Adult male C57BL/6 mice (n=40, 6-8 weeks old, 22-26 g) were obtained from the Shanxi Dayi Hospital (Taiyuan, China). All mice were allowed to have free access to food and water and maintained in a specific pathogen-free animal facility under a 12-h light/dark cycle at a controlled temperature (20-24°C) and in a humid (50±5%) environment. Mice were randomly divided into 4 groups. In the control group, a total of 8 mice were intraperitoneally injected with normal saline (Control). In the lipopolysaccharide (LPS) group, a total of 32 mice were injected with a single dose of LPS (20 mg/kg; Sigma-Aldrich; Merck KGaA). Mice in the LPS and Control group were intraperitoneally anesthetized with 50 mg/kg of 5% barbitone and subjected to a thoracic incision 12, 24 and 48 h after the injection. The blood samples (~0.3 ml per mouse) were collected by heart puncture and maintained at 37°C for 30 min. After 10 min of centrifugation at 3,500 × g at 4°C, the serum was used for measurement of renal function and ELISA. Kidney tissues were collected by bilateral nephrectomy. After removing the kidney capsule and perirenal fat, the left kidney tissues were fixed in 10% neutral formalin at 4°C for 24 h for histopathology, while the right kidney tissues were segmented for gene expression analyses. Animal experimental procedures were approved by the Ethics Committee of Committee of Shanxi Dayi Hospital and conducted in accordance with Guide for the Care and Use of Laboratory Animal.

The kidney tissues were fixed with 10% neutral formalin for <24 h and then washed with PBS. Mice kidneys were dehydrated with a series of graded ethanol and then embedded in paraffin and sliced into 3-μm-thick sections for H&E. The paraffin-embedded sections were stained with hematoxylin and eosin for 5 min at room temperature (Beijing Solarbio Science & Technology Co., Ltd.) and visualized under a light microscope (Leica Microsystems GmbH).

The collected serum was analyzed on the Roche molecular P800 autobiochemical analyzer (Roche Diagnostics GmbH). Blood urea nitrogen (BUN) levels were determined using Urease GLDH method, according to Urea Nitrogen Assay kits (Ningbo Purebio Biotechnology Co., Ltd.) and the levels of serum creatinine (Cr) were assayed following the protocol of the Creatinine Detection kits (Whitman Biotechnology Co., Ltd.).

The renal tubular epithelial HK-2 cells (American Type Culture Collection) were cultured in 5% CO₂ at 37°C in a humidified incubator with Dulbecco's modified Eagle's medium (HyClone/Pierce; Thermo Fisher Scientific, Inc.), which was supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% ampicillin. The culture medium was refreshed every 2 days until cell confluence reached 70-80%. Then, the cells were resuspended by digestion with 0.25% trypsin/EDTA. The cells were exposed to LPS (Sigma-Aldrich; Merck KGaA) in concentrations of 0, 0.1, 1, 10 and 100 μg/ml. A total of 24 h after LPS exposure, changes in cell viability and inflammation-associated factor levels were assessed to determine appropriate LPS concentration.

In order to investigate the effects of CD39 on the LPS-induced injury in HK-2 cells, the full-length mouse CD39 in pcDNA3.1 overexpression vector, empty pcDNA3.1 vector (Mock), small interfering RNA of CD39 (siCD39; 5′-GCG ATT GTC AGT GAA ACT T-3′) and small interfering-negative control RNA (siNC; 5′-CCT ATC TGG TCA ACA CGT ATT-3′) were obtained by GenePharma (Shanghai GenePharma Co., Ltd). A total of ~4×10⁵ HK-2 cells were incubated in each well of 6-well plates with complete medium until the cells reached 70% confluence. Cell transfection with 50 nmol/l vector was conducted according to the manufacturer's protocol for Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfected cells were subjected to 10 μg/ml LPS (Sigma-Aldrich; Merck KGaA) treatment for subsequent experiments. The activity was measured 24 h after the transfection experiment.

The cell viability was investigated using Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. All types of transfected cells treated with or without LPS exposure were cultured in 96-well plates (4×10³ cells/well). Then, 10 μl CCK-8 solution was added to each well and the absorbance was determined using a microplate reader (Bio-Rad Laboratories, Inc.) at a wavelength of 450 nm.

The levels of IL-1β, IL-18, IL-6 and TNF-α in blood serum and HK-2 cell supernatant were determined using commercially available ELISA kits (cat. nos. H002, H007, H015 and H052; Nanjing Jiancheng Bioengineering Institute). HK-2 cell culture supernatant was collected by a 5-min centrifugation at 10,000 × g at 4°C. In all cases, the ELISA was performed according to the manufacturer's protocols. In brief, the ELISA plates were coated with capture antibodies (100 μl/well). The serum or supernatant (serum/supernatant: 1:4) was incubated in the ELISA plate supplemented with the prepared standard liquid. The horseradish peroxidase-labeled antibodies were added into each well and maintained at 37°C for 1 h. After the reaction was terminated by phosphoric acid, optical density was determined by a microplate reader at 450 nm (Biotek, Synergy HT).

