Human renal proximal tubule epithelial cells (HK-2 cells, ATCC^® CRL-2190) were cultured in RPMI-1640 medium containing 2,000 mg/l NaHCO₃, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin in 5% CO₂ at 37°C. The cells were then stimulated for different periods of time with human recombinant TGF-β1 (1 ng/ml; PeproTech, Rocky Hill, NJ, USA). Cell culture reagents were obtained from Life Technologies Inc., Carlsbad, CA, USA.

Male CD-1 mice were obtained from the Xiangya Hospital of Central South University, Changsha, China. The mice in the present study were divided into 3 groups as follows: i) the mice in the control group (Con) were fed without surgical manipulation; ii) the mice in the sham-operated group were subjected to surgical manipulation without ureteral ligation; and iii) the mice in the unilateral ureteral obstruction (UUO) group were subjected to left ureteral ligation. UUO was performed as previously described (18). All mice were sacrificed 14 days following UUO and the kidney tissues were collected for various analyses. The protocols for animal experimentation and maintenance were approved by the Animal Ethics Committee at our institute and carried out in accordance with institutional guidelines.

MSP was carried out on bisulfate-treated DNA using the EZ DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Methylation-specific primers for the KLF4 promoter were designed using the MethPrimer program, as previously described (19). Bisulfite converted genomic DNA was PCR-amplified using methylation-specific primers and SYBR-Green reaction mix. The human primers used were unmethylated and were as follows: KLF4 forward, 5′-tgtagggtttaaataggtgataatga-3′ and reverse, 5′-aaataataaaaa ctcaaacaccaaa-3′; and methylated KLF4 forward, 5′-cgta gggtttaaataggtgataacg-3′ and reverse, 5′-aaataataaaaactcg aacaccgaa-3′. The mouse primers used were unmethylated and were as follows: KLF4 forward, 5′-ttgtaaagagttttgagatttttgg-3′ and reverse, 5′-caaaattaaacacataatcatcatt-3′; and methylated KLF4 forward, 5′-gttcgtaaagagttttgagattttc-3′ and reverse, 5′-gaaattaaacacgtaatcatcgtt-3.

The cells were treated with the demethylation drug, 5-Aza-2′-deoxycytidine (5-Aza), which was purchased from Sigma-Aldrich (St. Louis, MO, USA; A3656) at 15.5 nM for 72 h.

The total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the RNA was subsequently reverse transcribed using the PrimeScript RT Master Mix Perfect Real-Time kit (Takara, Dalian, China) to obtain the cDNA. Using the cDNA as a template, a real-time PCR assay was performed using the following pairs of primers: KLF-4 forward, 5′-ctgagcagcagggactg-3′ and reverse, 5′-ttgagatgggaactctttgt-3′ in HK-2 cells, and KLF-4 forward, 5′-ttctccacgttcgcgtccgg-3′ and reverse, 5′-tctcgccaacggttagtcgggg-3′ in mice; and Dnmt1 forward, 5′-aaccttcacctagccccag-3′ and reverse, 5′-tgacaggtggt cactcctcatg-3′; Dnmt3A forward, 5′-tattgatgagcgcacaagagagc-3′ and reverse, 5′-gggtgttccagggtaacattgag-3′; Dnmt3B forward, 5′-tacacagacgtgtccaacatgggc-3′ and reverse, 5′-gaggaagctgct aaggactagttc-3′; E-cadherin forward, 5′-aatgccgccatcgcttacac-3′ and reverse, 5′-cgacgttagcctcgttctca-3′; α-smooth muscle actin (α-SMA) forward, 5′-tcccttgagaagagttacgagtt-3′ and reverse, 5′-catgatgctgttgtaggtggtt-3′; zonula occludens-l (ZO-1) forward, 5′-ggaagttacgtggcgaag-3′ and reverse, 5′-ctctggcggacatcttgt-3′; fibroblast-specific protein 1 (FSP-1) forward, 5′-cctctctacaa ccctctct-3′ and reverse, 5′-ggacaccatcacatccag-3′; and β-actin forward, 5′-aggggccggactcgtcatact-3′ and reverse, 5′-ggcggcacc accatgtaccct-3′. The 20 μl real-time PCR reaction included 0.5 μl of cDNA template, 0.25 μl of Primer F, 0.25 μl of Primer R, 10 μl of RNase-free dH₂O and 8 μl of 2.5X Real-Time Master Mix (SYBR-Green I). The reaction conditions included a pre-denaturation step at 95°C for 10 sec, and 40 cycles of 95°C for 15 sec and 60°C for 60 sec. After the reaction, the data were subjected to statistical analysis.

