The experimental animals included 10 male Fisher (F344) rats as the donors and 10 male Lewis (LEW) rats as the recipients. All animals were aged between 8–12 weeks and weighed 200–250 g. Animals were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The F344-LEW rat CAN model was established as previously described (8). The rats were divided randomly into the following two groups: The AMD3100 group and the CAN group. The rats in the AMD3100 group were administered subcutaneous injections of low-dose cyclosporine (1.5 mg/kg) every day in the first 10 days post-transplantation and AMD3100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; 1 mg/kg) at 0, 2, 4, 6, 8 and 10 days post-transplantation, and the rats in the CAN group were administered subcutaneous injections of low-dose cyclosporine (1.5 mg/kg/day) and saline at a dose equivalent to AMD3100 in the first 10 days following transplantation. At 12 weeks post-surgery, the kidney allografts in both the AMD3100 and CAN groups were harvested under anesthesia using 2% pentobarbital sodium (40 mg/kg), and the rats were subsequently administered pentobarbital sodium (200 mg/kg). The animals were protected by following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (19). The animal protocols were approved by the Medical Research Center, Beijing Chaoyang Hospital, Capital Medical University (Beijing, China). The housing conditions were as follows: Temperature, 22±1°C; relative humidity, 55±5% and free access to water and normal diet under an alternating 12:12-h light-dark cycle.

The kidneys were cut longitudinally along the coronal plane and fixed in 10% neutral formalin solution at room temperature for 24 h, followed by dehydration with alcohol, embedding in paraffin, and sectioning into 2-µm thick slices. Hematoxylin and eosin, and Masson's trichrome staining were performed to assess the renal injury. For hematoxylin and eosin staining, the sections were stained at room temperature with hematoxylin for 5 min and eosin for 2 min. For Masson's trichrome staining (all steps conducted at room temperature), the nuclei were stained with Weigert's iron hematoxylin for 10 min, followed by plasma membrane staining with acid fuchsin, Xylidine Ponceau, and glacial acetic acid for 10 min, phosphomolybdic acid staining for 5 min, and fiber staining with methyl blue for 10 min. A total of two experienced pathologists independently evaluated the results of the histopathological analysis, and the images were acquired at ×400 magnification. The chronic allograft damage index (CADI) scores of the kidney specimens in the two groups were determined. The CADI scoring system covers the following six pathological aspects of CAN: Renal interstitial inflammation, interstitial fibrosis, tubular atrophy, basement membrane matrix thickening, glomerular sclerosis and arterial intimal hyperplasia (20).

Immunohistochemistry was performed using the same procedures of dehydration, embedding and sectioning. Endogenous peroxidase was blocked with 3% H₂O₂ at room temperature for 10 min, and then the specimens were heated in a citrate buffer (pH 6.0) to 98°C for 15 min. The primary goat anti-CXCR4 antibody (cat. no. ab1670; Abcam, Cambridge, UK; 1:1,000) was added in a dropwise manner to the sections, which were incubated at 4°C overnight. Subsequently, the specimens were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-goat immunoglobulin G (cat. no. A0181; Beyotime Institute of Biotechnology, Haimen, China; 1:50) at 37°C for 30 min. A freshly prepared 0.05% 3,3′-diaminobenzidine solution was added to each specimen section. The color reaction was observed and controlled by light microscopy (Olympus Corporation, Tokyo, Japan), and terminated by washing with tap water. Images were acquired at ×400 magnification.

Total RNA was extracted from the kidney tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) reagent, and the purity and concentration of the RNA was determined from the OD_260/280 readings of a spectrophotometer. The RNA integrity was determined by capillary electrophoresis using the RNA 6000 Nano Lab-on-a-Chip kit and a Bioanalyzer 2100 (both Agilent Technologies, Inc., Santa Clara, CA, USA). Only RNA extracts with RNA integrity values of >6 were subjected to further analysis. Higher yields of cDNA were labeled with a fluorescent dye (Cy5 and Cy3-dCTP) using the CapitalBio cRNA Amplification and Labeling kit (CapitalBio, Beijing, China). The labeled cRNAs from long noncoding (lnc)RNAs and mRNAs were purified and hybridized to the Agilent Rat lncRNA + mRNA Array V1.0 (Agilent Technologies, Inc.). Images were captured by the Agilent microarray scanner, gridded and then analyzed using Agilent Feature Extraction software, version 10.10 (Agilent Technologies, Inc.). The raw data were summarized and normalized using GeneSpring software V12.0 (Agilent Technologies, Inc.).

