The antioxidant peptide SS31 prevents oxidative stress, downregulates CD36 and improves renal function in diabetic nephropathy

The antioxidant peptide SS31 prevents oxidative stress, downregulates CD36 and improves renal... Abstract Background Oxidative stress plays an independent role in the pathogenesis of diabetic nephropathy (DN). CD36, a class B scavenger receptor, mediates reactive oxygen species (ROS) production in DN. SS31 is a mitochondria-targeted antioxidant peptide that can scavenge mitochondrial ROS. The antioxidative effects of SS31 on DN and the interaction between SS31 and CD36 remain poorly understood. Herein, we examined the effects of SS31 and investigated whether SS31 treatment attenuates CD36 expression in db/db diabetic mice and high glucose (HG)-induced HK-2 cells. Methods Eight-week-old db/m mice and db/db mice were administered with SS31 (3 mg/kg/day) for 12 weeks by intraperitoneal injection. For the in vitro studies, HG-cultured HK-2 cells were used. Biochemical parameters, body weight and histological changes in the mice were measured. The levels of oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Mn superoxide dismutase (MnSOD) and catalase (CAT), and the expression of CD36, nuclear factor-κB (NF-κB) p65 in mice and HK-2 cells were also analyzed. Results The results showed that SS31 alleviated proteinuria, glomerular hypertrophy and tubular injury, and affected creatinine level in db/db mice. SS31 suppressed the levels of oxidative stress, NADPH oxidase subunits, CD36 and NF-κB p65, and activated MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Conclusion Taken together, these data demonstrated a renoprotective role of SS31 in DN by suppression of enhanced oxidative stress and CD36 expression. CD36, diabetic nephropathy, NADPH, ROS, SS31 INTRODUCTION Diabetic nephropathy (DN) is the most severe microvascular complication of diabetes mellitus worldwide, and has become the largest single cause of end-stage renal failure [1]. The specific renal morphology of DN is an increase in kidney size, glomerular and tubular hypertrophy, glomerular basement membrane thickening and mesangial expansion, followed by the accumulation of glomerular extracellular matrix [2]. Alterations of diabetic renal function include excessive urinary albumin excretion, and reduced creatinine clearance or glomerular filtration rate [3]. Many studies have shown that oxidative stress plays an independent role in the progression and severity of DN [4]. Prolonged hyperglycemia, activated transforming growth factor (TGF)-β1 and accumulated advanced glycation end products in the glomerular and tubular epithelial cells of the kidney all cause the production of reactive oxygen species (ROS), which contribute to oxidative stress [3]. ROS can damage renal cells by oxidizing membrane phospholipids, proteins, carbohydrates and nucleic acids. In addition, ROS are also secondary messengers that activate many signaling cascade events, ultimately leading to cell damage and deterioration of kidney functions in the diabetic kidney [5, 6]. Thus, it is believed that protecting renal cells by suppressing oxidative stress is a potential therapeutic strategy for DN. CD36, which belongs to a class B scavenger receptor family, is a glycosylated surface receptor that is present in the plasma membrane and mitochondria of renal tubular cells, macrophages, endothelial cells, skeletal muscle, adipocytes and platelets [7]. CD36 has a role in mediating oxidative stress injury in type 2 diabetes [8]. CD36 deficiency prevents high glucose (HG)-induced ROS production in chronic kidney disease [9]. Furthermore, Susztak et al. [10] reported that increased CD36 protein expression was induced by d-glucose in proximal tubular epithelial cells and mediates apoptosis, which might contribute to the development of DN. Previously, we showed that the CD36 level is increased in HG-induced HK-2 cells and is associated with oxidative stress [11]. In addition, metformin can downregulate the oxidative stress-induced increase in the CD36 level in pancreatic beta cells [12]. These findings suggested that CD36 might be a therapeutic target against oxidative stress in DN. SS31 is a cell-permeable, mitochondrion-targeted antioxidant peptide. Several studies have revealed that SS31 can partition readily to the mitochondrial inner membrane, and can protect mitochondria against ROS production, mitochondrial permeability transition, swelling and cytochrome c release, in a wide variety of cell types [13–18]. Our recent study demonstrated that SS31 could alleviate renal morphological and functional alterations, inhibit renal cell apoptosis and alleviate the alteration of mitochondrial potential and ATP in uninephrectomy, streptozotocin (STZ)-induced diabetic mice and HG-induced mesangial cells [18]. Previous studies have demonstrated that the ROS scavenging activity of SS31 is mediated by its dimethyltyrosine residue [19]. Furthermore, SS31 could attenuate ischemic injury by downregulating CD36 [14]. In the hypoxia/reoxygenation-stressed human renal tubular cell line NRK52E, the protective role of SS31 was p66Shc-dependent [20]. However, the mechanisms underlying the renoprotective effects of SS31 remain unclear. In the present study, we investigated the therapeutic potential of SS31 against oxidative stress and examined whether SS31-induced