A total of 70 specific-pathogen free healthy male Sprague-Dawley rats (age, 12 to 14 weeks; weight, 370±20 g) were purchased from Chengdu Dashuo Biological Technology Co., Ltd. (Chengdu, China). The rats were housed under a 12/12 h light/dark cycle in a temperature-(22±2°C) and humidity-(40 to 60%) controlled room with free access to fresh water and standard laboratory food. The procedures for animal care were approved by the Committee on the Ethics of Animal Experiments at Sichuan University (Sichuan, China).

The bipolar pacing electrode used for inducing ventricular fibrillation was the SelectSecure Model 3830 (Medtronic, Inc., Minneapolis, MN, USA). The PGE1 solution (5 µg/ml) was purchased from Beijing Tide Pharmaceutical Co., Ltd. (Beijing, China). The vascular endothelial (VE)-cadherin antibody (cat. no. #2500) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Vascular endothelial growth factor receptor (VEGFR) antibody (cat. no. #BS4205) was purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA). The RNA prep pure, Quant cDNA and SuperReal PreMix Plus (SYBR-Green) kits were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The rat TM ELISA kit (cat. no. #CSB-E07939r) was purchased from Cusabio Biotech Co., Ltd. (Wuhan, Hubei, China). The rat IL-6 (cat. no. #ab00772) and TNF-α ELISA kit (cat. no. #ab100785) were purchased from Abcam (Shanghai, China).

A total of 14 rats were included in each group. Rats without VF were considered to be the Sham group (S). VF was induced in the experimental rats by transesophageal cardiac pacing with an alternating current (50 Hz, 6 mA, 4 msec) for 150 s (13,14). After 4 min, the rats with cardiac arrest were resuscitated with 200 times/min chest compression using a home-made animal cardiopulmonary resuscitator. ROSC for the rats was defined as the return of supraventricular rhythm with a mean aortic pressure of ≥60 mmHg for a minimum of 10 min (15). A total of 56 rats with successful cardiopulmonary resuscitation (CPR) were established as the ROSC model and then randomly divided into 4 groups: ROSC control group (R), PGE1 group [P; 1 ml PGE1 solution was administered intravenously with a syringe pump in 1 min (high injection speed would decrease the success rate of resuscitation)], TTM group (T; body temperature was decreased to 33±1°C within 30 min by placing ice around the rats) and PGE1/TTM group (PT; combined treatment with PGE1 and mild hypothermia). Blood samples were drawn from the left femoral vein of 5 rats from each group at 0.5, 4 and 8 h to evaluate the concentration of TM, TNF-α and IL-6. In every group, 3 rats were sacrificed at each time point (0.5, 4 and 8 h) for VE-cadherin and VCAM-1 mRNA expression analysis with the renal tissue homogenate. At 8 h post treatment, hematoxylin and eosin (H&E) and VE-cadherin/VEGFR double fluorescent immunohistochemistry (IHC) staining was performed (Fig. 1).

H&E staining was performed on tissue slides as described previously (16). Cell edema, inflammatory cell infiltration and micro-thrombus formation were examined by microscopy using the blind method. Tubular injury was scored according to Pallor's method (17): In total, 100 tubules from 10 different high power fields were scored. Higher scores represented more severe damage (maximum score/tubule was 10), with points given for the presence and extent of tubular epithelial cell flattening (1 point), brush border loss (1 point), cell membrane bleb formation (1 or 2 points), interstitial edema (1 point), cytoplasmic vacuolization (1 point), cell necrosis (1 or 2 points) and tubular lumen obstruction (1 or 2 points). Two technicians calculated these scores and the average values of the two sets of scores were recorded.

VE-cadherin/VEGFR double fluorescent IHC staining was performed according to the manufacturer's instructions (18). I/R injury to the RMEC was assessed by the extent of the damage exhibited by VE-cadherin immunostaining in the majority of the renal microvascular endothelium.

Renal total RNA was extracted using the RNA prep pure kit according to manufacturer's instructions. The quantity of RNA product was determined using a UV-Visible spectrophotometer (Thermo Scientific Evolution 201; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the ratio of 28S, 18S and 5S bands on a 2% agarose gel. Then, total RNA was reverse transcribed to cDNA using the Quant cDNA kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's instructions. cDNA was used as a template for quantification of the genes of interest by their primers on the CFX-96 Touch Real-time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The Super Real PreMix Plus (SYBR-Green) kit (Tiangen Biotech Co., Ltd.) was used for qPCR, according to the manufacturer's instructions. The thermocycling conditions for qPCR were as follows: 95°C for 15 min, then 40 cycles of 95°C for 10 sec, 55°C for 20 sec and 72°C for 30 sec. Actin served as the internal control. The primers used were as follows: VE-cadherin, forward, 5′-CATCCGCAAGACCAGTGAC-3′ and reverse, 5′-ACCACGTCCTTGTCTGTTGC-3′; VACM-1, forward, 5′-TACATTGGCACCATCTCA-3′ and reverse, 5′-GTTCAGCATCAGGGAGTT-3′; β-actin, forward, 5′-CCCATCTATGAGGGTTACGC-3′ and reverse, 5′-TTTAATGTCACGCACGATTTC-3′.

