Toxicology Letters 234 (2015) 110–119

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Indoxyl sulfate induces oxidative stress and hypertrophy in cardiomyocytes by inhibiting the AMPK/UCP2 signaling pathway Ke Yang 1, Xinli Xu 1, Ling Nie, Tangli Xiao, Xu Guan, Ting He, Yanlin Yu, Liang Liu, Yunjian Huang, Jingbo Zhang, Jinghong Zhao * Department of Nephrology, Institute of Nephrology of Chongqing and Kidney Center of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, PR China

H I G H L I G H T S


IS induces ROS generation in cardiomyocytes by down-regulation of UCP2.
UCP2 attenuates IS-induced cardiomyocyte hypertrophy by inhibiting ROS production.
AMPK inactivation contributes to IS-induced UCP2 down-regulation.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 September 2014 Received in revised form 24 January 2015 Accepted 28 January 2015 Available online 19 February 2015

As a typical protein-bound uremic toxin, indoxyl sulfate is considered to be able to induce cardiomyocytes hypertrophy by promoting oxidative stress in chronic kidney disease (CKD). Uncoupling protein 2 (UCP2), a member of the uncoupling protein family, may protect cardiomyocytes from oxidative stress by suppressing mitochondrial reactive oxygen species (ROS). In the present study, we aimed to determine whether UCP2 was involved in indoxyl sulfate-induced cardiomyocytes hypertrophy. We demonstrated that indoxyl sulfate could increase the ROS levels in a time and dose-dependent manner in cultured neonatal rat cardiomyocytes. Significant increases in [3H]-leucine incorporation, cell volume, and the mRNA expression levels of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and beta myosin heavy chain (b-MHC) were detected in cardiomyocytes after treatement with indoxyl sulfate at the concentration of 500 mM for 48 h, accompanied by a decreased expression of UCP2. In contrast, cardiomyocytes transfected with the lentiviral vector carrying UCP2 gene were resistant to indoxyl sulfate-induced ROS production and cell hypertrophy. Additionally, indoxyl sulfate-induced UCP2 reduction was correlated with the inhibition of AMP-activated protein kinase (AMPK) activity, while pretreatment with AICAR, an AMPK activator, effectively attenuated indoxyl sulfate-induced UCP2 down-regulation and hypertrophy in cardiomyocytes. Taken together, these results suggest that indoxyl sulfate-induced cardiomyocytes hypertrophy was partly due to the inhibition of AMPK/ UCP2 signaling and the enhancement of oxidative stress. ã 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cardiomyopathy Indoxyl sulfate Uncoupling protein 2 Oxidative stress AMPK

1. Introduction Cardiovascular disease (CVD) is the leading cause of mortality in patients with chronic kidney disease (CKD) (Go et al., 2004; Keith et al., 2004; Sarnak et al., 2003). Left ventricular hypertrophy (LVH) has been reported in up to 72% of adults and children with

* Corresponding author. Tel.: +86 23 68774321; fax: +86 23 68774321. E-mail address: [email protected] (J. Zhao). Ke Yang and Xinli Xu contribute equally to this work.

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http://dx.doi.org/10.1016/j.toxlet.2015.01.021 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.

end-stage renal disease (ESRD) (Johnstone et al., 1996; Levin et al., 1996; Mitsnefes et al., 2000; Rasic et al., 2004; Shin et al., 2007). Extensive studies have been performed to identify factors contributing to uremic cardiomyopathy in CKD patients, such as activation of the renin-angiotensin-aldosterone and sympathetic nervous systems and hemodynamic alterations, as well as hypertension (Gross and Ritz, 2008; Vlahakos et al., 1997). Nevertheless, these factors cannot fully explain the high prevalence of LVH in CKD patients. A growing body of evidence indicates that uremic toxins, particularly protein-bound subgroups such as indoxyl sulfate (IS),