To generate the KLF4-overexpression plasmid, KLF4-CDS (NM_004235.4) was amplified by PCR using the following primers: forward, 5′-gaattcatgaggcagccacctggc-3′ (including the EcoRI site, shown in bold font) and reverse, 5′-ggatccttaaaaat gcctcttcatgtg-3′, (including the BamHI site, shown in bold font); the sequence was then cloned into the BamHI/EcoRI sites of the pLV-Neo vector (Inovogen, Beijin, China). The pLV-Neo-KLF4 or pLV-Neo vectors were co-transfected with packaging plasmids into 293T cells using Lipofectamine 2000. The viral supernatant of pLV-Neo-KLF4 or pLV-Neo was filtered through a 0.45-μm filter and renamed as Lv-KLF4 and Lv-Con, respectively. All the supernatants were used to infect the target cells with 10 μg/ml of polybrene (Sigma-Aldrich). After the target cells were infected for 96 h, KLF4 expression was confirmed by quantitative (real-time) PCR and western blot analysis (Fig. 3). The cells infected with Lv-KLF4 and Lv-Con were used for further analyses.

ChIP assay was carried out as previously described (20). ChIP assays were performed using the ChIP-IT kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The HK-2 cells were conventionally cultured to approximately 70–80% confluency, the medium was decanted, and the medium containing 1% formaldehyde was then added to fix the cells. The cells were washed with cold PBS, and then glycine was added to terminate the fixation, and the cells were washed again with cold PBS. After the cells were scaped from the culture plate, the cold cell lysate was centrifuged to collect the nuclei. The pelleted nuclei were resuspended, and chromatin was cleaved into approximately 500-bp fragments by ultrasonication. The cleaved chromatin was treated with RNase and proteinase K, and the extent of cleavage was then assessed by 1% agarose gel electrophoresis. Protein G beads were used to remove the non-specific antibodies from the chromatin preparation, Subsequently, an anti-Dnmt1 antibody (Cat. no. ab13537; Abcam, Cambridge, UK), a positive control RNA polymerase (pol) II antibody (Cat. no. ab5131; Abcam) and a negative control IgG antibody (Cat. no. ab2410; Abcam) were added. The cells were incubated overnight at 4°C. Protein G beads were added to the solution containing the antibody/chromatin complexes, which were then washed and eluted, de-cross-linked and the co-precipitated DNA was purified. Finally, the co-precipitated DNA was used as a template in a PCR amplification using the following primers: forward, 5′-cag gagaatcgcttggacg-3′ and reverse, 5′-gaacaggcggaaagaggc-3′.

The cells were lysed in cell lysate, and then centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected and denatured. Proteins were separated by 10% SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were treated with TBST containing 50 g/l skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies, anti-KLF4 (Cat. no. BM0485; Abzoom Biolabs, Dallas, TX, USA), anti-E-cadherin (Cat. no. BM0530; Abzoom Biolabs), anti-α-SMA (Cat. no. YT5053; ImmunoWay Biotechnology Company, Newark, DE, USA), anti-ZO-1 (1:1,000 dilution; Cat. no. ab59720; Abcam), anti-FSP-1 (1:5,000 dilution; Cat. no. ab27957; Abcam) and anti-β-actin (Cat. no. BM0272; Abzoom Biolabs), respectively, at 37°C for 1 h. The membranes were rinsed and incubated for 1 h with the corresponding peroxidase-conjugated secondary antibodies. Chemiluminescent detection was performed using the ECL kit (Pierce Chemical Co., Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of β-actin.

