A total of 30 6-month-old healthy male adult New Zealand White rabbits (body weight, 1.8–2.2 kg) were obtained from the Experimental Animal Center of Yunnan College of Traditional Chinese Medicine. On arrival, the rabbits were allowed to acclimatize to their new environment for one week before any procedure was performed. The rabbits were fed separately, and had free access to 200 g of food and water in an undisturbed and clean environment. The animals were housed in a room that had a 12-h light/dark cycle, and was maintained at a temperature of 18–24°C and a relative humidity ranging from 40 to 70%. All experiments were performed in accordance with the recommendations provided in Guidelines for the Care and Use of Laboratory Animals, and the study protocol was approved by the Animal Research Committee of Yunnan College of Traditional Chinese Medicine (Cumming, China). Animals were anesthetized by pentobarbital (i.v., 40 mg/kg) and sacrificed by exsanguination (blood volume, ~85 ml each).

The CRF model was created by gavaging the rabbits with adenine for 3 weeks, as previously described (26). Adenine (purity >98%; FW: 135.14) was purchased from Guangzhou Weijia Technology Co., Ltd. (Guangzhou, China). New Zealand White rabbits (n=6) in the control group were gavaged with an equivalent amount of distilled water (10 ml/kg/day) for 21 days. The other New Zealand White rabbits (n=24) were used to construct the CRF rabbit model. In brief, the rabbits were gavaged with 25% adenine (150 mg/kg/day) dissolved in normal saline for 21 consecutive days. After 21 days, their kidney function, blood pressure and urinary protein levels were measured.

Serum creatinine (SCr) and blood urea nitrogen (BUN) were detected with an IDEXX Catalyst One Chemistry Analyzer (IDEXX Laboratories, Inc., Westbrook ME, USA).

The CRF rabbits were randomly assigned to a CRF model group (n=6), a losartan potassium positive control group (Posi, CRF model treated with losartan potassium; n=6), an acupuncture group (Acup, CRF model administered acupuncture; n=6), or an acupuncture+SB 431542 group (Acup+inhibitor, CRF model administered acupuncture and SB 431542; n=6). Rabbits in the CRF model group were fed a standard diet without drugs. CRF rabbits in the Posi group were fed a standard diet and also received 2.33 mg/kg/day of losartan potassium (100 mg/tablet, produced by Hangzhou MSD Pharmaceutical Co., Ltd., Hangzhou, China) by gavage, once a day. CRF rabbits in the acupuncture group were fed a standard diet and were stimulated by acupuncture treatment (Shenshu, Mingmen and Pishu) for 30 min, once a day. CRF rabbits in the Acup+inhibitor group were fed a standard diet and received SB 431542 (2.5 mg/kg/2 days; Tocris Bioscience, Bristol, UK) by intraperitoneal injection. Acupuncture was administered with a sterilized acupuncture needle (diameter: 0.25 mm, length: 40 mm; TCM Supply Co., Ltd. Yokohama, Japan). During acupuncture, sterilized acupuncture needles were gently inserted to a depth of 5 mm below the surface of acupuncture points at Shenshu, Mingmen and Pishu. The treatments were continued for 36 days, and each animal's diet, urine volume, mental state, hair color and weight were monitored.

