Curcumin [1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of curcumin were prepared in dimethyl sulfoxide (Sigma-Aldrich). The primary and secondary antibodies used were as follows: KL rabbit anti-human monoclonal (1:1,000; ab181373; Abcam, Cambridge, MA, USA), DNMT1 rabbit anti-human monoclonal (1:2,000; #5032; Cell Signaling Technology, Inc., Danvers, MA, USA), horseradish peroxidase-conjugated anti-rabbit IgG (1:3,000; #7074; Cell Signaling Technology). Invitrogen TRIzol reagent was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

The HK-2 human proximal tubule epithelial cell line was obtained from the American Type Culture Collection (Manassas, VA, USA), and maintained in Hyclone Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin (100 U/ml) and 1% streptomycin (100 µg/ml) (Roche Diagnostics, Indianapolis, IN, USA).

A total of 72 female C57BL/6 mice (age, 6–8 weeks; weight, 19.1±0.7 g) were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China) and all mouse experiments were approved by the Animal Welfare & Ethics Committee of Zhejiang University (Hangzhou, China). The mice were maintained in a specific-pathogen-free animal facility, which was controlled at 23–25°C with a 12-h light/dark cycle. The mice were caged 5 mice per cage and were fed with normal chow and sterilized water. Cyclosporine A (CsA; Novartis, Basel, Switzerland) was dissolved with olive oil at a concentration of 20 mg/ml. Mice were treated with the vehicle alone (olive oil; 1 ml/kg/day), CsA alone (15 mg/kg, s.c.), or CsA + curcumin (15 mg/kg/day, i.p.) for 14 days. An s.c. injection of olive oil (15 ml/kg/day) and i.p. injection of normal saline (0.9% w/v NaCl) served as controls.

Kidney tissues [5×5×5 mm; obtained from mice under 70 mg/kg intraperitoneal sodium pentobarbital anesthesia (Sigma-Aldrich) and stored at −80°C], or 1×10⁷ kidney cells were homogenized with 1 ml TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., San Diego, CA, USA). Chloroform (200 µl) was added to 1 ml TRIzol, and following vigorous shaking for 30 sec and centrifugation at 12,000 × g for 20 min at 4°C, the aqueous phase was collected. An equal volume of isopropanol was added and mixed thoroughly, and was maintained at room temperature for 15 min prior to centrifugation again at 12,000 × g for 20 min at 4°C. Ethanol was used to prepare the RNA. The total RNA was reverse-transcribed using an RT-qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc., San Diego, CA, USA). The polymerase used was Taq DNA polymerase (Invitrogen; Thermo Fisher Scientific, Inc.), and the DNA ladder and ethidium bromide used were also from Invitrogen (Thermo Fisher Scientific, Inc.). qPCR was conducted on an ABI Prism 7900HT (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SuperScript III Platinum SYBR Green One-step qRT-PCR system from Invitrogen (Thermo Fisher Scientific, Inc), which included SuperScript III, Platinum Taq, reaction mix and dNTPs. The cycling conditions were as follows: Initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 15 sec and 60°C for 60 sec, then 72°C for 5 min. The primers were as follows: Forward, 5′-CCCTA CCCAG CACCT TCAAA-3′ and reverse, 5′-GTGGC CGATG TTTCC AGTCT-3′ for Col I; forward, 5′-GGGCA CTACC ATGTA CCCAG-3′ and reverse, 5′-TGAAG GCGCT GATCC ACAAA A-3′ for α-SMA; forward, 5′-GGCCA CCAAC TTCGG AGTAA-3′ and reverse, 5′-TGTTC CATGA CCCCA TGAGC-3′ for PAI-1; forward, 5′-AGTGG CCGAG AGAGT TTTGG-3′ and reverse, 5′-GGTGT AATGA CTCAC CGCCA-3′ for KL; forwrd, 5′-GCTGT TCCTT GTAGG CGAGT-3′ and reverse, 5′-GGGGA CTCAA ACCTT GCGTA-3′ for Dnmt1. GAPDH served as an internal control to normalize for differences in the quantity of total RNA in each sample, and expression was calculated using the 2^−ΔΔCt method (21). A total of 3 independent experiments were performed.