The expression of E-cadherin, ZO-1, α-SMA and FSP-1 in the control HK-2 cells (infected with Lv-Con) and stimulated or not with TGF-β1, and in the HK-2 cells infected with Lv-KLF4 and stimulated with TGF-β1, was determined by immunocytochemistry. A standard immunostaining procedure was performed using antibodies against E-cadherin (1:500 dilutions), α-SMA (1:500 dilutions), FSP-1 (1:200 dilutions), ZO-1 (1:100 dilutions). Protein expression was graded on a scale of ‘±’ to ‘+++’ as previously described (21).

Data are expressed as the means ± SD from at least 3 separate experiments. Statistical analysis was carried out using SPSS 15.0 software. Differences between 2 groups were analyzed using the Student’s t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

To determine whether KLF4 is involved in renal fibrosis, an animal model of renal disease was created by subjecting mice to UUO. RT-qPCR and western blot analysis revealed that the KLF4 mRNA and protein levels were decreased in the renal tissues from the mice subjected to UUO compared with the mice in the control or sham-operated groups (Fig. 1A and B). MSP analysis revealed that KLF4 methylation was observed in the renal tissues of mice subjected to UUO, while KLF4 methylation was not observed in the mice in the control group or sham-operated group (Fig. 1C). These results suggest that KLF4 is downregulated by hypermethylation, thus contributing to renal fibrogenesis.

To further determine whether KLF4 is involved in renal fibrosis by affecting EMT in renal tubular epithelial cells, human renal proximal tubule epithelial cells (HK-2 cells) were stimulated with TGF-β1, which induces EMT in epithelial cells. Following stimulation for 36 h, a decrease in KLF4 and E-cadherin expression accompanied by an increase in α-SMA expression were observed in the HK-2 cells (Fig. 2), suggesting that KLF4 functions as a suppressor of EMT induced by TGF-β1 in tubular epithelial cells. Moreover, 5-aza-2′-deoxycytidine, as a demethylating agent, attenuated the TGF-β1-induced downregulation in KLF4 expression (Fig. 2). MSP analysis indicated that both methylated and unmethylated KLF4 were detected in the HK-2 cells stimulated with TGF-β1; however, only unmethylated KLF4 was detected in the controls (untreated cells) (Fig. 1C). These results demonstrate that the downregulation of KLF4 is at least partly induced by hypermethylation alternation, and that this may promote EMT in tubular epithelial cells in the process of renal fibrogenesis.

The results from RT-qPCR revealed that the Dnmt1 and Dnmt3A expression levels were significantly increased in the HK-2 cells stimulated with TGF-β1; however, Dnmt3B expression was decreased (Fig. 3A). ChIP assay indicated that after Dnmt1 protein-KLF4 promoter complexes were captured with Dnmt1-specific antibodies, KLF4-promoter-specific segments were detected (Fig. 3B). These data demonstrate that Dnmt1 plays a direct role in the TGF-β1-mediated hypermethylation of KLF4 in HK-2 cells.

To validate whether KLF4 plays a role in EMT in renal epithelial cells, the expression of KLF4 was enforced in the HK-2 cells. The results from RT-qPCR and western blot analysis revealed that the transfection of the HK-2 cells with Lv-KLF4 induced an increase in the expression of KLF4, E-cadherin and ZO-1, and a decrease in the expression of α-SMA and FSP-1, and that this attenuated the TGF-β1-induced downregulation in KLF4, E-cadherin and ZO-1 expression and the upregulation in α-SMA and FSP-1 expression (Fig. 4). In addition, immunocytochemistry of the HK-2 cells infected with Lv-KLF4 or Lv-KLF4 + TGF-β1 revealed similar results as regards the expression of these factors (Fig. 5). These data confirm that KLF4 inhibits the progression of EMT induced by TGF-β1 in renal epithelial cells.