K. Yang et al. / Toxicology Letters 234 (2015) 110–119

cannot be effectively removed by daily hemodialysis. Deteriorative renal function is often accompanied by excessive serum IS accumulation, which is hazardous to the cardiovascular system in patients with CKD (Itoh et al., 2012). A previous clinical study showed that a high serum level of IS was associated with cardiovascular mortality in CKD patients (Barreto et al., 2009). It has been demonstrated that increased oxidative stress is involved in the development and progression of LVH in CKD patients (Chade et al., 2005). Recently, the pro-hypertrophy and pro-inflammation effects of IS on cardiomyocytes by enhancing oxidative stress were confirmed in vitro (Lekawanvijit et al., 2010; Niwa, 2011). Additionally, IS can exacerbate cardiomyocytes hypertrophy with enhanced oxidative stress in hypertensive rats (Yisireyili et al., 2013). However, the deeper mechanisms underlying IS-induced cardiomyocytes hypertrophy remain unclear. Uncoupling proteins (UCPs), which are located in the inner mitochondrial membrane, have been considered as regulators of transmembrane proton electrochemical gradients in numerous human tissues. UCP2, a member of the UCP family, is expressed widely in certain tissues. Several lines of evidence have indicated that UCP2 has the abilities to protect the nervous system, modulate insulin secretion in the pancreas, promote the immune response to infection, and provide possible age-defying effects (Arsenijevic et al., 2000; Bechmann et al., 2002; Fleury et al., 1997; Fridell et al., 2005). Moreover, UCP2 can also protect cardiomyocytes against oxidative stress by inhibiting ROS generation (Teshima et al., 2003). Increasing evidence suggests that UCP2 has a beneficial effect on the cardiovascular system (Dromparis et al., 2013; Sun et al., 2013), However, the role of UCP2 in the progression of IS-induced cardiomyocytes hypertrophy remains unknown. In the present study, we investigated the expression changes of UCP2 in IS-induced hypertrophy in cardiomyocytes and further assessed the signaling pathway involved in this process. Our study indicated that IS increased oxidative stress and induced cardiomyocytes hypertrophy by suppressing UCP2 expression, while up-regulation of UCP2 could counteract against IS-induced cardiomyocytes hypertrophy. In addition, we also found that IS-induced UCP2 down-regulation and cardiomyocyte hypertrophy were related to the inhibition of Adenosine 50 -monophosphate (AMP)-activated protein kinase (AMPK) activity.

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saline, PBS) was added into each well and incubated for 5 h at 37  C. The formazan was dissolved by 150 ml dimethylsulfoxide (DMSO) for 10 min. The absorbance was detected at wavelength of 570 nm with background substration at 630 nm using a microplate reader (Microplate Fluorescence Reader FL-600, BioTek USA). In order to validate the data, each assay was performed at least three times. 2.3. Measurement of cytotoxicy To analyze the cytotoxic effect of IS on NRCMs, LDH release was examined using Cell-Mediated Cytotoxicity Assay (LDH measurement Kit Promega, USA). Briefly, NRCMs were isolated and incubated with different concentrations of IS (100, 250, 500, 1000 mM) for 48 h prior to cell lysis and supernatant harvest. After transfer 50 ml of the supernatant to a 96-well enzymatic assay plate, 50 ml of the reconstituted substrate Mix was added to each well. Then, the plate was covered and incubated at room temperature with protecting from light for 30 min. Finally, the stop solution was added and the absorbance was recorded at 490 nm with microplate reader. The experiment was performed three times to validate the results. 2.4. Real-time PCR Real-time PCR was performed to determine the mRNA expression levels of hypertrophy-related markers (e.g., ANF, BNP, and b-MHC). Briefly, total RNA was extracted from 106 cells using TRIzol reagent (Takara Biochemical, Osaka, Japan). cDNA synthesis was performed with approximately 2 mg RNA using a PrimeScriptTM RT Reagent Kit (Takara Biochemical, Osaka, Japan) with a gDNA Eraser (Perfect Real-time) Kit. Real-time amplification was performed using a 7500 Sequence Detection Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with the primers which were listed in Table 1. The mRNA levels were normalized using GAPDH. PCR reactions were performed in a total volume of 20 ml, which contained 2 SYBR1 Premix Ex Taq 10 mmol/L of each primer, 50 ROX Reference Dye and 2.0 ml cDNA template. PCR amplification reactions were performed as follows: 95  C for 30 s, followed by 45 cycles of 95  C for 5 s, 65  C for 30 s, and 72  C for 60 s. The amplification results were obtained using the 2–DDCt method. All experiments were repeated at least three times.