The samples obtained from all groups were washed and then fixed in 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min. Next, the tissues were dehydrated in a gradient alcohol series, treated in xylene, imbedded with paraffin, and cut into 4-µm sections. For the hematoxylin and eosin (H&E) staining assay, the sections were stained with hematoxylin for 5 min and then dehydrated with 70 and 90% ethanol; after which, they were treated with eosin for 3 min. Each section was then evaluated under a light microscope (CX41; Olympus Corporation, Tokyo, Japan; magnification, ×200). For Masson's trichrome staining, the sections were stained with reagents in a Masson's Trichrome Stain kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. Masson staining results were quantified using Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). Briefly, integral optical density of three areas-of-interest was assessed for staining quantification.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to extract total RNA from the tissue samples of all groups as described in the manufacturer's instructions. RNA concentrations were determined by measuring absorbance at 260/280 nm. cDNA was obtained by using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). Specific genes were amplified by using SYBR-Green PCR Master Mix (Takala Biotechnology, Co., Ltd., Dalian, China) and an ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). β-actin served as an endogenous housekeeping gene. Amplification data were analyzed using the 2^−ΔΔCq method (27). The following primers were used for amplification: TGF-β: 5′-CAAGTGGACATCAACGGGA-3′ (forward) and 5′-GCAGAAGTTGGCGTGGTAG-3′ (reverse); Smad3: 5′-GAAGGATGAGGTTTGCGTGA-3′ (forward) and 5′-GGATGGAATGGCTGTAGTCGT-3′ (reverse); ILK: 5′-TCACCCAACCCTCATTACG-3′ (forward) and 5′-TCATCAATCATTACACTACGGCTAT-3′ (reverse); β-actin: 5′-CCAGGTCATCACCATCGG-3′ (forward) and 5′-TGTCCACGTCGCACTTCA-3′ (reverse).

The total proteins were extracted from all groups of tissue samples by using a radio-immunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai, China). The concentration of total protein in each sample was measured by using a protein reagent (Bio-Rad Laboratories, Hercules, CA, USA). Next, aliquots of total protein (30 µg per sample) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels, and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (cat. no. IPVH00010; Millipore, Burlington, MA, USA). The membranes were then blocked with 5% non-fat dry milk for 1 h, and subsequently incubated overnight at 4°C with specific primary antibodies. After being washed, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000, cat. no. ab97057; Abcam, Cambridge, UK) at room temperature for 1.5 h. The immunoprecipitated proteins were detected by using an enhanced chemiluminescence substrate kit that was included in an enhanced chemiluminescence detection system (Amersham Biosciences Inc., Piscataway, NJ, USA). The primary antibodies were anti-TNF-α (dilution 1:1,000; cat. no. ab6671; Abcam), anti-Smad3 (dilution 1:1,000; cat. no. ab84177; Abcam), anti-ILK (dilution 1:1,000; cat. no. ab227154; Abcam), anti-TGF-β (dilution 1:1,000; cat. no. ab92486; Abcam), anti-eNOS (dilution 1:1,000; cat. no. ab76198; Abcam) and anti-GAPDH (dilution 1:3,000, cat. no. ab9485; Abcam).

Samples of blood from the right common carotid artery of each rabbit were collected into anticoagulant tubes and centrifuged. TGF-β, IL-8, TNF-α and IL-1β were measured by using specific ELISA kits according to the manufacturer's instructions. The ELISA kits used in the present study were for TGF-β (cat. no. CSB-E06900Rb; Cusabio, Wuhan, China), IL-8 (cat. no. SB-E13942Rb; Cusabio), TNF-α (cat. no. CSB-E06998Rb; Cusabio) and IL-1β (cat. no. E-EL-RB0385c; Elabscience Biotechnology Co., Ltd., Wuhan, China).

The tissues in all groups were fixed in 4% formalin (cat. no. SF100-20; Thermo Fisher Scientific, Inc.), embedded in paraffin, and cut into 4-µm thick sections. The sections were then deparaffinized, rehydrated and treated with 10 mM citrate buffer for 5 min at 100°C; after which, they were incubated with 10% fetal bovine serum (FBS) for 2 h at 37°C. Next, the sections were incubated overnight with anti-TGF-β (dilution 1:25; cat. no. ab92486; Abcam) at 4°C. After being washed with phosphate-buffered saline (PBS), the sections were incubated with an anti-rabbit secondary antibody (cat. no. ab150077; Abcam) for 2 h at room temperature. The immunostained tissues were then photographed under a light microscope (CX41; Olympus Corporation).

Each experiment was repeated at least 3 times, and results represent the mean ± standard deviation (SD). Student's t-test was used in data analysis between two groups. Statistical analyses of multiple groups were performed using the one-way analysis of variance (ANOVA) followed by post-hoc Tukey's test through IBM SPSS Statistics for Windows (version 23.0 software; IBM Corp, Armonk, NY, USA). P-values <0.05 were considered statistically significant.