For apoptosis analysis, HK-2 cells from each experimental group were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) using Annexin-V-FITC cell apoptosis assay kit (Sigma-Aldrich; Merck KGaA) in the dark at 25°C for 15 min. Then, apoptosis rate was analyzed under the FACSCalibur flow cytometer (BD Biosciences) equipped with Cell Quest software (version 3.3, BD Biosciences). The proportions of early and late apoptotic cells were counted and compared.

After 24 h of exposure to LPS, the ROS levels of each type of transfected HK-2 cells was assessed by the 2′,7′-dichlorofluorescein diace-tate (DCFDA) Cellular Reactive Oxygen Species Detection Assay kit (Abcam). In brief, DCFDA was dissolved in PBS at a final concentration of 20 μl/ml. The cells were cultured with DCFDA solution for 30 min at 37°C. After being washed with PBS, the levels of ROS were analyzed by flow cytometry (BD Biosciences).

Total RNA was extracted from the collected kidney tissues and transfected HK-2 cells using a high-purity total RNA Rapid Extraction kit (BioTeke Corporation). The isolated RNAs (1 μg) were mixed with nuclease-free water for cDNA synthesis using a Script cDNA Synthesis kit (Bio-Rad Laboratories, Inc.) at 37°C for 15 min and at 85°C for 5 sec. RT-qPCR was performed using LightCycler technology (Roche Diagnostics GmbH) with FastStart DNA MasterPLUS SYBR-Green I (Roche Diagnostics GmbH) detection. The qPCR reaction volume was 20 μl and contained template cDNA, 250 nM of each primer, and 4 μl of 5X SYBR-Green Master Mix. The PCR was performed by 50 cycles at 95°C for 10 sec, at 95°C for 10 sec, at 60°C for 20 sec and at 72°C for 30 sec. Relative gene expression was normalized to GAPDH and calculated using the 2^-ΔΔCq method (21). All primers are listed in Table I.

Following treatment, the protein expression in kidney tissues and HK-2 cells was detected by a western blot assay. Total protein was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology), following the protocols of the manufacturer. Protein concentrations were quantified using a bicinchoninic protein assay kit (Beyotime Institute of Biotechnology). The lysates (20 μg/lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvi-nylidene fluoride membranes. Blots were blocked with non-fat milk (5%) at room temperature for 2 h and then incubated with various primary antibodies at 4°C overnight. The membranes were then washed and cultured with secondary antibodies (1:2,000; cat. nos. ab205718 and ab205719; Abcam) for 2 h at room temperature. The blots were detected using enhanced chemiluminescence-plus reagents (GE Healthcare Life Sciences). The relative proteins were normalized to GAPDH. The primary antibodies used in this paper were NLRP3 (1:100; cat. no. ab214185; Abcam), cleaved Caspase-1 (1:1,000; cat. no. ab10836; Abcam) and CD39 (1:1,000; cat. no. ab108248; Abcam).

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc.). Data were expressed as mean ± standard error of mean. A Student's t-test was used for analyzing the difference between two groups. One-way analysis of variance followed by Dunnett's t-test was used to calculate the statistical significance between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To determine kidney function and pathological changes after LPS exposure, kidney tissue sections were subjected to H&E staining (Fig. 1A). In the control group, the kidney tissues had clear structures without degeneration, atrophy, swelling or necrosis of the renal tubular epithelial cells or inflammatory infiltration. In the LPS group, the kidney tissues showed obvious kidney injury at 12 h. A total of 24 h after LPS administration, kidney pathological changes were more significant and were characterized by vacuolar degeneration, luminal narrowing, loss of the brush border, tubule dilation and infiltration of interstitial inflammatory cells. After 48 h, the histopathology of the kidney was improved, however, inflammatory cell infiltration was still observed in the renal interstitium. In addition, as shown in Fig. 1B and C, the Cr and BUN levels in mice were significantly increased within 24 h after LPS administration (P<0.01), while both Cr and BUN levels decreased at 48 h, indicating that the levels of Cr and BUN were positively correlated with kidney pathological severity.

Moreover, the levels of IL-1β, IL-18, IL-6 and TNF-α peaked at 12 h and then reduced gradually as the time of LPS administration increased (Fig. 1D-G). However, the levels of these inflammatory cytokines were still much increased compared with the control group. The expression of CD39, inflammasome NLRP3 and cleaved caspase-1 were also measured by RT-qPCR and western blotting. As presented in Fig. 2A, C and D, both NLRP3 mRNA and protein levels increased significantly at 24 h after LPS injection (P<0.01) but reduced at 48 h. The expression of CD39 peaked at 12 h and then continuously reduced at 24 and 48 h (Fig. 2B, C and F). The changes in protein level of cleaved caspase-1 were consistent with the expression of CD39 (Fig. 2C and E). Taken together, the kidney injury and inflammation were the most serious at 48 h of LPS administration.