2. Materials and methods 2.5. [3H]-leucine incorporation 2.1. Cell culture Neonatal rat cardiomyocytes (NRCMs) were isolated from 1- to 2-day-old Sprague-Dawley rats (purchased from the Laboratory Animal Center of the Third Military Medical University, Chongqing, China), which were housed under controlled environments where food and water were provided ad libitum with a 12 h light–dark cycle. All animal usage and experimental procedures were approved by the Animal Ethical and Welfare Committee of the Third Military Medical University. NRCMs were cultured in DMEMH medium supplemented with 10% fetal bovine serum (FBS) as previously described (Woodcock et al., 2002). 2.2. Cell proliferation assay The viability of cells was detected using the 3- (4,5-dimethyl-2thiazolyl)- 2,5-diphenyl- 2H-tetrazolium bromide (MTT, Sigma–Aldrich, St. Louis, MO, USA) uptake assay. NRCMs were seeded in 96-well plate and subsequently exposed to serial concentrations of IS (100, 250, 500, 1000 mM, Sigma–Aldrich, USA) for 48 h. In another experiment, NRCMs were incubated with 500 mM IS for 24, 48, 72 h. After treatment with IS, 20 ml of MTT working solution (5 mg MTT/ml dissolved in phosphate buffer

For further evaluate hypertrophy, NRCMs treated with different conditions were seeded onto 96-well plates and exposed to 1 mCi [3H]-leucine (PerkinElmer, Waltham, MA, USA) per well for 24 h. After treatment with 5% trichloroacetic acid (Sigma–Aldrich, USA) for 30 min, the cells were scraped off and centrifuged. Then, a pellet of each sample was dissolved in 0.5 mol/L NaOH overnight at 4  C. The level of [3H]-leucine incorporation was measured with a

Table 1 The primer sequences. Genes

Forward

Reverse

ANF (rat)

50 -ATGGGCTCCTTCTCCATCAC30 50 -TGGGAAGTCCTAGCCAGTCT30 50 -TGACAGATCGGGAGAACCAG30 50 -CTGCACCACCAACTGCTTAG30

50 -TTCATCGGTATGCTCGCTCA30 50 -GATCCGGTCTATCTTCTGCC30 50 -CCGAACTGTCTTGGCATTGC30 50 -TCAGCTCTGGGATGACCTTG30

BNP (rat)

b-MHC (rat) GAPDH (rat)

ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; b-MHC, b-myosin heavy chain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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scintillation counter (Triathler LSC, Hidex, experiments were repeated three times.

Finland).

The

transfected with empty lentiviral vector were designated as the Vehicle group.

2.6. Delivery of UCP2 into NRCMs

2.7. Western blotting

The cDNA for human UCP2 was cloned into a lentiviral vector (designated as LeUCP2) to induce UCP2 overexpression using a lentivirus kit according to the manufacturer’s instructions. Transfection was performed in DMEM-H medium for 2 days after cardiomyocytes isolation. Then, NRCMs were washed with virus-free medium and treated with ponasterone A (Invitrogen China Limited, Beijing, China) to prompt transfection efficiency. Next, cardiomyocytes were incubated with normal medium. Cells

Protein was extracted as previously described (Yisireyili et al., 2013). The concentration of the protein was measured using a spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA, USA). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Amersham, UK). Subsequently, the membranes were blocked with 5% skim milk (Cell Signaling Technology, Danvers,

[(Fig._1)TD$IG]

