = 6)
a Significant differences compared with baseline: P b .05 (1-way repeated-measures ANOVA). b Significant differences between the VFCA group and the ASCA group: P b .01 (Student t test). c Significant differences between the VFCA group and the ASCA group: P b .05 (Student t test). d Significant differences compared with baseline: P b .01 (1-way repeated-measures ANOVA).
3.6. Correlations of NGAL and CysC between serum and urine High correlations between serum and urine were found in both NGAL and CysC. The correlation of NGAL (r = 0.956; P b .01) was better than CysC (r = 0.788; P b .01).
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Fig. 3. Serum and urine AKI biomarkers in VFCA and ASCA group at baseline and throughout ROSC 6 h. A, Serum CysC. B, Urine CysC. C, Serum NGAL. D, Urine NGAL. E, Urine KIM-1. F, Urine NAG. G, Serum Cr. The values are presented as mean (SD). ⁎P b .05 and ⁎⁎P b .01 vs baseline (1-way repeated-measures ANOVA). #P b .05 and ##P b .01 vs VFCA group (Student t test).
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3.7. Renal histopathology Under the LM, interstitial edema, renal tubular necrosis, glomerular capillaries angiectasis, mesangial proliferation, and inflammatory cells infiltration could be seen in both groups (Fig. 4A and B). Compared with the VFCA group, interstitial edema and acute renal tubular necrosis were more serious. Glomerular capillaries were highly dilated, with more inflammatory cells degenerating in the ASCA group. Moreover, irregular brush border of the renal tubular epithelial cells with more serious
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erosion was found in the ASCA group (Fig. 4B). Under the TEM, there were characteristic morphological changes of apoptosis in both groups, but the changes were more serious in the ASCA group than those in the VFCA group. In the VFCA group, nuclei were in different sizes and shapes, and chromatin was agglutinated. Abundant mitochondria were observed with crista swelling (Fig. 4C and E). However, in the ASCA group, nuclei deformation, chromatin pyknosis, and heterochromatin marginalized were extensive; the mitochondria vacuolization and autophagy could be observed (Fig. 4D and F).
Fig. 4. Renal ultramicrostructure of VFCA (A, C, and E) and ASCA (B, D, and F) groups at 6 hours after ROSC. A, Interstitial edema (①) and mesangial inflammatory cells infiltration (②). B, More severe than A, with serious brush border of the renal tubular epithelial cells erosion and necrosis (③) and glomerular capillaries angiectasis (④). C, Abundant mitochondria (⑤), nuclei in different sizes and shapes (⑥), chromatin agglutinated. D, Nuclei deformation (⑦), chromatin pyknosis, and heterochromatin marginalized. E, Abundant mitochondria (⑧) with crista damage accidentally. F, Mitochondria vacuolization and autophagy (⑨).
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4. Discussion The high mortality of patients who initially achieve ROSC after CA can be attributed to PCAS that involves multiple organs, and epidemiological data suggested that the mortality of PCAS was nearly 70% [5]. Because there is no doubt about the importance of heart and brain, post-CA myocardial dysfunction and brain injury as well as the systemic IRI are always paid close attention. However, the kidney is ignored, although the injured kidneys may play a key role in the systemic derangement present during multiorgan failure. Post-CA AKI is so common in the survivors of CA with a reported incidence of 23.2% [16], but there is limited information on the epidemiology. Incomplete recovery of renal function from AKI may cause excessive long-term morbidity and mortality and may have a higher risk of chronic kidney disease [17-19]. Many survivors need renal replacement therapy during the advanced life support phase or for lifelong time. So it is worth paying more attention to post-CA AKI. In this study, we measured 4 previously identified candidate biomarkers combined with the Cr to predict the AKI in 2 CA models and found that all the survival animals in both groups got abnormalities in AKI biomarkers. Serum NGAL and uNGAL increased much earlier than other biomarkers. Cystatin C increased within ROSC 2 to 3 hours in serum and urine. Urine KIM-1 increased at ROSC 4 h as well as NAG. Creatinine remained stable until ROSC 6 h. Seventeen live animals had an oliguria and developed to RIFLE injury or KDIGO/AKIN Stage 1. Combining with the kidneys' histopathology evidence, both indicated that animals with different causes of CA did suffer AKI, due to glomerular dysfunction and tubular injury. The mechanism of post-CA AKI is thought to be associated with IRI. Milder ischemia can lead to pre-renal dysfunction, which is relatively slight in all AKIs and may be reversed. When ischemia is more severe and renal tubular injury evolves to tubular necrosis [20], the prognosis of AKI will be disappointing. After ischemia, the whole-body reperfusion will further aggravate the kidney injury. Moreover, it has recently been demonstrated that AKI may not just be a consequence of CA but of time without spontaneous circulation. The underlying cause of CA, PCAS, and cardiogenic shock may strongly influence the prognosis of AKI [21]. In addition, we could also get knowledge that novel biomarkers had a higher sensitivity in diagnosis of AKI. As the international criterion standard for identification and classification of AKI was still dependent on Cr and urine output measurements, which