According to the previous research, ApoE^−/− mice were selected as the experimental animals (20,21). ApoE^−/− mice (n=18) were provided from Beijing Vital River Lab Animal Technology Co., Ltd. All the ApoE^−/− mice had free access to feed on a conventional and standard mice chow diet with clean water, and were caged under constant conditions, including a 12-h dark/light cycle, and 40–60% humidity at 24°C. At 8 weeks of age, male mice (23.7±0.9 g) were randomly divided into three groups as follows: i) Control group (n=6); ii) AngII group (n=6); and iii) AngII + Lut group (n=6). Lut (cat. no. MB2172; Dalian Meilun Biology Technology Co., Ltd.) was administered by gavage (100 mg/kg/d) (8,22). ApoE^−/− mice were implanted with Alzet osmotic minipumps (Model2004; Durect Corporation), filled with either saline vehicle or AngII solution (1,000 ng/kg/min) for a maximum period of 4 weeks (23,24). Each group of mice was subjected to its respective treatment for 4 weeks. The health and behaviour of the mice were monitored every day, and after 4 weeks, all mice were alive. The mice were anesthetized with a high dose of pentobarbital (100 mg/kg, intraperitoneally), blood samples (0.5–0.6 ml) were collected from the abdominal aorta, and then the mice were euthanized via cervical dislocation. The cessation of respiration and heartbeat indicated the death of the mice. Kidney tissues were harvested for further analysis. Serum samples were subsequently stored at −80°C until further use. The kidneys were frozen in liquid nitrogen for mRNA, histopathological and immunoblotting analyses. The current study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) (25) and was approved by the Ethics Committee of Dalian Municipal Central Hospital Affiliated of Dalian Medical University.

Serum concentrations of creatinine (CRE) were measured using an ELISA kit (cat. no. C011-2-1; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocols.

The kidney tissues at room temperature were fixed in 10% buffered formalin solution and subsequently dehydrated in 75% ethanol overnight, followed by paraffin embedding. Serial sections (thickness, 4 µm) were subjected to hematoxylin staining for 15 min and eosin staining for 5 min at room temperature to assess the pathological changes. Renal injury scores were determined by two blinded researchers according to the extent of the kidney injury, as previously described (13,26). The score grading was mainly based on the presence or absence of hemorrhage, tubular cell necrosis, tubular dilatation and cytoplasmic vacuole formation. The grading system was scored as follows: i) 0, normal kidney; ii) 1, 0–5% injury (minimal damage); iii) 2, 5–25% injury (mild damage); iv) 3, 25–75% injury (moderate damage); and v) 4, 75–100% injury (severe damage). All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation; scale bar, 100 µm).

Kidney tissues from each group were at room temperature stored in 10% formalin, dehydrated in an ascending series of alcohol (75, 85, 90 and 100%; 5 min each) and finally embedded in paraffin wax. Then, 4 µm-thick paraffin sections were sliced from these paraffin-embedded tissue blocks. The tissue sections were subsequently deparaffinized via immersion in xylene (3 times; 5 min each) and rehydrated using a descending series of alcohol (100, 90, 85 and 75%; 5 min each). Biopsy samples were stained at room temperature using Masson's trichrome stain to investigate changes in the kidney morphology and fibrosis. Blue staining represented collagen accumulation. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation).

Total RNA was extracted with TransZol Up Plus RNA Kit (cat. no. ER501-01; TransGen Biotech Co., Ltd.), and then RNA concentration and purity were measured using ISOGEN (cat. no. 317-02503; Nippon Gene Co., Ltd.). cDNA was synthesized using a first-strand cDNA synthesis kit (SuperScript VILO cDNA synthesis kit; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. RT-qPCR was performed using fluorescent SYBR-Green technology (Light Cycler; Roche Molecular Diagnostics). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 20 sec; followed by 45 cycles of 60°C for 30 sec and 72°C for 60 sec; and a cooling step at 4°C. The relative expression of the target genes was normalized to that of β-actin. The relative gene expression was determined using the 2^−ΔΔCq method (27). The primer sequences used for amplification are listed in Table I.

Total protein was extracted from kidney tissues using RIPA lysis buffer (cat. no. R0020; Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocols. Kidney tissue total protein concentrations were determined using a BCA Protein assay reagent kit (cat. no. DQ111-01; Beijing Transgen Biotech Co., Ltd.). Protein samples (30 µg per lane) were separated by SDS-PAGE on 10–15% gels, and were subsequently transferred to PVDF membranes. Subsequently, membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk, and then incubated at room temperature for 2 h. The membranes were then incubated with primary antibody diluent (cat. no. P0023A; Beyotime Institute of Biotechnology) and lightly shaken overnight at 4°C. Primary antibody rabbit against collagen I (cat. no. 14695-1-AP; 1:1,000; ProteinTech Group, Inc.), collagen III (cat. no. 22734-1-AP; 1:1,000; ProteinTech Group, Inc.), microtubule associated protein 1 light chain 3α (LC3; cat. no. 14600-1-AP; 1:1,000; ProteinTech Group, Inc.), p62 (cat. no. 55274-1-AP; 1:1,000; ProteinTech Group, Inc.) and β-actin (cat. no. 20536-1-AP; 1:1,000; ProteinTech Group, Inc.) was performed. Briefly, the kidney tissues were harvested, and the protein extracts were prepared according to established methods. The extracts were separated via SDS-PAGE (8–15%) and transferred to a PVDF membrane (EMD Millipore). The membranes were blocked overnight with 5% milk blocking reagent at 4°C. The proteins were visualized by the addition of the respective antibodies, followed by the incubation with species-specific peroxidase-labeled secondary antibodies (cat. no. 7074; anti-rabbit IgG; 1:2,000; Cell Signaling Technology, Inc.) for 1 h. β-actin was used as a control of the equal protein loading. The protein levels were expressed as protein/β-actin ratios to minimize loading Chemiluminescence reagent (ProteinTech Group, Inc.) was used to visualize bands. This analysis was performed three times independently. The intensity of the bands was semi-quantified using ImageJ software version 1.8.0 (National Institutes of Health).

