Plasticizer Di-(2-Ethylhexyl)Phthalate Induces Epithelial-to-Mesenchymal Transition and Renal Fibrosis In Vitro and In Vivo

Plasticizer Di-(2-Ethylhexyl)Phthalate Induces Epithelial-to-Mesenchymal Transition and Renal... Abstract Plasticizer di-(2-ethylhexyl)phthalate (DEHP) is known as an endocrine disruptor and a peroxisome proliferator. A currently epidemiological study has suggested that daily high DEHP intake from phthalate-tainted foods in children may be a risk factor for renal dysfunction. DEHP can leach from medical devices such as blood storage bags and the tubing. Long-term exposure to DEHP is associated with nephropathy and exacerbates chronic kidney diseases (CKDs) progression. However, the detailed effects and molecular mechanisms remain unclear. Here, we hypothesized that DEHP and its major metabolite mono-(2-ethylhexyl)phthalate (MEHP) incited epithelial-to-mesenchymal transition (EMT) and lead to aggravate renal fibrosis progression. Treatment with low-cytotoxic concentration DEHP, but not MEHP, for 72 h obviously induced the morphological and phenotypic changes and EMT markers induction in normal rat renal tubular epithelial cells (NRK-52E). AKT inhibitor MK-2206 inhibited DEHP-induced EMT features and signals of AKT phosphorylation and downstream NF-κB and GSK3β. DEHP did not affect the expression of transforming growth factor-β1 mRNA. DEHP down-regulated the peroxisome proliferator-activated receptor (PPAR)α and PPARγ protein expressions. PPARγ agonist pioglitazone partially and significantly inhibited DEHP-induced EMT induction. In vivo DEHP exposure for 6 weeks enhanced the renal dysfunction and renal fibrosis and mortality rate, but decreased the PPARα and PPARγ protein expressions, in a folic acid-induced kidney fibrosis mouse model. Taken together, these results demonstrate for the first time that DEHP arouses EMT induction and renal fibrosis progression in renal tubular cells and is associated with PPARs downregulation. DEHP exposure potentially exacerbated renal fibrosis/nephropathy in a kidney disease condition. di-(2-ethylhexyl) phthalate, epithelial-to-mesenchymal transition, renal fibrosis, peroxisome proliferator-activated receptors Di-(2-ethylhexyl)phthalate (DEHP), an endocrine disrupt chemical, is current most widely used plasticizer for polyvinyl chloride (PVC) production. The widespread use of the plastic products leads to broadly exposures in daily life of the humans. The dominant sources are considered from food packaging process, medical products and procedures, and contaminant foods (Fromme et al., 2007; Nabae et al., 2006). DEHP is known to rapid hydrolyze to its secondary product, mono-(2-ethylhexyl)phthalate (MEHP). It has been reported that DEHP can be fast excreted from urine and more than 75% metabolic products are cleared after 4 h (Barr et al., 2003; Koch et al., 2005). DEHP and its metabolic products have been shown to cause damages in heart, liver, kidney, and reproductive system and carcinogenesis (Fromme et al., 2007; Martinez-Arguelles et al., 2013; Posnack, 2014; Shih et al., 2015; Zhang et al., 2016). Nowadays, chronic kidney diseases (CKDs) are thought as a socioeconomic burden and a major health problem in the world (Lindquist and Mertens, 2013; Sun et al., 2017). The common pathway in CKD pathogenesis is known as renal fibrosis, which is associated with end-stage renal disease (Atzler et al., 2014; Lindquist and Mertens, 2013). The inflammation response can be traced during kidney injury, which activates renal cells to release the proinflammatory cytokines and recruits the inflammatory cells to synthesize profibrotic cytokines (Sun et al., 2017). Following, the matrix-producing cells recruitment and epithelial-mesenchymal transition (EMT) may occur and lead to renal fibrosis (Carew et al., 2012; Lamouille et al., 2014). During the progression of EMT, the renal proximal tubular cells lose their epithelial cell adhesion in which the loss of epithelial surface markers like as E-cadherin and the upregulation of mesenchymal markers like as vimentin and N-cadherin occur (Huang et al., 2012). The cells gradually changed to spindle mesenchymal morphology. In the meanwhile, the expression of α-smooth muscle actin (α-SMA), an early marker of chronic renal dysfunction, is increased in tubular cells that finding has been considered as evidence for EMT (Badid et al., 2002; Carew et al., 2012). On the late stage, the accumulation of abnormal extracellular matrix (ECM) proteins like as fibronectin, laminin, types I and VI collagen occurs in interstitial region and/or glomerular mesangium to cause renal fibrosis and loss renal function (Mason and Wahab, 2003). A previous study showed that DEHP exposure (3000–12 000 ppm) for 2–18 months caused renal tubular damage in mice in a time- and dose-dependent manner (Ward et al., 1986). Raymond et al. indicated that long-term exposure to DEHP (12 500 ppm) exacerbated the chronic progressive nephropathy in male rats (David et al., 2000). Similarly, Wood et al. showed that DEHP exposure (1.2%; 3147 mg/kg/day) lowered kidney weight and caused renal tubular degeneration at ≥ 52 weeks (Wood et al., 2014). It has also been found that maternal DEHP (0.25 and 6.25 mg/kg/day) exposure induces the offspring kidney injury, including nephron deficit at weaning as well as CKD in adulthood of rats (Wei et al., 2012). In addition, a currently epidemiological study has suggested that daily high DEHP intake (>0.05 mg/kg/day) from phthalate-tainted foods in children may be a risk factor for renal dysfunction (Tsai et al., 2016). These results indicated that long-term DEHP exposure possessed the ability to incite renal toxicity or chronic kidney lesion. However, the molecular mechanism for DEHP-related CKD remained unclear. DEHP is known as a peroxisome proliferator. The toxicity of DEHP and its metabolites has been to be considered to involve peroxisome proliferator-activated receptors (PPARs), but it still remains controversial (Gao et al., 2017; Kamijo et al., 2007; Melnick, 2001). In this study, we hypothesized that DEHP and/or its metabolite MEHP incite EMT in the renal tubular cells and enhance ECM accumulation and renal fibrosis. We used an in vitro renal proximal tubular cell model and a folic acid-induced kidney fibrosis mouse model (Yang et al., 2010; Yi et al., 2018) to verify this working hypothesis and investigate the involved molecular mechanisms. MATERIALS AND METHODS Cell culture NRK-52E, a rat renal proximal tubular cell line, was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). NRK-52E cells have been suggested to exhibit functions similar to those of the rat renal proximal tubule in vivo (Lash et al., 2002). Cells were cultured in media of DMEM supplemented with fetal bovine serum (5%), penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37°C in 5% CO2. Cell viability assay Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St Louis, Missouri) assay. Cells (5 × 103) were seeded in 96-well plates at 37°C and 5% CO2 overnight. Subsequently, the cells were treated with 5–100 μM DEHP or MEHP (Sigma-Aldrich) for 24 or 72 h, and then added MTT medium (0.5 mg/ml) to each well. After 2 h incubation, dimethyl sulfoxide was added and the plates were incubated at room temperature for 30 min. The absorbance at 570 nm was detected by SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, California). Cell morphology analysis Cells (1.5 × 105) were seeded on 6-well plates for 16 h and were exposed to DEHP (5–25 μM) or MEHP (10–50 μM) for 72 h. Cell morphology changes were observed by the microscope in the bright-fields. The cell shapes were recognized at a 40× to 200× magnification. Immunofluorescence staining Cells (5 × 104) were cultured in the 4-well chamber slides. After 16 h, cells were treated with 25 μM DEHP for 72 h and then fixed with 4% paraformaldehyde for 20 min at room temperature. Washed 2 times with 0.2% triton X-100/phosphate-buffered saline solution and blocking samples with 5% bovine serum albumin. The primary antibodies for E-cadherin, vimentin, and fibronectin (Cell Signaling Technology, Danvers, Massachusetts) were used. Western blotting The protein expressions in NRK 52E cells and renal cortex tissues were performed by Western blotting as described previously in Wu et al. (2013). The protein samples of NRK-52E cells or renal cortex tissue were lysed by the ice-cold RIPA buffer supplemented with protease inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, California) and separated by SDS-PAGE and transferred onto the Immobilon P membranes (Millipore, Temecula, California). After blocking with for 5% skim milk solution for 4 h, the membranes were incubated overnight at 4°C with primary antibodies for α-SMA (Sigma-Aldrich), E-cadherin (BD Biosciences, San Jose, California), vimentin, phosphorylated AKT, phosphorylated mammalian TOR (mTOR), mTOR, phosphorylated GSK3β, GSK3β, phosphorylated NF-κB, NF-κB, caspase-3 (Cell Signaling Technology), fibronectin, AKT, PPAR-α, PPAR-γ, and connective tissue growth factor (CTGF) (Santa Cruz Biotechnology). The antimouse or antirabbit secondary antibody (Santa Cruz Biotechnology) was incubated for 1 h and the membranes were detected by enhanced chemiluminescence (Thermo Fisher Scientific, Grand Island, New York) on Fuji Film LAS-4000 mini performing system. Quantification was performed by GelDoc (Bio-Rad, Espoo, Finland). Real-time RT-PCR For each RT reaction, 5 μg of total RNA was added in a reaction volume of 30 μl using the Promega reverse transcriptase reagent mix. The 100 ng of RT products were used as template for amplification using the SYBR Green PCR amplification reagent (Qiagen). The mRNA expression was normalized by the β-actin signals amplified in 1 separate reaction. The primer sets of TGF-β1 (forward: 5’-GCAACAATTCCTGGCGTTAC-3’, reverse: 5’-GTATTCCGTCTCCTTGGTTCAG-3’) and β-actin (forward: 5’-CCTGTATGCCTCTGGCGTA-3’, reverse: 5’-CCATCTCTTGCTCGAAGTCT-3’) were used. Cells were collected and detected the expressions of mRNA by Bio-Rad iQ5 Real-time RT-PCR Detection System. Animal experiment C57BL/6 male mice (20–25 g) were purchased from BioLASCO (Ilan, Taiwan). The Animal Research Committee of the College of Medicine, National Taiwan University, approved and conducted the study in accordance with the regulations of Taiwan and NIH guidelines on the care and welfare of laboratory animals. The animals were treated humanely and with regard for alleviation of suffering. Mice were housed in the rooms under pathogen-free conditions with a 12-h light-dark cycle. Mice were administered with DEHP (50 mg/kg/day) by oral gavage for 4 weeks, and then folic acid (200 mg/kg body weight) was intraperitoneally injected to induce the kidney injury (control, n = 11; DEHP, n = 11; folic acid, n = 11; DEHP + folic acid, n = 14). Subsequently, mice were continually administered with DEHP (50 mg/kg/day) for 2 weeks. Blood was collected from the tail vein at 1 day before and days 1, 3, 7, and 14 after folic acid injection. All animals were sacrificed 6 weeks after DEHP administration. Folic acid nephropathy mouse model has been suggested that renal function can be assessed as a measure of CKD (Yang et al., 2010). Histopathological analysis The paraffin-embedded renal tissue sections with 4 μm thickness was mounted on slides and stained with hematoxylin and eosin for assessment of renal morphology. The histopathological scoring was performed by a nephropathologist in a blinded manner. The tubular cell necrosis, tubular dilation, and intratubular cell detachment in sections were observed and analyzed as described previously in Wu et al. (2011). Abnormalities were graded by a semiquantitative score from 0 to 4: 0, no abnormalities; 1+, changes affecting <25% of the sample; 2+, changes affecting 25% to 50%; 3+, changes affecting 50%–75%; 4+, changes affecting >75%. In some experiments, the sections were stained with Masson’s trichrome for evaluation of interstitial collagen deposition. Stained sections were graded (0, no staining; 1, <25% staining; 2, 25%–50% staining; 3, 50%–75% staining; 4, 75 to 100% staining of the section) as described previously in Liu et al. (2016). Twenty fields were randomly selected at 400× magnification and assessed in each mouse. Statistical analysis Data are expressed as mean ± SEM for at least 3 independent experiments. The significant difference from the respective controls for each experimental test condition was assessed by 1-way analysis of variance followed by post hoc analysis with Bonferroni’s test. The difference is significant if the p-value is < .05. Statistical analysis was performed using GraphPad Prism V5.01 software. RESULTS Effects of DEHP and MEHP on Cell Viability and EMT Induction in Renal Proximal Tubular Cells We first investigated whether DEHP or its metabolite MEHP would affect cell viability and EMT induction in normal rat renal tubular epithelial cell line NRK-52E. As shown in Figure 1A, both DEHP and MEHP at the concentrations of 10–100 μM for 72 h slightly suppressed the cell viability. In addition, DEHP at the concentrations of 5–25 μM for 72 h did not induce apoptosis in NRK-52E cells (Figure 1B). NRK-52E cells treated with DEHP, but not MEHP, at the concentrations of 10–25 μM for 72 h became spindle-shaped and morphologically similar to mesenchymal/myofibroblast cells (Figure 1C). Therefore, exposure of DEHP at the concentrations of 10–25 μM may induce the EMT in NRK-52E cells. The signaling molecules including CTGF, E-cadherin, α-SMA, fibronectin, and vimentin are known to be involved in the EMT process (Cheng et al., 2015; Iwano et al., 2002; Masszi et al., 2003). As shown in Figure 2A, the protein expressions of CTGF, α-SMA, vimentin, and fibronectin were significantly and dose-dependently up-regulated and the protein expression of E-cadherin was significantly and down-regulated after DEHP treatment. However, MEHP treatment did not significantly upregulate or downregulate these proteins in NRK-52E cells (Figure 2B). Moreover, the immunocytochemistry staining also showed that the expressions of vimentin and fibronectin were conspicuously increased and the expression of E-cadherin was markedly decreased after DEHP treatment (Figure 3). These results indicate that prolonged exposure to DEHP, but not MEHP, elicited EMT induction in NRK-52E cells. Figure 1. View largeDownload slide Effects of DEHP and MEHP on cell viability and morphology in renal tubular cells. NRK-52E cells were treated with DEHP (5–100 μM) or MEHP (10–100 μM) for 72 h. Cell viability was determined by MTT assay (A). Cell apoptosis was detected by Annexin-V/propidium iodide staining (B). The protein expression of cleaved caspase-3 was measured by Western blot (B). Cell morphology was observed by a bright-field microscopy at low magnification (40× magnification) and (inserted) high magnification (200× magnification) (C). Data are presented as mean ± SEM for 3 independent experiments. Each assay is performed in triplicate. *p < .05, control versus DEHP or MEHP. Figure 1. View largeDownload slide Effects of DEHP and MEHP on cell viability and morphology in renal tubular cells. NRK-52E cells were treated with DEHP (5–100 μM) or MEHP (10–100 μM) for 72 h. Cell viability was determined by MTT assay (A). Cell apoptosis was detected by Annexin-V/propidium iodide staining (B). The protein expression of cleaved caspase-3 was measured by Western blot (B). Cell morphology was observed by a bright-field microscopy at low magnification (40× magnification) and (inserted) high magnification (200× magnification) (C). Data are presented as mean ± SEM for 3 independent experiments. Each assay is performed in triplicate. *p < .05, control versus DEHP or MEHP. Figure 2. View largeDownload slide Effects of DEHP and MEHP on EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) or MEHP (10–100 μM) for 72 h. The EMT and fibrotic markers were determined by Western blotting (A, DEHP and B, MEHP). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. Figure 2. View largeDownload slide Effects of DEHP and MEHP on EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) or MEHP (10–100 μM) for 72 h. The EMT and fibrotic markers were determined by Western blotting (A, DEHP and B, MEHP). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. Figure 3. View largeDownload slide DEHP-induced EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The EMT and fibrotic markers were determined by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Figure 3. View largeDownload slide DEHP-induced EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The EMT and fibrotic markers were determined by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Involvement of AKT-Related Signaling Pathway in DEHP-Evoked EMT Several signal cascades including TGF-β and receptor tyrosine kinase are known to contribute to induction of EMT (Lamouille et al., 2014). We next elucidated the possible pathways involved in the DEHP-evoked EMT induction. As shown in Figure 4A, the protein expression of phosphorylated AKT was dramatically increased by treatment with 25-μM DEHP for 72 h in NRK-52E cells in a dose-dependent manner. Following, we confirmed whether AKT activation contributed to DEHP-induced EMT induction in NRK-52E cells. AKT inhibitor MK-2206 effectively inhibited the increased phosphorylated AKT, vimentin and fibronectin expressions and the decreased E-cadherin expression in DEHP (25 μM)-treated NRK-52E cells (Figure 4B). The immunocytochemistry staining also showed that MK-2206 treatment effectively suppressed the increased vimentin and fibronectin in DEHP-treated NRK-52E cells (Figure 5). Furthermore, DEHP significantly increased the phosphorylation of NF-κB and GSK3-β in NRK-52E cells, which could be significantly reversed by MK-2206 treatment (Figure 4C). However, DEHP did not affect the phosphorylation of mTOR in NRK-52E cells (Figure 4C). These results suggest that DEHP incites EMT induction through the AKT-regulated NF-κB and GSK3-β signal cascades. Figure 4. View largeDownload slide Role of AKT in the DEHP-induced EMT and signaling molecules in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-α, PPAR-γ, EMT markers, mTOR, NF-κB, and GSK3β were detected by Western blotting (A–C). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP versus DEHP + MK-2206. Figure 4. View largeDownload slide Role of AKT in the DEHP-induced EMT and signaling molecules in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-α, PPAR-γ, EMT markers, mTOR, NF-κB, and GSK3β were detected by Western blotting (A–C). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP versus DEHP + MK-2206. Figure 5. View largeDownload slide AKT inhibitor inhibited DEHP-induced EMT in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The expressions of vimentin and fibronectin were performed by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Figure 5. View largeDownload slide AKT inhibitor inhibited DEHP-induced EMT in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The expressions of vimentin and fibronectin were performed by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. DEHP Inhibited PPAR-γ Expression to Incite the Fibrotic Signal PPAR-γ has been reported to contribute in several organ fibrosis including kidney through the regulation of TGF-β expression (Deng et al., 2012; Zhao et al., 2016). PPAR-α has also been shown to protect against DEHP-induced glomerulonephritis in mice (Kamijo et al., 2007). We hypothesized that DEHP induces EMT via suppression of PPAR-α or PPAR-γ. We next investigated the effects of DEHP on PPAR-α and PPAR-γ signals. Expectedly, the protein expressions of PPAR-α and PPAR-γ were down-regulated by treatment with 25 μM DEHP for 72 h in NRK-52E cells (Figure 4A). Treatment with DEHP (25 μM) for 24 h slightly increased the PPAR-γ expression, but the PPAR-γ expression was markedly decreased 72 h after treatment with DEHP (Figure 6A). PPAR-γ agonist pioglitazone treatment significantly reversed the DEHP-decreased PPAR-γ expression (Figure 6A). The increased α-SMA and fibronectin expressions and the decreased E-cadherin expression in DEHP-treated NRK-52E cells were partially but significantly reversed by pioglitazone treatment (Figure 6B). However, pioglitazone treatment did not affect the AKT phosphorylation in DEHP-treated NRK-52E cells (Figure 6A). Moreover, administration with fenofibrate (20 μM), a PPAR-α agonist, to NRK-52E cells significantly increased the protein expression of PPAR-α, and could significantly reverse the decreased PPAR-α protein expression induced by DEHP (Figure 7A). Fenofibrate could also significantly reverse the increased α-SMA and Fibronectin protein expressions and the decreased Ecadherin protein expression induced by DEHP (Figure 7B). In addition, the mRNA expression of TGF-β1 in NRK-52E cells was not affected 24 and 72 h after DEHP exposure (Figure 7C). These results suggest that the suppression of PPAR-α or PPAR-γ may be involved in the DEHP-induced EMT induction in renal proximal tubular cells. Figure 6. View largeDownload slide Effects of DEHP on PPAR-γ signaling in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-γ (A), and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-γ agonist (pioglitazone, 1–5 μM) on the protein expressions of phospho-AKT, AKT, PPAR-γ, and EMT markers in DEHP-treated NRK-52E cells were detected (A and B). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + pioglitazone. Figure 6. View largeDownload slide Effects of DEHP on PPAR-γ signaling in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-γ (A), and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-γ agonist (pioglitazone, 1–5 μM) on the protein expressions of phospho-AKT, AKT, PPAR-γ, and EMT markers in DEHP-treated NRK-52E cells were detected (A and B). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + pioglitazone. Figure 7. View largeDownload slide Effects of DEHP on PPAR-α and TGF-β signals in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of PPAR-α (A) and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-α agonist (fenofibrate, 10 and 20 μM) on the protein expressions of PPAR-α and EMT markers in DEHP-treated NRK-52E cells were detected. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + fenofibrate. Figure 7. View largeDownload slide Effects of DEHP on PPAR-α and TGF-β signals in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of PPAR-α (A) and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-α agonist (fenofibrate, 10 and 20 μM) on the protein expressions of PPAR-α and EMT markers in DEHP-treated NRK-52E cells were detected. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + fenofibrate. DEHP-Aggravated Renal Fibrosis in a Folic Acid-Induced Kidney Fibrosis Mouse Model We next investigated whether DEHP exposure induced or aggravated renal fibrosis in normal mice and folic acid-treated mice. Treatment with DEHP (50 mg/kg) for 6 weeks did not affect the survival rate (Figure 8A), but slightly and significantly increased the levels of creatinine and cystatin C (Figs. 8C and 8D) and induced renal injury (Figure 9A) and fibrosis (Figure 9B) in normal mice. In a folic acid-induced kidney fibrosis mouse model, DEHP administration significantly decreased the survival rate (Figure 8A) and increased kidney weight (Figure 8B) and cystatin C level (Figure 8D) and enhanced renal injury (Figure 9A) and fibrosis (Figure 9B) compared with folic acid alone group. Moreover, DEHP treatment significantly enhanced the protein expressions of vimentin, α-SMA, and fibronectin in the kidney of folic acid-treated mice (Figure 10A). The PPAR-α protein expressions in the kidneys were significantly decreased in mice treated with DEHP alone, folic acid alone, and DEHP + folic acid, (Figure 10B). The PPAR-γ protein expressions in the kidneys were also significantly decreased in mice treated with both DEHP alone and DEHP + folic acid, although it was increased in mice treated with folic acid alone (Figure 10B). In addition, the mRNA expressions of TGF-β1 in the kidneys were not changed in mice treated with DEHP alone, but markedly increased in mice treated with both folic acid alone and DEHP + folic acid (Figure 10C). DEHP exposure did not affect the folic acid-increased TGF-β1 mRNA expression. These results indicate that DEHP exposure can aggravate the process of CKD in a renal disease mouse model. Figure 8. View largeDownload slide The in vivo effects of DEHP on survival rate, kidney weight, and renal function markers in a folic acid-induced kidney fibrosis mouse model. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with folic acid (FA; 200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. Blood was collected at 1 day before and days 1, 3, 7, and 14 after folic acid injection. The survival rate (A), kidney weight (B), serum blood urea nitrogen (BUN) and creatinine (C), and serum cystatin C (D) were determined. Data are presented as mean ± SEM (n = 11–14). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 8. View largeDownload slide The in vivo effects of DEHP on survival rate, kidney weight, and renal function markers in a folic acid-induced kidney fibrosis mouse model. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with folic acid (FA; 200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. Blood was collected at 1 day before and days 1, 3, 7, and 14 after folic acid injection. The survival rate (A), kidney weight (B), serum blood urea nitrogen (BUN) and creatinine (C), and serum cystatin C (D) were determined. Data are presented as mean ± SEM (n = 11–14). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 9. View largeDownload slide The in vivo effects of DEHP on histopathological change and collagen deposition in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The renal histopathological examination and collagen deposition were performed by hematoxylin and eosin staining (A) and Masson’s trichrome staining (B), respectively. Data for scoring of tubule damage (A) and collagen deposition (B) are presented as mean ± SEM (n = 8). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 9. View largeDownload slide The in vivo effects of DEHP on histopathological change and collagen deposition in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The renal histopathological examination and collagen deposition were performed by hematoxylin and eosin staining (A) and Masson’s trichrome staining (B), respectively. Data for scoring of tubule damage (A) and collagen deposition (B) are presented as mean ± SEM (n = 8). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 10. View largeDownload slide The in vivo effects of DEHP on EMT induction and PPARs expression in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The protein expressions of EMT and fibrotic markers (A) and PPAR-α and PPAR-γ (B) in the kidneys from normal mice and FA-treated mice with or without DEHP exposure were performed by Western blotting. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM (n = 8). *p < 0.05, control versus DEHP or FA. #p < .05, FA versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 10. View largeDownload slide The in vivo effects of DEHP on EMT induction and PPARs expression in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The protein expressions of EMT and fibrotic markers (A) and PPAR-α and PPAR-γ (B) in the kidneys from normal mice and FA-treated mice with or without DEHP exposure were performed by Western blotting. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM (n = 8). *p < 0.05, control versus DEHP or FA. #p < .05, FA versus DEHP + FA. §p < .05, FA versus DEHP + FA. DISCUSSION DEHP has been the most common plasticizer used in the preparation of PVC. It has been used in PVC blood bags as a plasticizer for more than 50 years. Accumulation of evidence indicates that long-term exposure to DEHP disturbs kidney development, arouses glomerulonephritis, and damages renal function in animal and human studies (Kamijo et al., 2007; Tsai et al., 2016; Wei et al., 2012). The molecular mechanisms for DEHP-related renal injury still remain to be clarified. Moreover, less information is available about whether DEHP potentially accelerates the progress of CKD. In this study, the results showed that DEHP, but not its metabolite MEHP, induced renal tubular cell morphology change, EMT induction, and fibrotic signals. The in vivo results also indicated that DEHP effectively aggravated EMT induction and renal fibrosis in a folic acid-induced kidney fibrosis mouse model. DEHP and its metabolites exhibited the various effects in various cell types under various conditions. DEHP and its metabolites MEHP and 2-ethylhexanal have been shown to decrease mouse Leydig MA-10 cell viability and steroidogenic potential (Piché et al., 2012). Treatment with MEHP (1 × 10−3 M) for 24 h exhibited the strongest suppression in Leydig cell viability. Lucas et al. (2012) have found that MEHP at the concentrations of 0.5 and 0.75 μM for 24 h induced a decrease of spermatogonial stem cells-derived C18-4 cell viability. DEHP metabolite 2-ethylhexanoic acid at a concentration of 25 μM has been found to effectively induce PPARα expression in rat placental HRP-1 trophoblast cells, whereas both DEHP and MEHP (25 μM) had no significant effects (Xu et al., 2005). DEHP, but not MEHP, has been shown to decrease cell viability in Sertoli cell lines at a concentration of 100 μM (Nardelli et al., 2015). In this study, we found that DEHP, but not MEHP, at the concentrations of 10–25 μM induced renal proximal tubular cell morphology change and EMT induction. EMT is known as an important event to pathologically contribute to renal fibrosis progression. TGF-β1 is also known as a main inducer for EMT. TGF-β can activate the signaling pathway of PI3K/AKT/mTOR complex 1 (mTORC1) to promote the translation and cell size (Lamouille et al., 2014). Zhang et al. (2016) have indicated that the activation of PI3K/Akt/GSK-3β signaling contributes to tubulointerstitial fibrosis in diabetic nephropathy. The receptor tyrosine kinases-regulated PI3K/Akt/NF-κB signaling has also been suggested to be involved in the EMT progression (Badid et al., 2002). NF-κB signaling has been shown to contribute to indoxyl sulfate-induced fibrotic signals in renal proximal tubular cells (Wang et al., 2014). In this study, we also found that DEHP exposure lead to the activation of signals of AKT/GSK3-β and AKT/NF-κB in NRK-52E cells. AKT inhibitor MK-2206 effectively inhibited the activation of AKT and its downstream signals and EMT induction in DEHP-treated NRK-52E cells. However, both TGF-β1 mRNA expression and mTOR protein expression were not affected in NRK-52E cells treated with DEHP for 24–72 h. The TGF-β1 mRNA expression was also not significantly changed between DEHP and control group or folic acid and folic acid + DEHP groups. These findings suggest that DEHP in vitro exposure activate EMT induction in renal proximal tubular cells via a TGF-β-independent AKT signaling pathway. A previous study showed that oxidative stress-trigged mitochondrial destabilization was associated with increased PPAR-γ phosphorylation and decreased PPAR-γ expression in renal proximal tubular cells (Small et al., 2014). Troglitazone, a PPAR-γ agonist, has been found to suppress TGF-β1 signaling in human fibroblasts (Zhu et al., 2016). On the contrary, the fibrotic factors like as Snail and TGF-β can also inhibit the expression of PPAR-γ (Lee et al., 2013; Li et al., 2013). Curcumin has been shown to suppress TGF-β1-induced EMT in renal tubular cells via the up-regulation of PPAR-γ signaling (Li et al., 2013). The PPAR-γ agonists have been found to possess the protective effects on diabetic nephropathy, nondiabetic glomerulosclerosis, and renal tubulointerstitial fibrosis (Han et al., 2010; Ma et al., 2001; Yang et al., 2009). The protective role of PPAR-α against DEHP-induced glomerulonephritis in mice has also been mentioned (Kamijo et al., 2007). These previous findings indicated that the down-regulation of PPAR-γ and PPAR-α signaling may promote the induction of EMT and fibrosis. DEHP and its metabolites are known as the peroxisome proliferators. DEHP has been suggested that it can activate multiple signals through the activation of PPARs like as apoptosis suppression, but it can also incite the peroxisome proliferation-independent biological effects like as morphologic cell transformation (Melnick, 2001). In this study, we found that the protein expressions of PPAR-γ and PPAR-α were dramatically inhibited in NRK-52E cells after 72 h treatment with DEHP. Treatment with PPAR-γ agonist pioglitazone did not affect the AKT phosphorylation, but significantly reversed the EMT and fibrotic signals in DEHP-treated NRK-52E cells. Recently, Kang et al. (2015) have shown that the mRNA expressions of PPAR-α and PPAR-γ-coactivator 1α are lower and lipid accumulation and triglyceride content are higher in the kidneys of mice with folic acid-induced kidney fibrosis. In this study, we also found that the expressions of PPAR-α and PPAR-γ in the kidneys were significantly decreased in mice treated with both DEHP alone and DEHP + folic acid groups. In folic acid alone group, the PPAR-α expression was significantly decreased, however, the PPAR-γ expression was markedly elevated. It is not clear how folic acid alone causes the increase in renal PPAR-γ expression. It may consider the factor of time course for the effect of folic acid alone on renal PPAR-γ expression. This needs to be clarified in the future. DEHP exposure was capable of enhancing the renal fibrosis in a folic acid-induced kidney fibrosis mouse model. These results indicate that the downregulation of PPAR signaling may contribute to the activation of EMT process in renal tubular cells and the enhancement of renal fibrosis under DEHP exposure. The levels of DEHP in the bloods from replacement blood transfusions have been shown to be ranged from 10 to 650 μg/ml (Kavlock et al., 2002). The blood DEHP levels can reach up to several mg/kg/day during blood transfusions (Wittassek et al., 2011). The no observed adverse effect level (NOAEL) of DEHP from a 2-year chronic oral toxicity study in rats was 2500 ppm (147–182 mg/kg bw/day) for abnormalities on the pituitary and kidney (David et al., 2000). Kamijo et al. showed that dietary treatment to DEHP (0.01% [8–11 mg/kg/day] or 0.05% (42–55 mg/kg/day]) for 22 months induced prominent immune complex glomerulonephritis in PPAR-α-null mice (Kamijo et al., 2007). In this study, DEHP at the concentrations of 5–25 μM (about 19.5–97.5 μg/ml) was capable of inducing EMT in cultured renal tubular cells. Oral administration with DEHP 50 mg/kg/day for 6 weeks enhanced the renal function loss and renal tubular/interstitial fibrosis in folic acid-treated mice. These results indicate that DEHP incites EMT and promotes the progression of renal fibrosis at doses relevant to human exposure and the NOAEL. In conclusion, the results reported here demonstrate for the first time that DEHP treatment arouses EMT induction in renal proximal tubular cells via the up-regulation of AKT-related signaling and the down-regulation of PPARγ-related signaling. DEHP exposure exacerbated renal fibrosis/nephropathy in a kidney disease condition. 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Plasticizer Di-(2-Ethylhexyl)Phthalate Induces Epithelial-to-Mesenchymal Transition and Renal Fibrosis In Vitro and In Vivo

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Abstract