Kidneys harvested from the control, CsA and CsA + curcumin groups were fixed for 3–12 h in 10% formalin, followed by incubation at room temperature for 30 min in 30% sucrose (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in phosphate-buffered saline (PBS; Gibco; Thermo Fisher Scientific, Inc.), and flash frozen in cold isopentane (Sinopharm Chemical Reagent Co., Ltd.). A cryostat (CM3050S; Leica Microsystems, Inc., Buffalo Grove, IL, USA) was used to prepare 10-µm sections for staining. Alternatively, MAs were embedded in paraffin by Tissue-Tek VIP (Sakula Finetek, Flemingweg, Netherlands) automated instrument and sectioned at 6 µm for analysis. The sections were stained using hematoxylin and eosin (Beyotime Institute of Biotechnology, Nantong, China). The sections were assessed using a light microscope (DM6 B, Leica Microsystems, Inc.).

HK-2 cells were seeded into 96-well plates at a density of 1×10³ cells per well. Cell viability was quantified at 24 h in the absence or presence of curcumin using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), measuring absorbance at 450 nm according to the manufacturer's protocols.

HK-2 cells were plated in 24-well plates (2×10⁵ cells/well) and were transfected with 30 pmol KL siRNA (5′-GGU UGG AAU AAA CUU GUCA-dtdt-3′) or control siRNA (5′-UUC UCC GAA CGU GUC ACGU-dtdt-3′), using 2 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were used for further experiment after 48 hours.

Total protein was extracted using a Protein Extraction kit according to the manufacturer's instructions (EMD Millipore, Billerica, MA, USA) and western blotting was performed as previously described (22).

Genomic DNA was extracted from the cell lines and bisulfite modification of the DNA was performed using the EZ DNA Methylation kit (Zymo Research Corp, Irvine, CA, USA) according to the manufacturer's instructions. Methylation-specific primers were used to amplify a 161-bp region of KL promoter and the primers were as follows: Forward, AGCTG GGAGA AACAG GTGCC and reverse, TGGCA ATAAT TACCT GCGAG. The PCR products were purified and subcloned using pGEM-T Easy Vector Systems (Promega Corporation, Madison, WI, USA) for subsequent sequencing analysis. Methylation levels at each position were calculated from the quantified data of bisulfite sequencing according to the formula (height of C peak) / [(height of C peak) × (height of T peak)].

Various forward primers, in the presence or absence of a methyl group to one specific cytosine residue, were used in combination with the common RV4 reverse primer (Promega Corporation). PCR products from three independent reactions were extracted, and co-transfected with Renilla luciferase into HK-2 cells using Lipofectamine 2000. Luciferase activity was measured 48 h later using the Dual-Luciferase® Reporter Assay system (Promega Corporation) according to the manufacturer's instructions. Data were normalized to the level of Renilla expression for each well. Unmethylated control luciferase activity was defined as 100%.

All of the measurements were collected in triplicate for each independent preparation. The results were statistically analyzed using Student's t-test or one-way analysis of variance with SPSS software, version 20 (IBM SPSS, Armonk, NY, USA). P<0.05 were considered to indicate a statistically significant difference.

The mechanism of CsA-induced renal fibrosis has been demonstrated to be correlated with EMT (23); thus, to investigate the possible mechanism of the protective effects induced by curcumin against renal fibrosis, the expression of PAI-1 and α-SMA (considered as crucial proteins in the TGF-β signaling pathway) (24–26) were analyzed in the present study. The expression level of Col I served as a readout for the severity of renal toxicity. The HK-2 human proximal tubule epithelial cell line was treated with curcumin, and the mRNA expression levels of α-SMA, PAI-1 and Col I were determined by RT-qPCR. The results indicated that the expression levels of these three genes became elevated from 2 h and continued to increase for 22 h in the presence of CsA (8 µM). However, the increase in expression was less in the CsA (8 µM) + curcumin group (10 µM; Fig. 1A–C). In addition, curcumin significantly inhibited the proliferation of HK-2 cells in a concentration-dependent manner (Fig. 1D), suggesting that curcumin administered in a culture medium (DMEM supplemented with FBS and penicillin and streptomycin) could downregulate the expression of genes associated with TGF-β signaling. Furthermore, cell apoptosis was detected and no significant change was identified upon curcumin treatment.