Fig. 1. IS increases ROS production and induces cardiomyocyte hypertrophy. (A) ROS production was increased in NRCMs after treatment with IS at different concentrations (100, 250, 500, 1000 mM) for 6 h. The generation of intracellular ROS was detected using DCFH-DA. (B) ROS production were observed under laser confocal microscopy. Bar scale: 50 mm. (C) After treatment with 500 mM IS for 48 h, or further incubation with 2 mM IS for 48 h (500 mM ! 2 mM), the mRNA expression of ANF, BNP, and b-MHC were measured in NRCMs by real-time PCR. (D) The cell size was observed by immunofluorescence staining using a-actinin antibody (green). Bar scale: 50 mm. (E) [3H]-leucine incorporation was detected in NRCMs after treatment with 500 mM IS for 48 h, or further incubation with 2 mM IS for 48 h. (F) Quantification of the cell size. The data are presented as the mean  SEM. ***P < 0.001,**P < 0.01,*P < 0.05 vs. control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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MA, USA). After washing with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the membranes were incubated with mouse polyclonal anti-UCP2 (1:500, Abcam, Cambridge, MA, USA), mouse polyclonal anti-GAPDH (1:1000, Abcam, Cambridge, MA, USA) and rabbit monoclonal anti-phospho-AMPKa (Thr172) and antiAMPKa antibodies (1:1000, Cell Signaling Technology) overnight at 4  C. The membranes were washed using TBS-T and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2,000, GE Healthcare, Amersham, UK) and goat anti-mouse IgG (1:2,000, GE Healthcare) antibodies, and the signals were developed with ECL Plus system (GE Healthcare) and detected by ChemiDoc XRS System (Bio-Rad Hercules, CA, USA). The experiments were performed three times to validate the results. 2.8. Loading of cells with fluorescent indicators The generation of intracellular ROS was detected using DCFHDA (Sigma–Aldrich Pty. Ltd., Sydney, Australia) and MitoSOXTM Red mitochondrial superoxide indicator (Molecular Probes, Eugene, OR, USA) according to the manufacturers’ instructions. NRCMs were loaded with 10 mM DCFH-DA and 5 mM MitoSOXTM at 37  C for 30 min, respectively. ROS production was detected at 488 and 525 nm for DCFH and at 510 and 580 nm for MitosoxTM using microplate reader (Microplate Fluorescence Reader FL-600, BioTek USA). For the qualitative observation of myocardial hypertrophy, cells were loaded with a-actinin antibody (1:100, Santa Cruz Biotechnology, Inc.) overnight at 4  C and then incubated with FITC (1:300, ZSGB-BIO, Peking, China) or Cy3- (1:300, beyotime, Peking, China) conjugated goat anti-mouse secondary antibody (1:300, ZSGB-BIO, Peking, China) in the dark for 1 h. The nuclei were stained with DAPI (F. Hoffmann-La Roche Ltd. Basel, Switzerland) for 5 min. Image scanning was captured using a Leica TCSSP5 laser-scanning confocal microscope (Leica Microsystems, Inc., Bannockburn, IL, USA). According to a-actinin-positive staining, Image pro Plus software was used to measure the surface area of cells. The data were doubled to account for the portion of the cell area. The cells were selected randomly from two or three wells and a total of 20–25 cells were detected for each experimental group. 2.9. Statistical analysis The data analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 19.0 software (SPSS, Tokyo, Japan). The data are presented as the mean  SEM. Statistical significance was assessed using t-test between two groups or using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. P < 0.05 was considered statistically significant.

