Animals

Sixty male C57BL/6 N mice (7–8 weeks age) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All mice were housed in a 12-h dark/light cycle, temperature (22 ± 2 °C) and humidity-controlled environment with unlimited access to water and food. All the procedures were performed following the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and approved by the Ethics Committee of the Experimental Animals of the Navy Medical University (DWLL-202303011).

Chemicals and reagents

LPS (Escherichia coli serotype 055: B5, No. L-2880), dexamethasone (DXMS, No. D4902), DMSO (No. D2650), and Penicillin-Streptomycin solution (No. P4333) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Primary rabbit antibodies for NF-κB p-p65 (Ser536, No. 3033), NF-κB p65 (No. 8242), Nrf2 (No. 12721), HO-1 (No. 43966), and β-actin (No. 4970) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit antibodies for zonula occludens-1 (ZO-1, No. 21773-1-AP) and Ezrin (No. 67627-1-Ig) were purchased from Proteintech (Wuhan, China). HRP-conjugated goat anti-rabbit secondary antibody (No. SA00001-2) was purchased from Proteintech (Wuhan, China). UDCA (No. U104241) was purchased from Aladdin (Shanghai, China). ML385 (99.96%, No. HY-100523), a selective Nrf2 inhibitor, was obtained from MCE (Shanghai, China). ANNEXIN V-FITC/PI kit (No. CA1020), DMEM/F12 medium (No. D8437), and fetal bovine serum (FBS, No. S9010) were purchased from Solarbio Science & Technology CO., LTD. (Beijing, China). QuantiNova SYBR Green (No. 208054) was purchased from Qiagen (Hilden, Germany). Cell Counting Kit-8 (CCK-8, No. CK04) was purchased from DOJINDO Laboratories (Tokyo, Japan). HK-2 cells (No. CC4008) were obtained from Guangzhou Cellcook Biotech Co., Ltd (Guangzhou, China).

Experimental design

After one week of preliminary adaptation, the mice were randomly divided into 6 groups using a computerized random number generator (10 mice per group): Control (Saline), LPS-vehicle (10 mg/kg), DXMS (5 mg/kg dexamethasone), UDCA-15 (15 mg/kg), UDCA-30 (30 mg/kg), and UDCA-60 (60 mg/kg) groups. The randomization process ensured that each mouse had an equal chance of being assigned to any treatment group, thereby minimizing bias and enhancing the validity of the results. According to the 3R principles and statistical rigor (effect size 0.4, significance level 0.05, 80% power), a sample size of 10 mice per group ensures reliable results while minimizing animal use. This approach allowed for adequate statistical efficacy to detect significant differences between treatment groups while adhering to ethical guidelines for experimental animal research. DXMS was dissolved in sterile saline and UDCA was suspended in sterile saline. Before each injection, UDCA was thoroughly sonicated and vortexed to ensure a uniform suspension. DXMS and UDCA were injected intraperitoneally once daily for 7 days, and LPS was injected intravenously trough the tail vein of mice 1 h after the last injection of UDCA, and the control group was injected with equal volume of sterile saline (Fig. 1). Based on the strong anti-inflammatory and antiendotoxin effects, DXMS was selected as a positive control drug. The dose of LPS and DXMS was selected based on previous study [30]. Previous studies indicated that 40 mg/kg of UDCA can effectively alleviate diabetic nephropathy by reducing oxidative stress [27], and 60 mg/kg of UDCA can protect against cisplatin-induced AKI in mice [24]. Therefore, 15, 30 and 60 mg/kg of UDCA was chosen in our present study. After collecting blood and 24-h urine, all mice were sacrificed by decapitation under anesthesia using tibromethanol (250 mg/kg, ip) which purchased from Sigma-Aldrich (No. T48402). The experimental treatments were conducted by specialized technical staff at the Experimental Animal Center in No. 971st Hospital of the People’s Liberation Army Navy, who were blinded to the study’s purpose, content, and treatment drugs.