Abstract Plasticizer di-(2-ethylhexyl)phthalate (DEHP) is known as an endocrine disruptor and a peroxisome proliferator. A currently epidemiological study has suggested that daily high DEHP intake from phthalate-tainted foods in children may be a risk factor for renal dysfunction. DEHP can leach from medical devices such as blood storage bags and the tubing. Long-term exposure to DEHP is associated with nephropathy and exacerbates chronic kidney diseases (CKDs) progression. However, the detailed effects and molecular mechanisms remain unclear. Here, we hypothesized that DEHP and its major metabolite mono-(2-ethylhexyl)phthalate (MEHP) incited epithelial-to-mesenchymal transition (EMT) and lead to aggravate renal fibrosis progression. Treatment with low-cytotoxic concentration DEHP, but not MEHP, for 72 h obviously induced the morphological and phenotypic changes and EMT markers induction in normal rat renal tubular epithelial cells (NRK-52E). AKT inhibitor MK-2206 inhibited DEHP-induced EMT features and signals of AKT phosphorylation and downstream NF-κB and GSK3β. DEHP did not affect the expression of transforming growth factor-β1 mRNA. DEHP down-regulated the peroxisome proliferator-activated receptor (PPAR)α and PPARγ protein expressions. PPARγ agonist pioglitazone partially and significantly inhibited DEHP-induced EMT induction. In vivo DEHP exposure for 6 weeks enhanced the renal dysfunction and renal fibrosis and mortality rate, but decreased the PPARα and PPARγ protein expressions, in a folic acid-induced kidney fibrosis mouse model. Taken together, these results demonstrate for the first time that DEHP arouses EMT induction and renal fibrosis progression in renal tubular cells and is associated with PPARs downregulation. DEHP exposure potentially exacerbated renal fibrosis/nephropathy in a kidney disease condition. di-(2-ethylhexyl) phthalate, epithelial-to-mesenchymal transition, renal fibrosis, peroxisome proliferator-activated receptors Di-(2-ethylhexyl)phthalate (DEHP), an endocrine disrupt chemical, is current most widely used plasticizer for polyvinyl chloride (PVC) production. The widespread use of the plastic products leads to broadly exposures in daily life of the humans. The dominant sources are considered from food packaging process, medical products and procedures, and contaminant foods (Fromme et al., 2007; Nabae et al., 2006). DEHP is known to rapid hydrolyze to its secondary product, mono-(2-ethylhexyl)phthalate (MEHP). It has been reported that DEHP can be fast excreted from urine and more than 75% metabolic products are cleared after 4 h (Barr et al., 2003; Koch et al., 2005). DEHP and its metabolic products have been shown to cause damages in heart, liver, kidney, and reproductive system and carcinogenesis (Fromme et al., 2007; Martinez-Arguelles et al., 2013; Posnack, 2014; Shih et al., 2015; Zhang et al., 2016). Nowadays, chronic kidney diseases (CKDs) are thought as a socioeconomic burden and a major health problem in the world (Lindquist and Mertens, 2013; Sun et al., 2017). The common pathway in CKD pathogenesis is known as renal fibrosis, which is associated with end-stage renal disease (Atzler et al., 2014; Lindquist and Mertens, 2013). The inflammation response can be traced during kidney injury, which activates renal cells to release the proinflammatory cytokines and recruits the inflammatory cells to synthesize profibrotic cytokines (Sun et al., 2017). Following, the matrix-producing cells recruitment and epithelial-mesenchymal transition (EMT) may occur and lead to renal fibrosis (Carew et al., 2012; Lamouille et al., 2014). During the progression of EMT, the renal proximal tubular cells lose their epithelial cell adhesion in which the loss of epithelial surface markers like as E-cadherin and the upregulation of mesenchymal markers like as vimentin and N-cadherin occur (Huang et al., 2012). The cells gradually changed to spindle mesenchymal morphology. In the meanwhile, the expression of α-smooth muscle actin (α-SMA), an early marker of chronic renal dysfunction, is increased in tubular cells that finding has been considered as evidence for EMT (Badid et al., 2002; Carew et al., 2012). On the late stage, the accumulation of abnormal extracellular matrix (ECM) proteins like as fibronectin, laminin, types I and VI collagen occurs in interstitial region and/or glomerular mesangium to cause renal fibrosis and loss renal function (Mason and Wahab, 2003). A previous study showed that DEHP exposure (3000–12 000 ppm) for 2–18 months caused renal tubular damage in mice in a time- and dose-dependent manner (Ward et al., 1986). Raymond et al. indicated that long-term exposure to DEHP (12 500 ppm) exacerbated the chronic progressive nephropathy in male rats (David et al., 2000). Similarly, Wood et al. showed that DEHP exposure (1.2%; 3147 mg/kg/day) lowered kidney weight and caused renal tubular degeneration at ≥ 52 weeks (Wood et al., 2014). It has also been found that maternal DEHP (0.25 and 6.25 mg/kg/day) exposure induces the offspring kidney injury, including nephron deficit at weaning as well as CKD in adulthood of rats (Wei et al., 2012). In addition, a currently epidemiological study has suggested that daily high DEHP intake (>0.05 mg/kg/day) from phthalate-tainted foods in children may be a risk factor for renal dysfunction (Tsai et al., 2016). These results indicated that long-term DEHP exposure possessed the ability to incite renal toxicity or chronic kidney lesion. However, the molecular mechanism for DEHP-related CKD remained unclear. DEHP is known as a peroxisome proliferator. The toxicity of DEHP and its metabolites has been to be considered to involve peroxisome proliferator-activated receptors (PPARs), but it still remains controversial (Gao et al., 2017; Kamijo et al., 2007; Melnick, 2001). In this study, we hypothesized that DEHP and/or its metabolite MEHP incite EMT in the renal tubular cells and enhance ECM accumulation and renal fibrosis. We used an in vitro renal proximal tubular cell model and a folic acid-induced kidney fibrosis mouse model (Yang et al., 2010; Yi et al., 2018) to verify this working hypothesis and investigate the involved molecular mechanisms. MATERIALS AND METHODS Cell culture NRK-52E, a rat renal proximal tubular cell line, was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). NRK-52E cells have been suggested to exhibit functions similar to those of the rat renal proximal tubule in vivo (Lash et al., 2002). Cells were cultured in media of DMEM supplemented with fetal bovine serum (5%), penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37°C in 5% CO2. Cell viability assay Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St Louis, Missouri) assay. Cells (5 × 103) were seeded in 96-well plates at 37°C and 5% CO2 overnight. Subsequently, the cells were treated with 5–100 μM DEHP or MEHP (Sigma-Aldrich) for 24 or 72 h, and then added MTT medium (0.5 mg/ml) to each well. After 2 h incubation, dimethyl sulfoxide was added and the plates were incubated at room temperature for 30 min. The absorbance at 570 nm was detected by SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, California). Cell morphology analysis Cells (1.5 × 105) were seeded on 6-well plates for 16 h and were exposed to DEHP (5–25 μM) or MEHP (10–50 μM) for 72 h. Cell morphology changes were observed by the microscope in the bright-fields. The cell shapes were recognized at a 40× to 200× magnification. Immunofluorescence staining Cells (5 × 104) were cultured in the 4-well chamber slides. After 16 h, cells were treated with 25 μM DEHP for 72 h and then fixed with 4% paraformaldehyde for 20 min at room temperature. Washed 2 times with 0.2% triton X-100/phosphate-buffered saline solution and blocking samples with 5% bovine serum albumin. The primary antibodies for E-cadherin, vimentin, and fibronectin (Cell Signaling Technology, Danvers, Massachusetts) were used. Western blotting The protein expressions in NRK 52E cells and renal cortex tissues were performed by Western blotting as described previously in Wu et al. (2013). The protein samples of NRK-52E cells or renal cortex tissue were lysed by the ice-cold RIPA buffer supplemented with protease inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, California) and separated by SDS-PAGE and transferred onto the Immobilon P membranes (Millipore, Temecula, California). After blocking with for 5% skim milk solution for 4 h, the membranes were incubated overnight at 4°C with primary antibodies for α-SMA (Sigma-Aldrich), E-cadherin (BD Biosciences, San Jose, California), vimentin, phosphorylated AKT, phosphorylated mammalian TOR (mTOR), mTOR, phosphorylated GSK3β, GSK3β, phosphorylated NF-κB, NF-κB, caspase-3 (Cell Signaling Technology), fibronectin, AKT, PPAR-α, PPAR-γ, and connective tissue growth factor (CTGF) (Santa Cruz Biotechnology). The antimouse or antirabbit secondary antibody (Santa Cruz Biotechnology) was incubated for 1 h and the membranes were detected by enhanced chemiluminescence (Thermo Fisher Scientific, Grand Island, New York) on Fuji Film LAS-4000 mini performing system. Quantification was performed by GelDoc (Bio-Rad, Espoo, Finland). Real-time