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3. Results 3.1. IS induced excessive ROS production and hypertrophy in NRCMs Compared with the control group, ROS production was significantly increased in NRCMs in a dose and time-dependent manner after IS treatment at concentrations of 100, 250, 500, and 1000 mM (Fig. 1A and B). As shown in Fig. 1C–E, the mRNA expression levels of ANF, BNP and b-MHC were increased obviously in NRCMs after being cultured with 500 mM IS for 48 h, accompanied by the enhancement of [3H]-leucine uptake and the enlargement of cell size, whereas these changes could not be reversed by further incubation with lower concentration of IS (2 mM). These results suggest that IS at certain concentrations is able to induce hypertrophy in NRCMs by enhancing oxidative stress. 3.2. IS inhibited the proliferation and increased the cytotoxicity of NRCMs To further determine the effect of IS on the proliferation and cytotoxicity of NRCMs, we treated the cultured cardiomyocytes with various concentrations (100, 250, 500, and 1000 mM) of IS for 48 h or with fixed concentration (500 mM) of IS for 24, 48, and 72 h before the detection of cell viability and LDH release. It was found that IS could dose-dependently inhibit the proliferation of NRCMs (Fig. 2A). Moreover, the inhibition of NRCMs proliferation induced by 500 mM IS increased with the prolongation of incubation time (Fig. 2B). Meanwhile, treatment with 500 mM or 1000 mM IS for 48 h could remarkably increase the release of LDH in NRCMs (Fig. 2C). These results showed that high concentrations of IS can also exert cytotoxic effect on cardiomyocytes. 3.3. IS inhibited UCP2 expression in NRCMs To investigate the role of UCP2 in IS-induced cardiomyocytes hypertrophy, we detected UCP2 expression in NRCMs by western blotting. As shown in Fig. 3A, a significant decrease in the protein level of UCP2 was observed in NRCMs after treatment with IS at high concentrations (500 and 1000 mM) even for 60 min. With prolongation of the incubation time, 500 mM IS treatment resulted in a gradual reduction in the expression of UCP2 in NRCMs (Fig. 3B). These findings suggest that IS inhibits UCP2 expression in a dose and time-dependent manner. 3.4. UCP2 overexpression inhibited IS-induced ROS production We used a recombinant lentivirus containing the full-length UCP2 cDNA to induce UCP2 overexpression in NRCMs. As shown in

[(Fig._2)TD$IG]

Fig. 2. IS inhibits the proliferation and induced the cytotoxicity of NRCMs. (A) NRCMs were exposed to various concentration of IS (100, 250, 500, 1000 mM) for 48 h, and then the proliferation of cells were detected by MTT assay. (B) After treatment with 500 mM IS for 24, 48, 72 h, the proliferation of NRCMs were detected by MTT assay. (C) NRCMs were exposed to various concentration of IS (100, 250, 500, 1000 mM) for 48 h, and then the LDH release were detected using Cell-Mediated Cytotoxicity Assay (LDH measurement Kit). Data are expressed as mean  SEM. ***P < 0.001, **P < 0.01 vs. control group.

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Fig. 3. IS inhibits UCP2 expression in NRCMs. (A) After treatment with different concentrations of IS (100, 250, 500, and 1000 mM) for 1 h, the UCP2 expression in NRCMs was detected by western blotting. (B) After treatment with 500 mM IS for 0 min, 15 min, 30 min, 60 min, 3 h, 6 h, and 12 h, the UCP2 expression was assessed by western blotting. The data are presented as the mean  SEM (n = 3). ***P < 0.001, **P < 0.01, *P < 0.05 vs. control group.

[(Fig._4)TD$IG]

Fig. 4. Lentivirus-mediated delivery of UCP2 to NRCMs. (A) Cells were incubated with LeUCP2 (20 moi) or Vector for 48 h. Thereafter, NRCMs were observed by light microscopy or fluorescence microscopy, respectively (shown as LM and FM, respectively). Bar scale: 50 mm. (B) UCP2 expression were detected by western blotting of after treatment with LeUCP2 (20 moi) or Vector for 48 h. The protein expression of UCP2 was standardized by GAPDH. The data are presented as the mean  SEM (n = 3). *P < 0.05 vs. control group.

[(Fig._5)TD$IG]

Fig. 5. UCP2 overexpression inhibits IS-induced mitochondrial ROS production. (A and B) NRCMs were infected with LeUCP2 (20 moi) and Vehicle, or pretreatment with NAC (5 mM) before incubation with 500 mM IS, and then the cells were stained with MitosoxTM for 30 min. The mitochondrial ROS production were observed by fluorescence microscopy and the fluorescence intensity was measured using a microplate reader. Red represents mitochondrial ROS production, and green represents the lentivirustransduced cells. Bar scale: 50 mm. The data are presented as the mean  SEM (n = 3). ***P < 0.001 vs. control group; ###P < 0.001, ##P < 0.01 vs. IS group.