Fig. 1

Timeline of the experimental schedule

Renal function

After LPS injection, all mice were transferred into a metabolic cage to collect 24-h urine. The blood was collected from retro-orbital and centrifuged at 3000 rpm for 10 min to get serum. The urine protein (No. C035-2-1), serum creatine (Cr, No. C011-1-1), and blood urea nitrogen (BUN, No. C013-2-1) were analyzed using biochemical kits (Jiancheng, Nanjing, China). Briefly, urine protein was measured though a modified acidic dye-binding method, which determines concentration based on dye absorption at 595 nm. Serum Cr was analyzed using the alkaline picrate method, in which creatinine forms a colored complex with alkaline picrate, with absorbance measured at 520 nm. BUN was detected using the diacetyl monoxime method, which producing a colored product measured at 546 nm.

Histopathological examination

Three mice were randomly selected for renal histopathological examination. Kidney tissue were harvested and fixed in 10% formaldehyde for 24 h at room temperature. Tissue sections of 5 μm thickness were cut into the slides (4–6 sections were taken from each mouse) and stained with hematoxylin and eosin (H&E). The pathological changes in the cortex of kidney tissues were observed by an optical microscope (90i, Olympus, Japan).

Oxidative stress parameters

To determine oxidative stress parameters, kidney cortex tissues (approximately 100 mg) were homogenized in 1 mL of ice-cold physiological saline and centrifuged at 3 000 g for 10 min to obtain the supernatant. A 10 µL of the supernatant was used to measure protein concentration with a bicinchoninic acid (BCA) kit (No. PC0020, Solarbio, Beijing, China), and all oxidative stress parameters were normalized based on protein concentration. The BCA assay is based on proteins can reduce Cu²⁺ to Cu⁺, forming a purple complex with bicinchoninic acid. Absorbance of this complex was measured at 562 nm using a microplate reader (Imark, Bio-Rad, Hercules, CA, USA), and protein concentration was calculated from a standard curve.

SOD activity

SOD activity was assessed using the xanthine oxidase method, where SOD inhibits the reduction of nitroblue tetrazolium (NBT). Absorbance was measured at 560 nm using a microplate reader, and SOD activity was expressed as U/mg of protein (No. A001-3-2, Jiancheng, Nanjing, China).

MDA Content

MDA levels were measured using the thiobarbituric acid (TBA) reactive substances (TBARS) method, which forms a colored complex with MDA. The absorbance of this complex was measured at 532 nm using a microplate reader and expressed as nmol/mg of protein (No. A003-1-2, Jiancheng, Nanjing, China).

GSH Content

The GSH assay is based on a reaction with 2-nitrobenzoic acid, producing a yellow product with absorbance measurable at 412 nm using a microplate reader. GSH content was calculated and expressed as µmol/mg of protein (No. A006-2-1, Jiancheng, Nanjing, China).

Enzyme-linked immunosorbent assay (ELISA)

To measure the concentrations of IL-1β (No. EK0392), IL-6 (No. EK0410), and TNF-α (No. EK0526) in kidney and serum, the ELISA method was performed using commercially available kits for cytokine detection (Boster, Wuhan, China). The levels of neutrophil gelatinase-associated lipocalin (NGAL, No. CSB-E09447m) in serum and urine, kidney injury molecule-1 (KIM-1, No. CSB-E08519m), N-acetyl-β-D-glucosaminidase (NAG, No. CSB-E09412m), and retinol binding protein (RBP, No. CSB-E11722m) levels in urine were detected using ELISA kits (CUSABIO, Wuhan, China). The preparation of all reagents and protocol were followed according to the manufacturer’s instructions. The absorbance was read using ELISA reader at 450 nm and 570 nm dual filters.

