Male Sprague-Dawley rats (weighing 200–250 g) were obtained from the Changsheng Biotech Co., Ltd. (Beijing, China) and housed under specific pathogen-free conditions at a constant temperature of 20–22°C and humidity of 50–60% with a 12-h light/dark cycle, and were allowed free access to food and water. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of China Medical University, Shenyang, China.

CLP procedure was performed to initiate sepsis in rats according to previously published protocols (18). In short, the rats were first anaesthetized by the intraperitoneal (i.p.) administration of 10% chloral hydrate (350 mg/kg; Sinopharm, Beijing, China), and a ventral midline incision (1.5 cm in length) was made on the rats. The caecum was exposed, ligated, punctured with a gauge needle 3 times, and then placed back into the abdomen. Rats that underwent sham operation (the caecum was exposed by a ventral midline incision, but was not ligated or punctured) were used as the controls. Thereafter, the abdominal incision was closed in layers with 3-0 surgical sutures, and the rats were allowed to recover from the anaesthesia. These rats were randomly divided into 6 groups (n=8/group) as follows: i) group 1: the sham-operated group (Sham); ii) group 2: the sham-operated group administered the low dose of sivelestat (Sham + L-sivelestat); iii) group 3: the sham-operated group administered the high dose of sivelestat (Sham + H-sivelestat); iv) group 4: the rats with sepsis who were not treated (Sepsis); v) group 5: the rats with sepsis who were administered the low dose of sivelestat (Sepsis + L-sivelestat); and vi) group 6: the rats with sepsis who were administered the high dose of sivelestat (Sepsis + H-sivelestat). The rats in groups 2 and 3, and 5 and 6, rats were administered an i.p. injection of 50 or 100 mg/kg body weight sivelestat (Ono Pharmaceutical Co., Osaka, Japan) immediately after the sham-operation or the initiation of sepsis. The rats in groups 1 and 4 received an infusion of normal saline (vehicle) into the intraperitoneal cavity. The doses of sivelestat used in our study were selected based on our preliminary experiments (data not shown) and a previous study (12). Blood samples from each rat were obtained at 6 and 24 h post-surgery, and the rat kidneys were rapidly removed at 24 h post-surgery after sacrifice (by an overdose of anesthetics) and immediately frozen in liquid nitrogen or fixed in 10% formaldehyde (Sinopharm) for use in subsequent experiments.

In order to determine whether sivelestat affects animal survival, the mortalities were measured in another set of rats treated with or without sivelestat up to 108 h after the procedure (n=10/group). In addition, the mean arterial pressure (MAP) in these rats was assessed using a non-invasive blood pressure monitoring system at 24 h according to the manufacturer's instructions (ALC-NIBP, Alcott Biotech Co., Ltd., Shanghai, China).

Serum samples were centrifuged at 1,000 rpm for 20 min, and then subjected to the detection of blood urea nitrogen (BUN), neutrophil gelatinase-associated lipocalin (NGAL) levels, tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) levels and NE levels in the serum and renal tissues using commercially available kits (Boster, Uscn Life Science, Inc., Wuhan, China) according to the manufacturer's instructions.

The rats that underwent CLP or sham surgery were anaesthetized at 24 h and subjected to the following assessments. GFR was determined by measuring the inulin clearance. In brief, inulin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline to a concentration of 5 mg/ml, and then administered through the femoral vein at a dose of 1 ml/h/100 g body weight. Following equilibration, the urinary bladder was cannulated to collect a 30-min urine sample. In addition, a blood sample (1 ml) was collected at the midpoint of the urine collection period. Ten minutes later, urine and blood samples were collected again. Inulin concentrations in the urine and plasma were determined using the anthrone method, while sodium and creatinine concentrations were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively. The values of GFR and FE_Na were calculated through standard formulas, as previously described (19–21): GFR = [(urine inulin) x (volume of urine collected)]/[(plasma inulin) x (time of collection)], and expressed as ml/min/100 g body weight; FE_Na = 100 x [(urine sodium x plasma creatinine)/(plasma sodium x urine creatinine)].