RT-PCR For each RT reaction, 5 μg of total RNA was added in a reaction volume of 30 μl using the Promega reverse transcriptase reagent mix. The 100 ng of RT products were used as template for amplification using the SYBR Green PCR amplification reagent (Qiagen). The mRNA expression was normalized by the β-actin signals amplified in 1 separate reaction. The primer sets of TGF-β1 (forward: 5’-GCAACAATTCCTGGCGTTAC-3’, reverse: 5’-GTATTCCGTCTCCTTGGTTCAG-3’) and β-actin (forward: 5’-CCTGTATGCCTCTGGCGTA-3’, reverse: 5’-CCATCTCTTGCTCGAAGTCT-3’) were used. Cells were collected and detected the expressions of mRNA by Bio-Rad iQ5 Real-time RT-PCR Detection System. Animal experiment C57BL/6 male mice (20–25 g) were purchased from BioLASCO (Ilan, Taiwan). The Animal Research Committee of the College of Medicine, National Taiwan University, approved and conducted the study in accordance with the regulations of Taiwan and NIH guidelines on the care and welfare of laboratory animals. The animals were treated humanely and with regard for alleviation of suffering. Mice were housed in the rooms under pathogen-free conditions with a 12-h light-dark cycle. Mice were administered with DEHP (50 mg/kg/day) by oral gavage for 4 weeks, and then folic acid (200 mg/kg body weight) was intraperitoneally injected to induce the kidney injury (control, n = 11; DEHP, n = 11; folic acid, n = 11; DEHP + folic acid, n = 14). Subsequently, mice were continually administered with DEHP (50 mg/kg/day) for 2 weeks. Blood was collected from the tail vein at 1 day before and days 1, 3, 7, and 14 after folic acid injection. All animals were sacrificed 6 weeks after DEHP administration. Folic acid nephropathy mouse model has been suggested that renal function can be assessed as a measure of CKD (Yang et al., 2010). Histopathological analysis The paraffin-embedded renal tissue sections with 4 μm thickness was mounted on slides and stained with hematoxylin and eosin for assessment of renal morphology. The histopathological scoring was performed by a nephropathologist in a blinded manner. The tubular cell necrosis, tubular dilation, and intratubular cell detachment in sections were observed and analyzed as described previously in Wu et al. (2011). Abnormalities were graded by a semiquantitative score from 0 to 4: 0, no abnormalities; 1+, changes affecting <25% of the sample; 2+, changes affecting 25% to 50%; 3+, changes affecting 50%–75%; 4+, changes affecting >75%. In some experiments, the sections were stained with Masson’s trichrome for evaluation of interstitial collagen deposition. Stained sections were graded (0, no staining; 1, <25% staining; 2, 25%–50% staining; 3, 50%–75% staining; 4, 75 to 100% staining of the section) as described previously in Liu et al. (2016). Twenty fields were randomly selected at 400× magnification and assessed in each mouse. Statistical analysis Data are expressed as mean ± SEM for at least 3 independent experiments. The significant difference from the respective controls for each experimental test condition was assessed by 1-way analysis of variance followed by post hoc analysis with Bonferroni’s test. The difference is significant if the p-value is < .05. Statistical analysis was performed using GraphPad Prism V5.01 software. RESULTS Effects of DEHP and MEHP on Cell Viability and EMT Induction in Renal Proximal Tubular Cells We first investigated whether DEHP or its metabolite MEHP would affect cell viability and EMT induction in normal rat renal tubular epithelial cell line NRK-52E. As shown in Figure 1A, both DEHP and MEHP at the concentrations of 10–100 μM for 72 h slightly suppressed the cell viability. In addition, DEHP at the concentrations of 5–25 μM for 72 h did not induce apoptosis in NRK-52E cells (Figure 1B). NRK-52E cells treated with DEHP, but not MEHP, at the concentrations of 10–25 μM for 72 h became spindle-shaped and morphologically similar to mesenchymal/myofibroblast cells (Figure 1C). Therefore, exposure of DEHP at the concentrations of 10–25 μM may induce the EMT in NRK-52E cells. The signaling molecules including CTGF, E-cadherin, α-SMA, fibronectin, and vimentin are known to be involved in the EMT process (Cheng et al., 2015; Iwano et al., 2002; Masszi et al., 2003). As shown in Figure 2A, the protein expressions of CTGF, α-SMA, vimentin, and fibronectin were significantly and dose-dependently up-regulated and the protein expression of E-cadherin was significantly and down-regulated after DEHP treatment. However, MEHP treatment did not significantly upregulate or downregulate these proteins in NRK-52E cells (Figure 2B). Moreover, the immunocytochemistry staining also showed that the expressions of vimentin and fibronectin were conspicuously increased and the expression of E-cadherin was markedly decreased after DEHP treatment (Figure 3). These results indicate that prolonged exposure to DEHP, but not MEHP, elicited EMT induction in NRK-52E cells. Figure 1. View largeDownload slide Effects of DEHP and MEHP on cell viability and morphology in renal tubular cells. NRK-52E cells were treated with DEHP (5–100 μM) or MEHP (10–100 μM) for 72 h. Cell viability was determined by MTT assay (A). Cell apoptosis was detected by Annexin-V/propidium iodide staining (B). The protein expression of cleaved caspase-3 was measured by Western blot (B). Cell morphology was observed by a bright-field microscopy at low magnification (40× magnification) and (inserted) high magnification (200× magnification) (C). Data are presented as mean ± SEM for 3 independent experiments. Each assay is performed in triplicate. *p < .05, control versus DEHP or MEHP. Figure 1. View largeDownload slide Effects of DEHP and MEHP on cell viability and morphology in renal tubular cells. NRK-52E cells were treated with DEHP (5–100 μM) or MEHP (10–100 μM) for 72 h. Cell viability was determined by MTT assay (A). Cell apoptosis was detected by Annexin-V/propidium iodide staining (B). The protein expression of cleaved caspase-3 was measured by Western blot (B). Cell morphology was observed by a bright-field microscopy at low magnification (40× magnification) and (inserted) high magnification (200× magnification) (C). Data are presented as mean ± SEM for 3 independent experiments. Each assay is performed in triplicate. *p < .05, control versus DEHP or MEHP. Figure 2. View largeDownload slide Effects of DEHP and MEHP on EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) or MEHP (10–100 μM) for 72 h. The EMT and fibrotic markers were determined by Western blotting (A, DEHP and B, MEHP). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. Figure 2. View largeDownload slide Effects of DEHP and MEHP on EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) or MEHP (10–100 μM) for 72 h. The EMT and fibrotic markers were determined by Western blotting (A, DEHP and B, MEHP). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. Figure 3. View largeDownload slide DEHP-induced EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The EMT and fibrotic markers were determined by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Figure 3. View largeDownload slide DEHP-induced EMT induction in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The EMT and fibrotic markers were determined by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Involvement of AKT-Related Signaling Pathway in DEHP-Evoked EMT Several signal cascades including TGF-β and receptor tyrosine kinase are known to contribute to induction of EMT (Lamouille et al., 2014). We next elucidated the possible pathways involved in the DEHP-evoked EMT induction. As shown in Figure 4A, the protein expression of phosphorylated AKT was dramatically increased by treatment with 25-μM DEHP for 72 h in NRK-52E cells in a dose-dependent manner. Following, we confirmed whether AKT activation contributed to DEHP-induced EMT induction in NRK-52E cells. AKT inhibitor MK-2206 effectively inhibited the increased phosphorylated AKT, vimentin and fibronectin expressions and the decreased E-cadherin expression in DEHP (25 μM)-treated NRK-52E cells (Figure 4B). The immunocytochemistry staining also showed that MK-2206 treatment effectively suppressed the increased vimentin and fibronectin in DEHP-treated NRK-52E cells (Figure 5). Furthermore, DEHP significantly increased the phosphorylation of NF-κB and GSK3-β in NRK-52E cells, which could be significantly reversed by MK-2206 treatment (Figure 4C). However, DEHP did not affect the phosphorylation of mTOR in NRK-52E cells (Figure 4C). These results suggest that DEHP incites EMT induction through the AKT-regulated NF-κB and GSK3-β signal cascades. Figure 4. View largeDownload slide Role of AKT in the DEHP-induced EMT and signaling molecules in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-α, PPAR-γ, EMT markers, mTOR, NF-κB, and GSK3β were detected by Western blotting (A–C). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP versus DEHP + MK-2206. Figure 4. View largeDownload slide Role of AKT in the DEHP-induced EMT and signaling molecules in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-α, PPAR-γ, EMT markers, mTOR, NF-κB, and GSK3β were detected by Western blotting (A–C). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP versus DEHP + MK-2206. Figure 5. View largeDownload slide AKT inhibitor inhibited DEHP-induced EMT in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The expressions of vimentin and fibronectin were performed by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. Figure 5. View largeDownload slide AKT inhibitor inhibited DEHP-induced EMT in renal tubular cells. NRK-52E cells were treated with DEHP (5–25 μM) in the presence or absence of AKT inhibitor MK-2206 (0.5 and 1 μM) for 72 h. The expressions of vimentin and fibronectin were performed by immunocytochemistry staining. The results were representative of at least 3 independent experiments performed in triplicate. DEHP Inhibited PPAR-γ Expression to Incite the Fibrotic Signal PPAR-γ has been reported to contribute in several organ fibrosis including kidney through the regulation of TGF-β expression (Deng et al., 2012; Zhao et al., 2016). PPAR-α has also been shown to protect against DEHP-induced glomerulonephritis in mice (Kamijo et al., 2007). We hypothesized that DEHP induces EMT via suppression of PPAR-α or PPAR-γ. We next investigated the effects of DEHP on PPAR-α and PPAR-γ signals. Expectedly, the protein expressions of PPAR-α and PPAR-γ were down-regulated by treatment with 25 μM DEHP for 72 h in NRK-52E cells (Figure 4A). Treatment with DEHP (25 μM) for 24 h slightly increased the PPAR-γ expression, but the PPAR-γ expression was markedly decreased 72 h after treatment with DEHP (Figure 6A). PPAR-γ agonist pioglitazone treatment significantly reversed the DEHP-decreased PPAR-γ expression (Figure 6A). The increased α-SMA and fibronectin expressions and the decreased E-cadherin expression in DEHP-treated NRK-52E cells were partially but significantly reversed by pioglitazone treatment (Figure 6B). However, pioglitazone treatment did not affect the AKT phosphorylation in DEHP-treated NRK-52E cells (Figure 6A). Moreover, administration with fenofibrate (20 μM), a PPAR-α agonist, to NRK-52E cells significantly increased the protein expression of PPAR-α, and could significantly reverse the decreased PPAR-α protein expression induced by DEHP (Figure 7A). Fenofibrate could also significantly reverse the increased α-SMA and Fibronectin protein expressions and the decreased Ecadherin protein expression induced by DEHP (Figure 7B). In addition, the mRNA expression of TGF-β1 in NRK-52E cells was not affected 24 and 72 h after DEHP exposure (Figure 7C). These results suggest that the suppression of PPAR-α or PPAR-γ may be involved in the DEHP-induced EMT induction in renal proximal tubular cells. Figure 6. View largeDownload slide Effects of DEHP on PPAR-γ signaling in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-γ (A), and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-γ agonist (pioglitazone, 1–5 μM) on the protein expressions of phospho-AKT, AKT, PPAR-γ, and EMT markers in DEHP-treated NRK-52E cells were detected (A and B). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + pioglitazone. Figure 6. View largeDownload slide Effects of DEHP on PPAR-γ signaling in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of phospho-AKT, AKT, PPAR-γ (A), and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-γ agonist (pioglitazone, 1–5 μM) on the protein expressions of phospho-AKT, AKT, PPAR-γ, and EMT markers in DEHP-treated NRK-52E cells were detected (A and B). The quantification was performed by densitometry. Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + pioglitazone. Figure 7. View largeDownload slide Effects of DEHP on PPAR-α and TGF-β signals in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of PPAR-α (A) and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-α agonist (fenofibrate, 10 and 20 μM) on the protein expressions of PPAR-α and EMT markers in DEHP-treated NRK-52E cells were detected. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + fenofibrate. Figure 7. View largeDownload slide Effects of DEHP on PPAR-α and TGF-β signals in renal tubular cells. NRK-52E cells were treated with DEHP (25 μM) for 72 h. The protein expressions of PPAR-α (A) and EMT markers (B) were performed by Western blotting. In some experiments, the effects of PPAR-α agonist (fenofibrate, 10 and 20 μM) on the protein expressions of PPAR-α and EMT markers in DEHP-treated NRK-52E cells were detected. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM for 3 independent experiments performed in triplicate. *p < .05, control versus DEHP. #p < .05, DEHP group versus DEHP + fenofibrate. DEHP-Aggravated Renal Fibrosis in a Folic Acid-Induced Kidney Fibrosis Mouse Model We next investigated whether DEHP exposure induced or aggravated renal fibrosis in normal mice and folic acid-treated mice. Treatment with DEHP (50 mg/kg) for 6 weeks did not affect the survival rate (Figure 8A), but slightly and significantly increased the levels of creatinine and cystatin C (Figs. 8C and 8D) and induced renal injury (Figure 9A) and fibrosis (Figure 9B) in normal mice. In a folic acid-induced kidney fibrosis mouse model, DEHP administration significantly decreased the survival rate (Figure 8A) and increased kidney weight (Figure 8B) and cystatin C level (Figure 8D) and enhanced renal injury (Figure 9A) and fibrosis (Figure 9B) compared with folic acid alone group. Moreover, DEHP treatment significantly enhanced the protein expressions of vimentin, α-SMA, and fibronectin in the kidney of folic acid-treated mice (Figure 10A). The PPAR-α protein expressions in the kidneys were significantly decreased in mice treated with DEHP alone, folic acid alone, and DEHP + folic acid, (Figure 10B). The PPAR-γ protein expressions in the kidneys were also significantly decreased in mice treated with both DEHP alone and DEHP + folic acid, although it was increased in mice treated with folic acid alone (Figure 10B). In addition, the mRNA expressions of TGF-β1 in the kidneys were not changed in mice treated with DEHP alone, but markedly increased in mice treated with both folic acid alone and DEHP + folic acid (Figure 10C). DEHP exposure did not affect the folic acid-increased TGF-β1 mRNA expression. These results indicate that DEHP exposure can aggravate the process of CKD in a renal disease mouse model. Figure 8. View largeDownload slide The in vivo effects of DEHP on survival rate, kidney weight, and renal function markers in a folic acid-induced kidney fibrosis mouse model. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with folic acid (FA; 200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. Blood was collected at 1 day before and days 1, 3, 7, and 14 after folic acid injection. The survival rate (A), kidney weight (B), serum blood urea nitrogen (BUN) and creatinine (C), and serum cystatin C (D) were determined. Data are presented as mean ± SEM (n = 11–14). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 8. View largeDownload slide The in vivo effects of DEHP on survival rate, kidney weight, and renal function markers in a folic acid-induced kidney fibrosis mouse model. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with folic acid (FA; 200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. Blood was collected at 1 day before and days 1, 3, 7, and 14 after folic acid injection. The survival rate (A), kidney weight (B), serum blood urea nitrogen (BUN) and creatinine (C), and serum cystatin C (D) were determined. Data are presented as mean ± SEM (n = 11–14). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 9. View largeDownload slide The in vivo effects of DEHP on histopathological change and collagen deposition in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The renal histopathological examination and collagen deposition were performed by hematoxylin and eosin staining (A) and Masson’s trichrome staining (B), respectively. Data for scoring of tubule damage (A) and collagen deposition (B) are presented as mean ± SEM (n = 8). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 9. View largeDownload slide The in vivo effects of DEHP on histopathological change and collagen deposition in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The renal histopathological examination and collagen deposition were performed by hematoxylin and eosin staining (A) and Masson’s trichrome staining (B), respectively. Data for scoring of tubule damage (A) and collagen deposition (B) are presented as mean ± SEM (n = 8). *p < .05, control versus DEHP or FA. #p < .05, DEHP versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 10. View largeDownload slide The in vivo effects of DEHP on EMT induction and PPARs expression in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The protein expressions of EMT and fibrotic markers (A) and PPAR-α and PPAR-γ (B) in the kidneys from normal mice and FA-treated mice with or without DEHP exposure were performed by Western blotting. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM (n = 8). *p < 0.05, control versus DEHP or FA. #p < .05, FA versus DEHP + FA. §p < .05, FA versus DEHP + FA. Figure 10. View largeDownload slide The in vivo effects of DEHP on EMT induction and PPARs expression in the kidneys of folic acid-treated mice. C57BL/6 mice were fed with DEHP (50 mg/kg/day) for 4 weeks and then injected with FA (200 mg/kg) to induce nephropathy. Subsequently, mice were continually administered with DEHP for 2 weeks. The protein expressions of EMT and fibrotic markers (A) and PPAR-α and PPAR-γ (B) in the kidneys from normal mice and FA-treated mice with or without DEHP exposure were performed by Western blotting. The quantification was performed by densitometry. The mRNA of TGF-β was measured by qPCR (C). Data are presented as mean ± SEM (n = 8). *p < 0.05, control versus DEHP or FA. #p < .05, FA versus DEHP + FA. §p < .05, FA versus DEHP + FA. DISCUSSION DEHP has been the most common plasticizer used in the preparation of PVC. It has been used in PVC blood bags as a plasticizer for more than 50 years. Accumulation of evidence indicates that long-term exposure to DEHP disturbs kidney development, arouses glomerulonephritis, and damages renal function in animal and human studies (Kamijo et al., 2007; Tsai et al., 2016; Wei et al., 2012). The molecular mechanisms for DEHP-related renal injury still remain to be clarified. Moreover, less information is available about whether DEHP potentially accelerates the progress of CKD. In this study, the results showed that DEHP, but not its metabolite MEHP, induced renal tubular cell morphology change, EMT induction, and fibrotic signals. The in vivo results also indicated that DEHP effectively aggravated EMT induction and renal fibrosis in a folic acid-induced kidney fibrosis mouse model. DEHP and its metabolites exhibited the various effects in various cell types under various conditions. DEHP and its metabolites MEHP and 2-ethylhexanal have been shown to decrease mouse Leydig MA-10 cell viability and steroidogenic potential (Piché et al., 2012). Treatment with MEHP (1 × 10−3 M) for 24 h exhibited the strongest suppression in Leydig cell viability. Lucas et al. (2012) have found that MEHP at the concentrations of 0.5 and 0.75 μM for 24 h induced a decrease of spermatogonial stem cells-derived C18-4 cell viability. DEHP metabolite 2-ethylhexanoic acid at a concentration of 25 μM has been found to effectively induce PPARα expression in rat placental HRP-1 trophoblast cells, whereas both DEHP and MEHP (25 μM) had no significant effects (Xu et al., 2005). DEHP, but not MEHP, has been shown to decrease cell viability in Sertoli cell lines at a concentration of 100 μM (Nardelli et al., 2015). In this study, we found that DEHP, but not MEHP, at the concentrations of 10–25 μM induced renal proximal tubular cell morphology change and EMT induction. EMT is known as an important event to pathologically contribute to renal fibrosis progression. TGF-β1 is also known as a main inducer for EMT. TGF-β can activate the signaling pathway of PI3K/AKT/mTOR complex 1 (mTORC1) to promote the translation and cell size (Lamouille et al., 2014). Zhang et al. (2016) have indicated that the activation of PI3K/Akt/GSK-3β signaling contributes to tubulointerstitial fibrosis in diabetic nephropathy. The receptor tyrosine kinases-regulated PI3K/Akt/NF-κB signaling has also been suggested to be involved in the EMT progression (Badid et al., 2002). NF-κB signaling has been shown to contribute to indoxyl sulfate-induced fibrotic signals in renal proximal tubular cells (Wang et al., 2014). In this study, we also found that DEHP exposure lead to the activation of signals of AKT/GSK3-β and AKT/NF-κB in NRK-52E cells. AKT inhibitor MK-2206 effectively inhibited the activation of AKT and its downstream signals and EMT induction in DEHP-treated NRK-52E cells. However, both TGF-β1 mRNA expression and mTOR protein expression were not affected in NRK-52E cells treated with DEHP for 24–72 h. The TGF-β1 mRNA expression was also not significantly changed between DEHP and control group or folic acid and folic acid + DEHP groups. These findings suggest that DEHP in vitro exposure activate EMT induction in renal proximal tubular cells via a TGF-β-independent AKT signaling pathway. A previous study showed that oxidative stress-trigged mitochondrial destabilization was associated with increased PPAR-γ phosphorylation and decreased PPAR-γ expression in renal proximal tubular cells (Small et al., 2014). Troglitazone, a PPAR-γ agonist, has been found to suppress TGF-β1 signaling in human fibroblasts (Zhu et al., 2016). On the contrary, the fibrotic factors like as Snail and TGF-β can also inhibit the expression of PPAR-γ (Lee et al., 2013; Li et al., 2013). Curcumin has been shown to suppress TGF-β1-induced EMT in renal tubular cells via the up-regulation of PPAR-γ signaling (Li et al., 2013). The PPAR-γ agonists have been found to possess the protective effects on diabetic nephropathy, nondiabetic glomerulosclerosis, and renal tubulointerstitial fibrosis (Han et al., 2010; Ma et al., 2001; Yang et al., 2009). The protective role of PPAR-α against DEHP-induced glomerulonephritis in mice has also been mentioned (Kamijo et al., 2007). These previous findings indicated that the down-regulation of PPAR-γ and PPAR-α signaling may promote the induction of EMT and fibrosis. DEHP and its metabolites are known as the peroxisome proliferators. DEHP has been suggested that it can activate multiple signals through the activation of PPARs like as apoptosis suppression, but it can also incite the peroxisome proliferation-independent biological effects like as morphologic cell transformation (Melnick, 2001). In this study, we found that the protein expressions of PPAR-γ and PPAR-α were dramatically inhibited in NRK-52E cells after 72 h treatment with DEHP. Treatment with PPAR-γ agonist pioglitazone did not affect the AKT phosphorylation, but significantly reversed the EMT and fibrotic signals in DEHP-treated NRK-52E cells. Recently, Kang et al. (2015) have shown that the mRNA expressions of PPAR-α and PPAR-γ-coactivator 1α are lower and lipid accumulation and triglyceride content are higher in the kidneys of mice with folic acid-induced kidney fibrosis. In this study, we also found that the expressions of PPAR-α and PPAR-γ in the kidneys were significantly decreased in mice treated with both DEHP alone and DEHP + folic acid groups. In folic acid alone group, the PPAR-α expression was significantly decreased, however, the PPAR-γ expression was markedly elevated. It is not clear how folic acid alone causes the increase in renal PPAR-γ expression. It may consider the factor of time course for the effect of folic acid alone on renal PPAR-γ expression. This needs to be clarified in the future. DEHP exposure was capable of enhancing the renal fibrosis in a folic acid-induced kidney fibrosis mouse model. These results indicate that the downregulation of PPAR signaling may contribute to the activation of EMT process in renal tubular cells and the enhancement of renal fibrosis under DEHP exposure. The levels of DEHP in the bloods from replacement blood transfusions have been shown to be ranged from 10 to 650 μg/ml (Kavlock et al., 2002). The blood DEHP levels can reach up to several mg/kg/day during blood transfusions (Wittassek et al., 2011). The no observed adverse effect level (NOAEL) of DEHP from a 2-year chronic oral toxicity study in rats was 2500 ppm (147–182 mg/kg bw/day) for abnormalities on the pituitary and kidney (David et al., 2000). Kamijo et al. showed that dietary treatment to DEHP (0.01% [8–11 mg/kg/day] or 0.05% (42–55 mg/kg/day]) for 22 months induced prominent immune complex glomerulonephritis in PPAR-α-null mice (Kamijo et al., 2007). In this study, DEHP at the concentrations of 5–25 μM (about 19.5–97.5 μg/ml) was capable of inducing EMT in cultured renal tubular cells. Oral administration with DEHP 50 mg/kg/day for 6 weeks enhanced the renal function loss and renal tubular/interstitial fibrosis in folic acid-treated mice. These results indicate that DEHP incites EMT and promotes the progression of renal fibrosis at doses relevant to human exposure and the NOAEL. In conclusion, the results reported here demonstrate for the first time that DEHP treatment arouses EMT induction in renal proximal tubular cells via the up-regulation of AKT-related signaling and the down-regulation of PPARγ-related signaling. DEHP exposure exacerbated renal fibrosis/nephropathy in a kidney disease condition. 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Toxicological SciencesOxford University Press

Published: Apr 14, 2018

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