Western blot

Total proteins in kidney cortex tissues were extracted with RIPA buffer (No. R0010, Solarbio) containing phenylmethylsulfonyl fluoride (PMSF. No. P0010, Solarbio) and phosphatase inhibitor cocktail (No. P1260, Solarbio). The protein concentration was measured using the BCA method. Then, 50 µg of total proteins was separated on 10% SDS-PAGE, initially at 40 V for 30 min, followed by 80 V for 90 min. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (No. IPVH00010, Millipore, Shanghai, China) at 200 mA for 100 min using a wet transfer method. The membranes were blocked in 5% non-fat milk at room temperature for 2 h, followed by incubation with primary antibodies against NF-κB p65 (1:1000), phosphor-NF-κB p65 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), ZO-1 (1:2000), Ezrin (1:1000), and β-actin (1:5000) at 4 °C overnight. After three washes with PBST (5 min each), the membranes were incubated with an HRP-conjugated secondary antibody (1:30 000) for 1 h at room temperature. Finally, the bands were visualized using an enhanced chemiluminescence (ECL) kit (No. WBKLS0500, Merck, Darmstadt, Germany). The relative protein levels were normalized to β-actin, and Image J (v1.53, National Institutes of Health, Bethesda, MD, USA) was used to quantify relative optical density.

Cell culture

HK-2 cells were cultured in DMEM/F12 medium containing 10% FBS and 1% penicillin-streptomycin solution at 5% CO2 and 37 °C. The seventh to eighth generation cells were used in this study. The cell injury model was established by incubating the HK-2 cells with 5 µg/mL LPS [31]. (1) To screen for compounds that provide significant protection against LPS-induced injury in HK-2 cells, we randomly selected 12 different compounds (designated CC1 to CC12) from the drug library in our laboratory. HK-2 cells were seeded in a 96-well plate at a density of 104/well and cultured for 24 h. The cells were pre-treated with 20 µM of each compound for 1 h before being exposed to LPS for an additional 24 h. (2) To access the cytotoxicity of UDCA, HK-2 cells were seeded in 96-well plate at a density of 104/well and cultured for 24 h. They were then treated with various concentrations of UDCA (2.5, 5, 10, 20, 40, and 80 µM) for 24 h. (3) HK-2 cells were seeded in a 96-well plate at a density of 104/well and cultured for 24 h. The cells were pre-treated with DXMS (1 µM), UDCA ((10, 20, and 40 µM), or ML385 (10 µM) for 1 h before being treated with LPS for 24 h. The control group cells were treated with an equal concentration (0.1%) of DMSO as the solvent control.

Cell viability

After treatment with LPS for 24 h, the medium was replaced and 10 µL CCK-8 solution were added and cultured for 1 h. The absorbance was measured at 450 nm by microplate reader.

Cell apoptosis

For cell apoptosis assay, HK-2 cells were seeded in a 6-well plate at a density of 105/well and cultured for 24 h. The cells were pre-treated with UDCA (40 µM) or ML385 (10 µM) for 1 h, followed by treatment with LPS (5 µg/mL) for an additional 24 h. After collecting the cells by centrifugation at 1000 rpm for 10 min, cells were resuspended in PBS, and 5 µL Annexin V/FITC dye was added. After incubating at room temperature for 5 min in the dark, followed by the addition of 5 µL of PI dye, the apoptosis rate was detected using a flow cytometer (CytoFLEX, Beckman, Brea, CA, USA). The parameters were set as FITC (488 nm excitation, 525 nm emission) for Annexin V and PI (488 nm excitation, 610 nm emission). Gating was performed using forward and side scatter to identify viable, early apoptotic (Annexin V+/PI−), and late apoptotic (Annexin V+/PI+) cells. Analysis was conducted with FlowJo software (v10.8, FlowJo LLC, Ashland, OR, USA).

Statistical analyses

All data are represented as mean ± S.E.M. Prior to analysis of variance, the normality and homogeneity of variances were checked using Shapiro-Wilk test. If the assumptions of ANOVA were not met, appropriate data transformations were performed using the log transformation method. One-way ANOVA followed by Turkey’s HSD post hoc test using SPSS 18.0 (IBM, Chicago, IL, USA) was conducted to analyze the statistic differences. A value of P < 0.05 was considered statistically significant.