The formaldehyde-fixed kidney tissues were embedded in paraffin, cut into 5-µm-thick sections, and then deparaffinized in xylene and hydrated in graded ethanol. For the morphological examination, these slices were stained with hematoxylin (Solarbio, Beijing, China) and eosin (Sinopharm). For immunohistochemistry, these sections were heated in citrate buffer at 100°C for 10 min to retrieve the antigen and then treated with 3% hydrogen peroxide (both from Sinopharm) at room temperature for 15 min to block the endogenous peroxidase activity. After being blocked with normal goat serum at room temperature for 15 min, these sections were incubated with rabbit polyclonal antibodies against high-mobility group box 1 (HMGB1; 1:100 dilution, bs-0664R), inducible nitric oxide (NO) synthase (iNOS; 1:100 dilution, bs-0162R) (both from Bioss, Beijing, China) and a mouse monoclonal antibody against ED-1 (1:50 dilution, sc-59103; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. These sections were then incubated with biotin-labeled goat anti-rabbit IgG (1:200 dilution, A0277) or anti-mouse IgG (1:200 dilution, A0286) at 37°C for 30 min, and treated with horseradish peroxidase (HRP)-conjugated streptavidin (all from Beyotime, Shanghai, China) for an additional 30 min. Finally, the tissue sections visualized with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin (both from Solarbio), and evaluated under a light microscope (Olympus, Tokyo, Japan).

Total protein samples were extracted from the kidney tissues using RIPA buffer (Beyotime) and the protein concentrations were evaluated by bicinchoninic acid assay. An equivalent amount of each protein sample (40 µg) was loaded and separated through sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat milk, and incubated overnight at 4°C with rabbit polyclonal antibodies against NE (1:500 dilution, bs-6982R), HMGB1 (1:500 dilution, bs-0664R), iNOS (1:500 dilution, bs-0162R), serine/threonine kinase (Akt; 1:500 dilution, bs-6951R) and phosphorylated Akt (p-Akt; 1:500 dilution, bs-0876R). All primary antibodies were purchased from Bioss. Thereafter, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000 dilution, A0208; Beyotime) at 37°C for 45 min and visualized using an enhanced chemiluminescence (ECL) system (Seven Sea Pharmtech Co., Ltd., Shanghai, China). The protein expression levels were expressed as a ratio to the endogenous control, β-actin.

All data are presented as the means ± standard deviation (SD), and analyzed using GraphPad Prism version 6.0 or SPSS version 20.0. One-way analysis of variance (ANOVA) was performed on data obtained from the same time point, whilst two-way ANOVA was performed on data from different time points. The Bonferroni test was utilized for post hoc comparisons between different groups. A P-value <0.05 was considered to indicate a statistically significant difference. Survival data were analyzed by the Kaplan-Meier curve and log-rank (Mantel-Cox) test.

We first evaluated whether the NE levels were affected by the CLP procedure using an ELISA kit and western blot analysis. Our results revealed that the administration of sivelestat alone generated no changes in comparison with the sham-operated rats (Fig. 1). The serum NE level was significantly increased at both 6 and 24 h post-CLP surgery, whereas decreased after sivelestat treatment (Fig. 1A). In addition, renal NE expression was significantly enhanced at 24 h post-CLP surgery, whereas it was suppressed by treatment with sivelestat (Fig. 1B and C). The higher dose of sivelestat was more effective than the lower dose.