In the present study, no rabbit in any group died, and no abnormalities of feeding or activity were observed among rabbits in the control group. However, some rabbits in the other groups displayed loss of appetite, lethargy, slowed mobility and weight loss. Rabbits in the model group displayed the most significant symptoms.

Our chronic renal failure (CRF) model was created by gavaging rabbits with adenine for 3 weeks. In this study, the base line body weights, urine protein, SCr and BUN levels of all the rabbits were similar prior to establishment of the CRF model. After treatment, there were no significant changes in body weight, urine protein, SCr and BUN levels of rabbits in the control group when compared with the pre-treatment values. While the mean body weight in the model group after treatment was significantly lower than that in the control group (P<0.05; Fig. 1A), the urine protein levels in the model group were significantly higher than those in the control group (P<0.05; Fig. 1B). Furthermore, the SCr and BUN levels in the model group were also significantly higher than those in the control group (both P<0.05; Fig. 1C and D, respectively). These findings indicated that the CRF model had been successfully created.

To explore how acupuncture affects RIF, H&E and Masson's trichrome staining were performed to evaluate morphological features of the renal tissues. As shown in Fig. 2A, the sizes of the glomeruli in the model group were dramatically decreased, and the glomeruli displayed features of patchy tubulointerstitial injury, such as a fuzzy boundary. However, these features were absent in the Posi control group, and both the losartan potassium and acupuncture groups had glomeruli of significantly increased sizes. Acupuncture was able to attenuate these symptoms. As shown in Fig. 2B, interstitial fibrosis was observed more often in the model group than in the control group, and the symptoms were attenuated in the animals treated with losartan or acupuncture. Additionally, we found that the fibrogenesis was even further attenuated by TGF-β inhibitor (SB 431542) administration without significant difference (Fig. 2B).

Acupuncture downregulates TNF-α, Smad3, ILK and TGF-β expression, and upregulates eNOS in the CRF model via the TGF-β/Smad pathway. To explore the mechanism by which acupuncture affects RIF, New Zealand White rabbits were assigned to a control group, a CRF model group, a losartan potassium (Posi) group, an acupuncture (Acup) group, and an acupuncture+SB 431542 (Acup+inhibitor) group, respectively. As shown in Fig. 3A and B, the mRNA (Fig. 3A) and proteins (Fig. 3B) expression levels of TNF-α, Smad3, ILK and TGF-β expression were significantly upregulated in the model group when compared with the control group; TNF-α, Smad3, ILK and TGF-β expression levels were markedly downregulated in the Posi group and Acup group when compared with the model group; TNF-α and Smad3 expression were significantly decreased in the Acup+inhibitor group when compared with the Acup group (P<0.05 or P<0.01), while the levels of ILK and TGF-β expression did not significantly change. In addition, we also found that eNOS expression was markedly downregulated in the model group when compared with the control group; eNOS expression was dramatically upregulated in the Posi group and Acup group when compared with the model group; and eNOS expression was significantly increased in the Acup+inhibitor group when compared with the Acup group (Fig. 3A and B).

TGF-β expression was detected by an IHC assay, and the results showed that TGF-β was rarely found in the control group, but was highly expressed in renal tissues (mainly in injured tubulointerstitium and renal tubular epithelial cells). It was also found that TGF-β expression was markedly reduced in the Posi and Acup groups relative to its expression in the model group. Furthermore, TGF-β expression was also dramatically reduced in the Acup+inhibitor group when compared with its expression in the Acup group (Fig. 3C).

ELISA assays were performed to measure differences in TGF-β, IL-8, TNF-α and IL-1β concentrations in blood serum. The results revealed that the concentrations of TGF-β, IL-8, TNF-α and IL-1β in the model group were significantly increased when compared with those in the control group. Furthermore, the concentrations of TGF-β, IL-8, TNF-α and IL-1β in the Posi and Acup groups were significantly decreased relative to those in the model group. Finally, the concentrations of TGF-β, IL-8, TNF-α and IL-1β in the Acup+inhibitor group were significantly decreased relative to those in the Acup group (P<0.05 or P<0.01; Fig. 4).