To further confirm the effects of curcumin on renal fibrosis, CsA-induced renal fibrosis model was established using mice. Renal tissue samples were harvested from the 6 mice and evaluated the expression of the same genes in the tissue samples after 2 weeks. The expression of α-SMA, PAI-1 and Col I were elevated from 2 days for the next 12 days in the CsA alone and the CsA + curcumin groups. The findings demonstrated that in the CsA + curcumin group, the increasing expression levels were lower than in the CsA group (Fig. 1E–G). Furthermore, the renal fibrosis was markedly reduced in the CsA + curcumin group (Fig. 1H). Taken together, these results indicate that curcumin attenuated TGF-β signaling and exerts protective effects against renal fibrosis.

TGF-β is the master regulatory gene for EMT and is required for activation of diverse EMT biomarkers, such as α-SMA and β-catenin, which are significant in fibrosis (10). KL has been shown to suppress TGF-β signaling via binding to the type II TGF-β receptor, thus inhibiting the EMT (27). Therefore, the present study investigated whether curcumin inhibits TGF-β signaling via inducing KL expression, and used the expression of PAI-1, α-SMA and Col I as readouts for TGF-β signaling. To support our hypothesis, HK-2-Si KL (HK-2-Si) cells, with the KL gene knocked down, were used. The HK-2-Si cells were incubated with curcumin and the expression levels of α-SMA, PAI-1 and Col I were evaluated. The expression level of KL was significantly downregulated at the mRNA and protein levels (Fig. 2A and B), demonstrating the key role of curcumin in regulating the expression of KL. Knockdown of KL almost completely obviated the downregulation in PAI-1, α-SMA and Col I in mRNA expression levels (Fig. 2C–E), illustrating the essential role of KL in curcumin-mediated inhibition of TGF-β signaling. Thus, it was demonstrated that regulation of KL expression is crucial for the inhibitory affects on TGF-β signaling (a master regulator of fibrosis), thus indicating that regulating the expression of KL may be important for protection against CsA-induced renal fibrosis.

Hypermethylation of CpG islands rich in KL promoter causes a reduction in KL expression in various types of cells. Therefore, the present study hypothesized that curcumin inhibits methylation of the KL promoter in HK-2 cells. The KL promoter region −275 to −115 base pairs, which is particularly CpG-rich, was amplified and bisulfite sequencing was performed for statistical analysis. As shown in Table I, CsA promoted CpG methylation at various sites, while curcumin reversed the hypermethylation effects at the majority of the sites, particularly at site 249, 240, and 236, suggesting that these sites are pivotal to KL expression in HK-2 renal tubule epithelial cells. To further emphasize the important role of these sites, PCR was used to introduce methyl groups to individual C-terminal residues, and the promoter was amplified using forward primers differing only by containing a methyl group. The reverse primer was common to all PCR reactions amplifying from the 3′-end of the firefly luciferase gene. The linear DNA was subsequently co-transfected with Renilla luciferase into the HK-2 cells. After 48 h, luciferase activity was measured, and in vitro methylation of CpG 249, 240, and 236 was observed to cause decreased luciferase activity upon methylation (Fig. 3), which highlighted the hypermethylation effects of curcumin on HK-2 cells.

Previous studies have demonstrated that KL expression was negatively correlated with the expression of PAI-1, α-SMA and SMAD2/3 (19). Therefore, whether the expression of KL altered following the addition of curcumin to the culture medium was evaluated in HK-2 cells. KL expression levels were elevated in the CsA + curcumin group, whereas they were inhibited in the CsA group (Fig. 4A and B). Previous studies proposed that KL expression was primarily dominated by Dnmt1 (28). To further investigate the mechanism of KL expression in the presence of curcumin, MSP was performed to evaluate the methylation of KL, and RT-qPCR and western blot were conducted to analyze the expression of Dnmt1. Methylation of KL was inhibited in the presence of curcumin when compared with the CsA group (Fig. 4B). In addition, the Dnmt1 expression correlated with increased methylation, with methylation being downregulated in the CsA + curcumin group (Fig. 4C and D). These results indicate that curcumin may inhibit the methylation of KL and, as result, downregulate the expression of Dnmt1, regulate specific genes in the TGF-β signaling pathway and induce a protective effect against renal fibrosis.