ELISA kits were used for the evaluation of TM, TNF-α and IL-6 concentrations in the blood collected at 0.5, 4 and 8 h for each group and were performed according to manufacturer's instructions.

Data are presented as mean ± standard deviation. Comparisons between groups were analyzed by one-way analysis of variance. Comparisons between 2 groups with P<0.05 were further analyzed by Student-Newman-Keuls analysis. Using Pearson's correlation analysis, the correlation between TM peak concentration at 8 h and the concentration of TNFa, and between TM peak concentration and the concentration of IL-6 in each group was analyzed. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 19.0 software (IBM SPSS, Armonk, NY, USA).

Rat kidney sections were stained using H&E and examined using a light microscope (magnification, ×400; Fig. 1). The morphology of the glomeruli and tubules was assessed. The normal structure was preserved in the S group, while a decrease in glomerulus size and widespread degeneration of the tubular architecture, intratubular cast formation and luminal congestion with extensive loss of the brush border were observed in the R group. The P and T groups exhibited marked improvements in histological features associated with renal injury. In the PT group, there was no significant difference when compared with the sham group (Fig. 2).

Blind review of the specimens from different groups revealed greater tubular injury in the R group. The P and PT groups exhibited a significant protective effect; the cytoplasmic vacuolization, cellular necrosis, tubular luminal debris and obstruction were much more marked in the R group (P<0.05). Statistical analysis revealed that the PT group experienced greater benefits with respect to reductions in injury than the P and T groups (P<0.05; Fig. 3).

The kidney is a blood vessel-rich organ. The microvascular endothelial cells are primarily present in the glomerular and peritubular capillary bed. To determine the extent of the damage caused by I/R injury to the RMEC, the pattern of VE-cadherin immunostaining was examined. Under physiological conditions, VE-cadherin immunostaining was noted along the renal microvascular endothelium. Following I/R injury, the loss of VE-cadherin immunostaining in the majority of the renal microvascular endothelium suggested a disruption of the normal junctional complex. The destruction of VE-cadherin in the P, T and PT groups was markedly decreased compared with the R group, while the PT group was similar to the sham operation group under physiological conditions (Fig. 4).

Relative quantitative analysis of mRNA expression of VE-cadherin and VCAM-1 genes in renal tissue with different interventions was performed using RT-qPCR. In I/R injury, enhanced VE-cadherin mRNA expression was observed in the rat kidney within 8 h following ROSC. When compared to the ROSC control group, at the 0.5 and 4 h time points, the 3 intervention groups significantly slowed the rise of VE-cadherin mRNA expression (P<0.05). However, no statistical differences were identified between these 3 intervention groups (Fig. 5).

VCAM-1 mRNA expression was also examined to assess I/R injury to the RMEC following ROSC. When compared to the S group, all of the other groups exhibited significantly upregulated expression of VCAM-1 mRNA within 8 h (P<0.05). When VCAM-1 mRNA expression rose to the highest level at 4 h, the 3 different interventions demonstrated a marked ability to decrease the expression (P<0.05). There was no marked difference between the R group and P or T group at 8 h; however, the PT treatment did significantly reduce the mRNA expression of VCAM 1 (P<0.05; Fig. 6).

Circulating TM, a common early indicator of endothelial injury, was used as a cell marker in the present study. I/R injury induced a rapid increase in circulating TM concentration. All 3 of the interventions provided a protective effect, however, the PT group appeared to have a much greater advantage (Table I).

TNF-α and IL-6 concentrations were chosen as the parameters for the study of the anti-inflammatory mechanism. The concentrations did not alter significantly immediately following the I/R injury and ROSC. However, they gradually increased over time and peaked at 8 h. The 3 interventions provided beneficial effects by preventing the elevation of TNF-α concentration following 4 h. However, only the PT group demonstrated a much greater protective effect in TNF-α and IL-6 when compared with the other groups at 8 h (Table II).

Statistical analysis demonstrated that, at 8 h, I/R injury generated peak levels of circulating TM, TNF-α and IL-6 concentrations, and the interventions simultaneously induced the most significant effects. Pearson's correlation analysis demonstrated that peak TM levels were significantly associated with peak levels of TNF-α and IL-6 concentrations (Fig. 7).