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Fig. 4A and B, UCP2 expression increased up to a multiplicity of infection (moi) of 20, as determined by western blotting. Because no significant difference was observed between the virus volumes of 20 and 40 moi (data not shown), 20 moi was used for further experiments. As expected, the level of UCP2 protein expression was significantly increased in LeUCP2-transfected NRCMs, compared with those in the control and lentivirus-transduced cells (Vehicle). To further investigate the effect of UCP2 overexpression on IS-induced oxidative stress, we assessed the mitochondrial ROS production in LeUCP2-transfected NRCMs. After treatment with 500 mM IS for 6 h, the mitochondrial ROS production was remarkably increased in vehicle-treated NRCMs However, similar to the effect of N-acetyl-L-cysteine (NAC, 5 mM), a ROS scavenger, the overexpression of UCP2 significantly suppressed the IS-induced mitochondrial ROS production in NRCMs (Fig. 5A and B). These results indicate that IS increases mitochondrial ROS generation possibly by suppressing UCP2 expression in NRCMs, and up-regulatation of UCP2 can reverse the effect of IS.

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3.5. AMPK inactivation contributes to IS-induced UCP2 downregulation and ROS production in NRCMs Because AMPK activation is considered as a preventive therapeutic target for cardic injury and it may act as an inducer of UCP2 expression (Xie et al., 2008), we further investigated the role of AMPK signaling in IS-induced cardiomyocytes hypertrophy. Western blotting confirmed that the expression of phosphorylated AMPK in NRCMs was inhibited by 500 mM IS treatment in a timedependent manner (Fig. 6A). As shown in Fig. 6B, pretreatment with AICAR (500 mM), an AMPK activator, reversed the downregulation of UCP2 induced by IS in NRCMs. Moreover, preincubation with 500 mM AICAR obviously inhibited IS-induced ROS production in NRCMs, similar to the effects of 5 mM NAC. In contrast, Compound C, an AMPK inhibitor, displayed an ability to reduce the expression of UCP2 and promote ROS production in NRCMs, but there was no significant cooperative effect between Compound C and IS on the down-regulation of UCP2 and ROS

[(Fig._6)TD$IG]

Fig. 6. AMPK activation reverses IS-induced reduction of UCP2 expression and mitochondrial ROS production. (A) After treatment with 500 mM IS for 0 min, 15 min, 30 min, 60 min, 3 h, 6 h, and 12 h, the phosphorylated AMPK and AMPK expression was datected by western blotting. The protein expression of phosphorylated AMPK was standardized by AMPK. (B) The effect of 500 mM AICAR, 10 mM Compound C and 5 mM NAC on IS-induced the decrease of UCP2 expression, as assessed by western blotting. The protein expression of UCP2 was standardized by GAPDH. (C and D) The effect of 500 mM AICAR, 10 mM Compound C and 5 mM NAC on IS-induced mitochondrial ROS production. The mitochondrial ROS production were observed under inverted fluorescence microscope and fluorescence intensity was measured using a microplate reader. Bar scale: 50 mm. The data are presented as the mean  SEM (n = 3). ***P < 0.001 vs. control groups; ##P < 0.01, #P < 0.05 vs. IS group.

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production (Fig. 6B and D). These results suggest that the inhibition of AMPK signaling contributes to IS-induced down-regulation of UCP2 and ROS production in cardiomyocytes. 3.6. AMPK activation and UCP2 overexpression attenuated IS-induced cardiomyocyte hypertrophy To investigate the effects of AMPK activation and UCP2 overexpression on the protection of IS-induced cardiomyocyte hypertrophy, we observed the morphological changes of

cardiomyocytes using a-actinin immunostaining after treatment with AICAR and transduction with LeUCP. As shown in Fig. 7A and B, preincubation with 500 mM AICAR or 20 moi LeUCP2 remarkably inhibited IS-induced increase in cell size, similar to the effect of NAC. In addition, the expression levels of hypertrophy markers (ANF, BNP, and b-MHC) and [3H]-leucine incorporation were also significantly suppressed in the cells treated with AICAR and LeUCP2 (Fig. 7C and D). These results indicate that both UCP2 overexpression and AMPK activation can attenuate the IS-induced cardiomyocyte hypertrophy.