The threshold values of ≥2 absolute fold change (FC) and a Benjamini-Hochberg corrected P-value of ≤0.05 were used. The data were log2 transformed and median-centered using the Adjust Data function of Cluster 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/index.html). These genes were classified according to the Gene Ontology (GO) analysis provided by the Molecular Annotation System 3.0 (http://bioinfo.capitalbio.com/mas3/). Signaling pathway analysis of these genes was performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.kegg.jp/kegg/pathway.html).

The rat renal tubular epithelial cell line NRK-52E was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Lablead Biotech Co., Ltd., Beijing, China) at 37°C with 5% CO₂. The NRK-52E cells with optimal morphology were digested and seeded in a 60-mm dish at a concentration of ~1×10⁵ cells/dish.

Once the cells had attached, the medium was changed to DMEM without serum. After 24 h, the cells were divided into four groups and treated as follows: SDF-1 group, 100 ng/ml SDF-1 (cat. no. ab79959; Abcam); SDF-1 + AMD3100 group, 100 ng/ml SDF-1 and 5 µg/ml AMD3100 (Sigma-Aldrich; Merck KGaA); SDF-1 + DKK-1 group, 100 ng/ml SDF-1 and 200 ng/ml DKK-1 (R&D Systems, Inc., Minneapolis, MN, USA); and control group, PBS at a dose equivalent to the SDF-1, AMD3100 and DKK-1 in the other groups.

Following incubation at 37°C for 24 h, the cellular total protein and nuclear protein were extracted for western blotting (WB) and the total RNA was extracted for reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The cells were divided into two groups; one group was lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing phenylmethylsulfonyl fluoride and the other group was lysed using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology). The lysed cells were centrifuged at 14,000 × g for 20 min at 4°C. The protein concentration was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). The proteins were mixed with loading buffer and boiled for 10 min, and subjected to 10% SDS-PAGE, with 30 µg protein loaded per lane. The separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking of the nonspecific background in 5% milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-α-tubulin (cat. no. 2125; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:2,000); rabbit anti-α-SMA (cat. no. ab5694; Abcam; 1:4,000); mouse anti-E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.; 1:1,000); rabbit anti-CXCR4 (cat. no. ab124824; Abcam; 1:2,000); rabbit anti-lamin B1 (cat. no. 13435; Cell Signaling Technology, Inc.; 1:1,000); rabbit anti-CXXC finger protein 5 (CXXC5; cat. no. 84546; Cell Signaling Technology, Inc.; 1:1,000) and rabbit anti-β-catenin (cat. no. 8480; Cell Signaling Technology, Inc.; 1:1,000). Subsequently, the membranes were washed with TBS with Tween-20 and incubated with the HRP-conjugated secondary antibodies (cat. no. TA130003 and cat. no. TA140003; OriGene Technologies, Inc., Beijing, China; 1:1,000) at room temperature for 1 h. Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (EMD Millipore) was used to detect positive immune reactions. α-Tubulin was used as the reference for total protein and lamin B1 was used as the reference for nuclear protein. Densitometric analysis was performed using Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was extracted from the cells using TRIzol^® reagent and reverse-transcribed into cDNA using a SuperScript IV First-Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocols. The RT-qPCR reactions were performed on the ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SuperScript III Platinum SYBR Green One-Step RT-qPCR Kit (Invitrogen; Thermo Fisher Scientific, Inc.); the primers used are listed in Table I. The target mRNA levels were normalized against GAPDH mRNA standards and calculated using the 2^−ΔΔCq method (21).

The NRK-52E cells were cultured under standard conditions to 80–90% confluence, and were pretreated with DMEM without serum at 37°C for 24 h. Subsequently, the cells were treated with 100 mg/ml SDF-1 with or without 5 µg/ml AMD3100 and 200 ng/ml DKK-1 during the wound healing assay. Cell migration was assessed by measuring the movement of cells into the acellular area created by a sterile insert at 0, 12 and 24 h following scratching (magnification, ×400).