Kidney tissues were fixed in 10% buffered formalin solution for 30 min at room temperature and dehydrated in 75% ethanol overnight, followed by paraffin embedding. Immunohistochemical analysis was performed using the SP-9001 SPlink Detection kits (OriGene Technologies, Inc.), according to the manufacturers' instructions. Paraffin-embedded sections (4 µm) were deparaffinized with xylene and subsequently rehydrated in a descending series of ethanol washes (100, 90, 85 and 75%; 5 min each). The sections were treated for 15 min at 98°C with 3% H₂O₂ in methanol to inactivate the endogenous peroxidase activity and were subsequently incubated at room temperature for 1 h with primary antibodies rabbit against collagen I (cat. no. 14695-1-AP; 1:200; ProteinTech Group, Inc.), collagen III (cat. no. 22734-1-AP; 1:200; ProteinTech Group, Inc.), LC3 (cat. no. 14600-1-AP; 1:200; ProteinTech Group, Inc.) and p62 (cat. no. 55274-1-AP; 1:200; ProteinTech Group, Inc.). The secondary antibody from SPlink Detection kits (OriGene Technologies, Inc.) was incubated with the tissue for 30 min at room temperature. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation). For each staining, 3×7 sections (6 mice) per group were analyzed and the representative images were presented. All image analyses were performed by a blinded reviewer.

All data are presented as the mean ± SEM, and all experiments were repeated at least three times. SPSS version 19.0 software (IBM Corp.) was used for statistical analysis. The Kolmogorov-Smirnov test was used to detect the normality of all data. Inter-group variation was measured using a one-way ANOVA, followed by individual comparisons with a Tukey's tests. P<0.05 was considered to indicate a statistically significant difference.

The kidney/body weight ratio did not differ among the three groups. The metabolic characteristics of ApoE^−/− mice from the three groups following 4 weeks of treatment are presented in Table II. The CRE levels in the AngII-induced ApoE^−/− mice were increased by 198% compared with those of the control group (93.21±6.61 vs. 31.24±3.73 µmol/l; P<0.05), indicating a reduction of renal function. The CRE levels of the AngII + Lut group were markedly decreased by 60.5% following the treatment of the mice with Lut compared with those in the AngII group (36.86±4.53 vs. 93.21±6.61 µmol/l; P<0.05). No difference was noted between the AngII + Lut and control groups.

To evaluate the histopathological damage in the kidney tissue, H&E staining was performed (Fig. 2A). The AngII + Lut group had markedly reduced inflammatory cell infiltration in the kidney tissues compared with that observed in the AngII group. These results suggested that Lut decreased inflammatory cell infiltration in the AngII-induced renal damage of ApoE^−/− mice. Renal injury scoring (Fig. 2B) demonstrated that Lut treatment could significantly decrease renal injury caused by AngII in mice, which was consistent with the H&E results.

Masson's trichrome staining indicated that collagen deposition was significantly increased in the interstitium and the perivascular and subvascular areas, suggesting that AngII induced renal interstitial fibrosis. Lut treatment markedly attenuated the AngII-induced collagen deposition (Fig. 3). Taken together, these data demonstrated that Lut attenuated renal fibrosis in AngII-induced kidney tissues.

Collagen I and collagen III (Fig. 4A) immunostaining was performed. The AngII + Lut group exhibited markedly decreased collagen I and III expression levels in the kidney tissues compared with those observed in the AngII group (Fig. 4B). The expression levels of collagen I and III proteins in the kidney tissues were also determined via western blotting (Fig. 4C). The results indicated that the protein expression levels of collagen I and III in the mice of the AngII + Lut group were significantly suppressed compared with those in the AngII group (Fig. 4D).

To evaluate the expression levels of LC3 and p62 in kidney tissues, LC3 and p62 (Fig. 5A) immunostaining was performed. The AngII + Lut group exhibited markedly increased LC3 and decreased p62 expression levels in the kidney tissues compared with those observed in the AngII group (Fig. 5B). The protein expression levels of LC3 and p62 in the kidney tissues were also determined via western blotting (Fig. 5C). The results demonstrated that LC3 protein expression in the AngII + Lut group was significantly increased compared with that in the AngII group, whereas p62 protein expression in the AngII + Lut group was significantly decreased compared with those in the AngII group (Fig. 5D). These results indicated that Lut increased LC3 expression and decreased p62 expression in ApoE^−/− mice with AngII-induced renal damage.

The expression levels of IL-1β, IL-6, TNF-α and IL-10 were measured using RT-qPCR (Fig. 6). The expression levels of IL-1β, IL-6 and TNF-α were significantly elevated in the AngII group compared with those of the control group. However, Lut treatment decreased significantly the expression levels of IL-1β, IL-6 and TNF-α compared with the AngII group. The expression levels of IL-10 did not show these changes.