[(Fig._7)TD$IG]

Fig. 7. UCP2 overexpression and AMPK activation inhibit IS-induced cardiomyocyte hypertrophy. (A) The cell size was observed by immunofluorescence staining using aactinin antibody. Red represents the positive staining of a-actinin, and green represents the lentivirus-transduced cells. The cells were observed using a Leica confocal microscope. Bar scale: 50 mm. (B) Quantification of the cell size. (C) NRCMs were incubated with 500 mM IS for 48 h after being infected with LeUCP2 (20 moi), or pretreatment with 500 mM AICAR or 5 mM NAC, and then ANF, BNP, and b-MHC mRNA expressions were measured by real-time PCR. (D) [3H]-leucine incorporation was detected in NRCMs. The data are presented as the mean  SEM (n = 3). *P < 0.05 vs. control group; ***P < 0.001, **P < 0.01 vs. control group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. IS group.

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4. Discussion Uremic cardiomyopathy, which is the most common complication of CKD, results in a higher mortality in CKD patients (Chinnappa et al., 2014; London, 2003). The present study demonstrates that (i) IS induces ROS generation in cardiomyocytes by down-regulation of UCP2 expression, (ii) UCP2 overexpression protects cardiomyocytes against oxidative stress and hypertrophy by decreasing IS-induced ROS production, and (iii) IS-induced down-regulation of UCP2 and cardiomyocytes hypertrophy are associated with AMPK inactivation. Our findings uncovered the potential mechanisms involved in IS-induced cardiomyopathy. Left ventricular hypertrophy (LVH) is a typical pathological change of uremic cardiomyopathy and carries an ominous prognosis (Foley et al., 1995). LVH is frequently seen in patients with end-stage renal disease (Rassic et al., 2004), and the prevalence of LVH is increased even in the early stages of CKD (Shin et al., 2007). Conventional cardiovascular risk factors, such as hypertension and volume overload, have been considered to play prominent roles in the etiopathogenesis of uremic cardiomyopathy (Yamakawa et al., 2000). However, blood pressure correction and volume load reduction can not effectively prevent the development of uremic cardiomyopathy (Siedlecki et al., 2009). Recently, extensive studies have been performed to examine the contributions of non-traditional risk factors, including uremic toxins, particularly protein-binding toxins, such as IS, to the progress of uremic cardiomyopathy in CKD patients (Ito and Yoshida, 2014). IS has been proposed to elevate oxidative stress and cause a decline in renal function (Dou et al., 2007; Lekawanvijit et al., 2012; Shimoishi et al., 2007; Tumur and Niwa, 2009; Yamamoto et al., 2006). Because IS cannot be effectively removed by conventional dialysis, this toxin accumulates in the blood in CKD patients, which may finally result in severe impairment of the cardiovascular system. Previous studies have revealed that IS can increase ROS production, cause cellular toxicity, and impair the functions of glomerular mesangial cells (Gelasco and Raymond, 2006), renal tubular cells (Motojima et al., 2003), vascular smooth muscle cells (Muteliefu et al., 2009; Yamamoto et al., 2006), and human umbilical vein endothelial cells (Dou et al., 2007; Masai et al., 2010; Shimoishi et al., 2007; Tumur et al., 2010; Yang et al., 2012). Furthermore, Suree Lekawanbijit et al. found that IS has pro-hypertrophic, pro-fibrotic and pro-inflammatory effects on cardiomyocytes that are involved in cardiac dysfunction (Lekawanvijit et al., 2010). In this study, we aimed to investigate the pathogenesis involved in the deteriorative effects of IS on cardiomyocytes. Several studies have reported that serum levels of IS in uremic patients increased approximately fifty-fold (Meert et al., 2007; Vanholder et al., 2003), and the highest concentration of serum IS in CKD patients was above 500 mM (Lim et al., 1993). In line with the in vivo findings, the concentration of IS at the range of 100–1000 mM was usually used for in vitro studies (Ichii et al., 2014; Wang et al., 2014). Accordingly, here we also chose this concentration range of IS to perform the experiments. In our study, after incubation of cardiomyocytes with IS, the cell size and [3H]-leucine incorporation increased. Meanwhile, the mRNA expression levels of ANF, BNP, and b-MHC were up-regulated. All these results demonstrate that IS has pro-hypertrophic effect on cardiomyocytes. Besides, we also found that IS could suppress the proliferation and increase cytotoxicity in NRCMs, which is consistent with previous reports that IS has toxic effects on other kinds of cells (Wang et al., 2014; Yisireyili et al., 2014). Based on the results of DCFH-DA staining, we further found that IS treatment increased ROS generation in NRCMs in a dose-dependent manner. Oxidative stress caused by ROS