To determine an optimal LPS concentration for subsequent experiment, HK-2 cells were treated with a series of concentrations (0, 0.1, 1, 10 and 100 μg/ml) of LPS. As shown in Fig. 3A, the HK-2 cell viability was gradually decreased as the concentration of LPS increased and reduced significantly when the cells were treated with 10 μg/ml LPS (P<0.01). As listed in Fig. 3B-E, the inflammatory cytokines (IL-1β, IL-18, IL-6 and TNF-α) showed a positive association with the concentration of LPS. The treatment of LPS (1 μg/ml) was enough to cause a significant inflammation in HK-2 cells (P<0.01). Furthermore, the expression of NLRP3 and cleaved caspase-1 was increased under the treatment of 10 μg/ml LPS (Fig. 3F and H-J). Meanwhile, the concentration of 0.01 μg/ml LPS slightly affected the expression of CD39. However, 1 μg/ml LPS could induce an increase of the CD39 expression levels and both its mRNA and protein levels, which then decreased as the concentrations of LPS increased (Fig. 3G, H and K). Together, the concentration of LPS was positively correlated with the level of inflammation, while the expression of CD39 increased slightly under a low concentration of LPS but gradually decreased as the LPS concentration increased.

In order to study the functional effects of CD39 on the LPS-induced inflammation and apoptosis in HK-2 cells, the CD39 overexpression vector was constructed and transfected into HK-2 cells. As Fig. 4A shows, the CD39 vector was expressed stably in HK-2 cells. The CCK-8 assay showed that the cell viability in LPS + Mock group was significantly reduced in comparison with the Mock group, while the transfection of CD39 could enhance the HK-2 cell viability (P<0.01; Fig. 4B). In addition, the treatment of LPS could induce strong inflammation, as the levels of IL-1β, IL-18, IL-6 and TNF-α were significantly increased in the LPS group, however, increasing CD39 expression could effectively inhibit the levels of these inflammatory cytokines (P<0.01; Fig. 4C-F). The apoptosis and ROS level was also measured by flow cytometry. As shown in Fig. 4G and H, the apoptosis rate and ROS level significantly increased following LPS administration (P<0.01), while transfection of CD39 had the ability to decrease the apoptosis rate and ROS level. Therefore, the results of the present study indicated that elevated CD39 may contribute to the mitigation of the LPS-induced injury, inflammation and ROS accumulation in HK-2 cells.

As mentioned earlier, the overexpression of CD39 could significantly inhibit the LPS-induced inflammation in HK-2 cells. After transfection of CD39, the expression of NLRP3, which works as an inflammasome and whose activation serves an essential role in increasing inflammatory cytokines, was measured. As indicated in Fig. 5A, the expression of NLRP3 was increased under LPS administration, while the transfection of CD39 overexpression could significantly inhibit LPS-induced increased NLRP3 level. The transfection of CD39 induced a significant upregulation of CD39 mRNA, compared with LPS + Mock group (P<0.01; Fig. 5B). The changes of their protein levels are shown in Fig. 5C-F. The protein levels of NLRP3 and CD39 were basically consistent with their mRNA levels. In comparison to the LPS + Mock group, the protein level of cleaved caspase-1 was significantly reduced after the transfection with CD39 overexpression (P<0.01). Therefore, these results suggested that the inhibitory effects of CD39 overexpression on LPS-induced inflammation may rely on suppressing the activation of NLRP3.

In order to further verify the protective effects of CD39 on the HK-2 cells under the treatment of LPS, the siCD39 vector was transfected into HK-2 cells and the levels of inflammation and apoptosis rate were detected. Fig. 6A demonstrated that the expression of CD39 was effectively inhibited by siRNA in HK-2 cells. LPS treatment could induce the reduced cell viability, while the transfection of siCD39 could further decrease cell viability, compared with the LPS + NC group (P<0.05; Fig. 6B). The levels of those inflammatory cytokines (IL-1β, IL-18, IL-6 and TNF-α) are shown in Fig. 6C-F, and it was observed that the transfection of siCD39 aggravated the inflammation caused by LPS treatment. The levels of IL-1β, IL-18 and IL-6 in LPS + siCD39 group were significantly increased compared with those in LPS + NC group (P<0.05), while the TNF-α level was increased slightly. In addition, compared with cells transfected with the NC vector, the apoptosis rate and ROS level both had a slight increase in the cells transfected with siCD39 after LPS treatment (Fig. 6G and H). Collectively, the results of the present study indicated that in contrast to CD39 overexpression, the transfection of siCD39 could aggravate LPS-induced inflammation, cell apoptosis and ROS accumulation in HK-2 cells.

After transfection with siCD39, the NLRP3 mRNA levels were increased again, compared with the LPS + NC group (Fig. 7A). In the meantime, the transfection of siCD39 obviously decreased the expression of CD39 and further weakened the stress reaction of CD39 to LPS treatment (P<0.01; Fig. 7B). The protein levels of NLRP3 and CD39 were basically consistent with their mRNA levels, and the cleaved caspase-1 protein level had a slight upregulation under the effects of CD39 inhibition and LPS treatment (Fig. 7C-F). The results of the present study suggested that the inhibition of CD39 may promote the activation of NLRP3 and subsequent inflammation in HK-2 cells.