were not specific for renal tubular lesions, the incidence of post-CA AKI might be higher than previously anticipated. In our study, all of the novel AKI biomarkers indeed increased, which suggested that all survival animals had AKI more or less. Some animals had AKI so early and so mild that they had normal Cr. Furthermore, AKI might happen much earlier than expected because the NGAL increased as early as ROSC 1 h and the other novel biomarkers increased before ROSC 6 h. Because the study of the CA animal models is mainly on VF, there is still confusion whether the post-CA kidney function was the same in asphyxiation. In our study, we compared the biomarkers in the 2 different models and found that the concentration of sNGAL and uNGAL in the ASCA group was higher than those in the VFCA group after ROSC 1 h. The similar results were found in sCysC after ROSC 2 h and in uCysC, uKIM-1, and uNAG after ROSC 3 h. These differences might suggest that the animals in the ASCA group had more serious kidney injury, especially tubular necrosis, than the animals in the VFCA group. This conclusion was verified by the histopathologic evidence of LM and TEM. The mechanism of the difference post-CA kidney injury is yet unknown but is thought to be related to either IRI or anoxia injury. In both groups, the kidney had IRI after ROSC. Oxygen debt, generalized activation of immunologic and coagulation pathways, adrenal insufficiency, and
necrosis are all important pathophysiologic mechanisms that may contribute to microcirculatory reperfusion disorders and kidney injury [22]. It is obvious that the ASCA group had a severe anoxia besides the IRI. As known, kidney is sensitive to anoxia, especially the tubular. When asphyxiation occurs, the whole body suffers hypoxemia, hypercarbia, and acidemia, which may led to a severe and irreversible acute tubular necrosis. Furthermore, in the ASCA group, MAP and CO decreased significantly after resuscitation compared with the VFCA group, which might result in worse renal reperfusion after ROSC. All those would exacerbate AKI. Collectively, the results of ecsomatics and histology demonstrated that both VFCA and ASCA could result in a severe kidney injury, but the injury was more serious in ASCA. 4.1. Limitations The number of animals achieved ROSC in the ASCA group is relatively small, which may influence the outcome. The primary end point of the experiment was set at 6 hours after ROSC, so we could not get knowledge of the tendency of the AKI biomarkers after then, which is thought to be important. The experiment was conducted on apparently healthy animals, whereas most individuals who had CA have underlying pathologic findings. 5. Conclusions Our findings suggest that post-CA AKI that occurs at an early stage of postresuscitation is common in both VFCA and ASCA, but ASCA has more severe kidney injury and worse prognosis. The novel AKI biomarkers in serum and urine, such as NGAL, CysC, KIM-1, and NAG, are of significant importance as early predictors of post-CA AKI. Acknowledgments The authors thank Yi Zhang, Qin Yin, Zhi-Jun Guo, Shuo Wang, and Qian Zhang for excellent technical assistance. References [1] Nichol G, Thomas E, Callaway CW, et al. Regional variation in out-of-hospital cardiac arrest incidence and outcome. JAMA 2008;300:1423–31. [2] Sandroni C, Nolan J, Cavallaro F, et al. In-hospital cardiac arrest: incidence, prognosis and possible measures to improve survival. Intensive Care Med 2007;33:237–45. [3] Deasy C, Bray JE, Smith K, et al. Out-of-hospital cardiac arrests in the older age groups in Melbourne, Australia. Resuscitation 2011;82:398–403. [4] Deasy C, Bray JE, Smith K, et al. Out-of hospital cardiac arrests in young adults in Melbourne, Australia. Resuscitation 2011;82:830–4. [5] Neumar RW, Nolan JP, Adrie C, et al. Post-cardiac arrest syndrome: epidemiology, pathophysiology, treatment, and prognostication. A consensus statement from the International Liaison Committee on Resuscitation (American Heart Association, Australian and New Zealand Council on Resuscitation, European Resuscitation Council, Heart and Stroke Foundation of Canada, Inter American Heart Foundation, Resuscitation Council of Asia, and the Resuscitation Council of Southern Africa), the American Heart Association Emergency Cardiovascular Care Committee, the Council on Cardiovascular Surgery and Anesthesia, the Council on Cardiopulmonary, Perioperative, and Critical Care, the Council on Clinical Cardiology, and the Stroke Council. Circulation 2008; 118(23):2452–83. [6] Bellomo R, Kellum JA, Ronco C. Acute kidney injury. Lancet 2012;380:756–66. [7] KDIGO. Clinical practice guideline for acute kidney injury. Kidney Int Suppl 2012;2:1 e138. [8] Uchino S, Kellum JA, Bellomo R, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005;294:813–8. [9] Hewitt SM, Dear J, Star RA. Discovery of protein biomarkers for renal diseases. J Am Soc Nephrol 2004;15:1677–89. [10] Sharma RK. Biomarkers of acute kidney injury. Clin Queries: Nephrol 2012;0101: 13–7. [11] Adiyanti SS, Loho T. Acute kidney injury (AKI) biomarker. Acta Med IndonesIndones J Intern Med 2012;44(3):246–55. [12] Idris AH, Becker LB, Ornato JP, et al. Utstein-style guidelines for uniform reporting of laboratory CPR research. A statement for healthcare professionals from a task force of the American Heart Association, the American College of Emergency Physicians, the American College of Cardiology, the European
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