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over-production is closely associated with certain uremic complications, including uremic cardiomyopathy (Gross and Ritz, 2008). Accumulating evidence has demonstrated that increased ROS production can affect cardiovascular function and contribute to inflammation, atherosclerosis, and cardiovascular remodeling (Miyazaki et al., 2000; Schulman et al., 2005; Vaziri and Rodriguez-Iturbe, 2006). The majority of ROS are generated in mitochondria, and 90% of the oxygen that can be metabolized in humans is produced by the mitochondrial cytochrome oxidase enzyme (Gross and Ritz, 2008). UCPs, which are located in the mitochondrial inner membrane, affect ROS production by decreasing the electrochemical gradient. It has been revealed that UCP2 has protective effects on cardiovascular system by inhibiting ROS generation and suppressing oxidative stress (Teshima et al., 2003; Xu et al., 2011; Dromparis et al., 2013; Sun et al., 2013; Tian et al., 2012; Zhang et al., 2012). It is interesting to definite the possible relationship between UCP2 and IS-induced oxidative stress and cadiomyocytes hypertrophy. In this study we found that IS suppressed UCP2 expression in cardiomyocytes in a dose- and time-dependent manner. Similar to the previous findings that UCP2 overexpression could prevent the apoptosis of cardiomyocytes and neuronal cells by decreasing ROS production (Mattiasson et al., 2003; Vincent et al., 2004), our data showed that lentiviral UCP2 (LeUCP2) transfection could reduce IS-induced ROS generation in cardiomyocytes, suggesting that up-regulation of UCP2 is a compensatory way to counteract IS-induced cardiomyocyte hypertrophy. AMPK is a well-known putative metabolic and energy sensor in certain cells. Currently, the inactivation of AMPK signaling pathway is considered to be involved in cardiovascular dysfunction, especially under the conditions of hemodynamic overload and myocardial ischemia (Nagata and Hirata, 2010). AMPK has been shown to inhibit ROS generation by inhibiting NADPH oxidase activity and to stimulate nitric oxide production by enhancing endothelial nitric oxide synthase activity (Fisslthaler and Fleming, 2009). Furthermore, AMPK affects the cellular redox state by avoiding tyrosine nitration and inhibiting prostacyclin synthase in endothelial cells. This phenomenon has been confirmed by the fact that AICAR, an AMPK activator, can inhibit high glucose-induced nitration and inactivate prostacyclin synthase in HUVECs through the down-regulation of oxidative stress. In addition, the modulation of prostacyclin synthase nitration by AMPK was shown to be mediated by UCP2 up-regulation (Xie et al., 2008). Our present study showed that IS suppressed the expression of phosphorylated AMPK in a time-dependent manner, indicating that AMPK activation was inhibited by IS in NRCMs. We further found that pretreatment of NRCMs with AICAR could effectively reverse the IS-induced reduction in UCP2 expression and attenuate IS-induced ROS production and cells hypertrophy. Taken together, our results demonstrate that IS down-regulates the expression of UCP2 in NRCMs at least in part through the suppression of AMPK activation, which will finally lead to cardiomyocyte hypertrophy. In summary, we first uncover the crucial role of UCP2 in IS-induced cardiomyocyte hypertrophy, and demonstrate that AMPK/UCP2 signaling activation may protect against IS-induced cardiomyopathy by inhibition of oxidative stress. Although the present study is limited to the in vitro experiments, it may provide potential therapeutic targets for uremic cardiomyopathy.

Conflict of interest The authors declare that there are no conflicts of interest.

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