Statistical analysis and data processing were performed using SPSS version 20 (IBM Corp., Armonk, NY, USA). The normally distributed measurement data are expressed as the mean ± standard deviation of experiments that were replicated three times. The independent samples t-test was used to compare two groups when the data were normally distributed and exhibited homogeneity of variance. Comparison between multiple groups was conducted using one-way analysis of variance followed by a least significance difference test. If the data exhibited a skewed distribution or failed to present homogeneity of variance, the Mann-Whitney U test was used for the comparison of two groups. P<0.05 was considered to indicate a statistically significant difference.

The histopathological changes and CADI scores confirmed CAN in the rat kidney grafts at 12 weeks post-transplantation. The AMD3100 group exhibited milder tubular atrophy, inflammatory cell infiltration and interstitial fibrosis, and also had a lower CADI score compared with the CAN group (Fig. 1A and B). Subsequently, the expression levels of a number of fibrosis-associated markers were detected. Using immunohistochemistry, CXCR4 was observed in the tubules in the CAN group, but it was expressed at lower levels in the AMD3100 group (Fig. 1C). RT-qPCR demonstrated that the expression of α-SMA was downregulated and that of E-cadherin was upregulated in the AMD3100 group compared with the CAN group, which was confirmed at the protein level by WB (Fig. 1D-F). The expression of CXCR4 was downregulated following administration of AMD3100 (Fig. 1D and F). These findings indicated the protective effect of AMD3100 in rat CAN by inhibiting the SDF-1/CXCR4 axis, and the SDF-1/CXCR4 axis may serve an important role in CAN.

In order to analyze alterations in the expression of mRNAs and lncRNAs in the rat CAN model, the kidney allograft tissues of the AMD3100 group and the CAN group were analyzed using an mRNA + lncRNA microarray. The results of the microarray were uploaded to the Gene Expression Omnibus database (GSE114088). Clustering analysis revealed that 506 mRNAs and 404 lncRNAs were significantly differentially expressed between the two groups, including 81 mRNAs and 140 lncRNAs with an FC value >2.0 (Fig. 2A and B). KEGG pathway analysis revealed that one of the top ranked pathways was the Wnt signaling pathway (Fig. 2C), which has been widely reported to be an important mechanism in EMT and fibrosis (22,23). The results of the microarray also predicted the association of a number of lncRNAs and mRNAs. CXXC5 mRNA was demonstrated to be negatively correlated with the lncRNAs LOC100912353, LOC102551030 and LOC102556393, which, it may be considered, may be a probable target for regulating renal allograft fibrosis (Table II). The coding potential of these lncRNAs is low, with the PhyloCSF (24) scores <-100 (Table II).

Variation in the expression levels of EMT-associated markers in the different groups of NRK-52E cells was quantitated using RT-qPCR and WB. In the SDF-1 group, α-SMA was upregulated and E-cadherin was significantly downregulated (P<0.05) compared with the other groups (Fig. 3A-C). These results confirmed that EMT was induced by SDF-1 in renal tubular epithelial cells, and this was antagonized by AMD3100 and DKK-1.

The results of the wound healing assay revealed that SDF-1 promoted cell migration across the wound edge into the scratch area at 12 and 24 h compared with the other groups. In the SDF-1 + AMD3100 group and SDF-1 + DKK-1 group, extensive cell migration was not observed until 24 h post-treatment (Fig. 3D and E).

The protein expression levels of β-catenin in the nucleus were increased significantly in the SDF-1 group, while the increase in the levels of total β-catenin was not as significant (Fig. 4), indicating the nuclear accumulation of β-catenin. The WB and RT-qPCR results demonstrated a reduction in the levels of CXXC5 expression in the SDF-1 group compared with the control group; however, expression in the SDF-1 + AMD3100 group was not significantly different to that in the control group. Notably, CXXC5 was also downregulated in the SDF-1 + DKK-1 group (Fig. 4A-C). These results suggested that CXXC5 is involved in EMT induced by the SDF-1/CXCR4 axis, and CXXC5 may be a potential upstream regulator of the WNT/β-catenin pathway.

In order to detect the expression of the aforementioned lncRNAs, RT-qPCR was performed. Two of these lncRNAs were demonstrated to be significantly upregulated in the SDF-1 group and the SDF-1 + DKK-1 group, but not the SDF-1 + AMD3100 group, compared with the control group (Fig. 4D).