Another set of rats (10 per group) was subjected to survival analysis for 108 h. As indicated in Fig. 2A, no sham-operated rats treated with 50 or 100 mg/kg sivelestat or the vehicle died during the whole experimental period. All rats in the sepsis group were dead at 108 h post-CLP surgery, while 4 and 6 septic rats survived for 108 h when treated with 50 (P=0.062 vs. sepsis group) and 100 mg/kg sivelestat (P<0.01 vs. sepsis group), respectively (Fig. 2A). As compared with the sham-operated group (104±5.8 mmHg), CLP induced a reduction in MAP (89±7.2 mmHg) (P<0.001), which was restored by sivelestat (Fig. 2B). Morphological changes in the rat kidneys were determined by H&E staining and the parameters related to renal function were detected using commercial kits. Pathological alterations in the rat kidneys were characterized by the loss of the brush border, tubular cell flattening and tubular lumen dilation, while the administration of sivelestat partly reversed these changes (Fig. 2C). In addition, sivelestat did not affect the serum BUN levels, NGAL levels, inulin clearance and FE_Na in the rats that underwent sham surgery (P>0.05; Fig. 2D–G). However, CLP-induced a significant upregulation in serum BUN and NGAL levels in the rats and this effect was suppressed by sivelestat, with the higher dose being more effective (Fig. 2D and E). Additionally, the decreased inulin clearance (indicating reduced GFR) in the rats with sepsis was partly reversed by sivelestat (Fig. 2F). The CLP-induced increase in FE_Na (indicating tubular dysfunction) was slightly reduced by sivelestat, but failed to reach statistical significance (Fig. 2G). Collectively, the administration of sivelestat protected the rats against sepsis induced by multiple bacterial infection. Since the lower dose of sivelestat did not induce mortality, kidney distortion or renal dysfunction in the sham-operated rats, this group was excluded from the following mechanism experiments.

The overproduction of pro-inflammatory mediators in response to bacterial infection is one of the major characteristics of sepsis (22). Therefore, the serum levels of TNF-α and IL-1β were assessed in this study. We found that the levels of these two pro-inflammatory cytokines were significantly increased at 6 and 24 h post-CLP procedure (Fig. 3A). Although sivelestat alone generated no changes in the serum levels of TNF-α and IL-1β as compared to the sham-operated group (P>0.05), a marked inhibitory effect of sivelestat on these two pro-inflammatory cytokines in the rats with sepsis rats was observed (Fig. 3A). Immunohistochemistry was also performed in the renal tissues to detect the ED-1-positive macrophages (a marker of macrophage infiltration) at 24 h after the surgical procedures (Fig. 3B). As indicated in Fig. 3C, the number of renal ED-1-positive cells was increased from 1.63±1.92 to 13.38±5.29 per 0.095 mm² after CLP surgery, but was decreased by the administration of sivelestat (9.13±2.64 by L-sivelestat; 5.38±1.77 by H-sivelestat). Unlike TNF-α and IL-1β that are released immediately in response to bacterial infection, HMGB1 is a late pro-inflammatory mediator (23). We therefore wished to determine whether sivelestat can reduce HMGB1 expression by detecting its renal expression at 24 h after the sham or CLP procedure using immunohistochemical staining and western blot analysis. A weak nuclear expression of HMGB1 was observed in a few renal parenchymal cells in the control kidney tissues (Fig. 3B). However, the number of HMGB1-positive cells was significantly increased after the CLP procedure, whereas it was decreased by treatment with sivelestat at different doses (Fig. 3B). The results from western blot analysis confirmed the immunohistochemical results (Fig. 3D). The above-mentioned results indicate an anti-inflammatory effect of sivelestat in a model of sepsis-related AKI.

The polymicrobial infection induced-overproduction of pro-inflammatory mediators can accelerate the release of powerful secondary mediators, such as NO (24). Given the fact that the production of NO is predominantly mediated by iNOS (25), we evaluated renal iNOS protein expression by using immunohistochemical staining and western blot analysis. We found that iNOS was expressed in the cytoplasm of renal tubule epithelial cells (Fig. 4A). As compared with the normal kidney tissues, the more intense expression of iNOS was observed in the septic kidney tissues (Fig. 4A). However, the upregulation of iNOS expression was significantly inhibited by treatment with sivelestat (Fig. 4A). These results were confirmed by western blot analysis (Fig. 4B).

The activation of the Akt pathway was assessed by western blot analysis of the Akt phosphorylation product, p-Akt in the rat kidney tissues at 24 h post-surgery. We noted that total Akt protein expression in the kidney tissues remained unaltered in the different experimental groups (Fig. 5). Treatment with sivelestat alone had no significant effect on renal p-Akt expression in the rats subjected to sham operation (Fig. 5). However, a marked increase in the phosphorylation levels of Akt was observed in the septic renal tissues. The high phosphorylation levels of Akt induced by sepsis were suppressed after low-dose sivelestat treatment and the levels almost returned to normal after high-dose sivelestat treatment (Fig. 5). Our data thus suggest that the administration of sivelestat suppresses the CLP-induced activation of Akt in rat kidneys.