renoprotection is CD36-dependent in db/db mice and HG-induced HK-2 cells. MATERIALS AND METHODS Experimental animals Thirty-two male 8-week-old C57BLKS/J db/db diabetic and db/m normal male mice were purchased from the Model Animal Research Center of Nanjing University and housed in a temperature-controlled room in the animal center of the Shanxi Medical University. SS31 was provided by ChinaPeptides (Shanghai, China). Half of the db/db and db/m mice were injected with saline intraperitoneally and used as controls; the other half was injected with 3 mg/kg/day SS31 intraperitoneally for 12 weeks. The dosage (3 mg/kg/day) was based on related studies showing the efficacy of SS31 without adverse effect [16, 18]. All mice were given free access to food and water, and sacrificed at 20 weeks of age. Serum samples, 24-h urine samples and kidney tissues were collected from each mouse for further study. All experimental protocols were conducted according to the Ethics Review Committee for Animal Experimentation of Shanxi Medical University. Cell culture HK-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in an atmosphere of 5% CO2. d-glucose and mannitol were purchased from Sigma (St Louis, MO, USA). After fasting for 24 h, the HK-2 cells were stimulated with 5.6 mmol/L glucose (normal glucose, NG), NG plus 24.4 mM mannitol (M), 30 mmol/L glucose (high glucose, HG) and HG plus 100 nM SS31 (SS31) for 48 h. Metabolic data Urine volume, body weight, blood pressure, blood glucose and albumin concentrations were measured at 20 weeks of age. Urine was collected over a 24-h period, with each mouse placed individually in a metabolic cage. Urinary albumin, urinary creatinine, serum creatinine, serum total cholesterol and serum triglycerides were measured using reagent kits (BioSino Bio-technology and Science Inc., Beijing, China), according to the respective manufacturer's instructions. The 24-h urinary albumin excretion rate (UAER) = urinary albumin (μg/mL) × 24-h urine volume (mL). Renal pathology Renal tissues were fixed with 4% paraformaldehyde overnight at 4°C, dehydrated and embedded in paraffin. Sections (2-μm-thick) were prepared for periodic acid-Schiff (PAS) and Masson trichrome staining. Thirty glomeruli and approximately 80 ± 100 proximal tubules in each mouse (eight mice in each group) were measured for mesangial matrix fraction and tubular area using the image processing and analysis system, ImageJ. We analyzed the degree of glomerular and tubular injury semiquantitatively. The mean glomerular tuft volume (GV) was determined from the mean glomerular cross-sectional tuft area (GA), as described previously [18]. GV was calculated as GV = β/k × (GA)3/2, with β = 1.38, the shape coefficient for spheres, and k = 1.1, a size distribution coefficient. The fraction of the mesangial matrix was expressed as the ratio of the PAS-positive material in the mesangium to the glomerular tuft area. The glomerular and tubulointerstitial injury index was conducted by a pathologist in a blinded fashion, as described previously [21, 22]. The glomerular injury index was graded from 0 to 4 on the basis of the degree of glomerulosclerosis and mesangial matrix expansion: grade 0 represented normal glomeruli; grade 1 represented a mesangial matrix expansion area up to 25%; grade 2 represented mesangial matrix expansion of >25–50%; grade 3 represented mesangial matrix expansion of >50–75%; and grade 4 represented >75% mesangial matrix expansion. The percentage of damaged tubules (interstitial inflammation and fibrosis, tubular dilation and cast formation) was graded from 0 to 3 as follows: 0, normal; 1, tubular lesion <25%; 2, 25–50% lesion and 3, lesion >50%. Immunohistochemistry The kidney sections (4 µm) were dewaxed and rehydrated in graded ethanol. After 15 min of antigen retrieval by microwave treatment in 10 mM citrate (pH 6.0) buffer, nonspecific binding was blocked with phosphate-buffered saline containing 10% goat serum at room temperature for 30 min. The sections were stained overnight with antibodies against collagen IV (1:200), TGF-β1 (1:150), fibronectin (1:200) and CD36 (1:150), respectively, at 4°C. The sections were further incubated with biotinylated secondary antibody for 1 h. Labeling was visualized with 3,3-diaminobenzidine to produce a brown color. Measurement of urine malondialdehyde and 8-hydroxydeoxyguanosine levels by an enzyme-linked immunosorbent assay Urine measurement of urine malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from the samples using an mRNA extraction kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction. cDNA was prepared using PrimeScript RT Master Mix Kit (Takara Bio Inc., Shiga, Japan). Real-time PCR was performed with a SYBR Premix ExTaq™ (Takara Bio Inc.) in the Agilent Mx3000P QPCR Systems (Agilent, Palo Alto, CA, USA). The sequences of the primers used were as follows: mouse Mnsod, forward: 5′-CAGGATGCCGCTCCGTTAT-3′ and reverse: 5′-TGAGGTTTACACGACCGCTG-3′; mouse Cat, forward: 5′-CAGCGACCAGATGAAGCAGTG-3′ and reverse: 5′-GTACCACTCTCTCAGGAATCCG-3′; mouse Nox4, forward: 5′-GCATCTGCATCTGTCCTG AA-3′ and reverse: 5′-TGGAACTTGGGTTCT TCCAG-3′; mouse P22, forward: 5′-TGG ACGTTTCACACAGTGGT-3′ and reverse: 5′-TAGGCTCAATGGGAGTCCAC-3; mouse Cd36, forward: 5′-CTCCTAGTAGGCGTGGGTCT-3′ and reverse: 5′-CACGGGGTCTCAACCATTCA-3′; human Mn superoxide dismutase (MnSOD), forward: 5′-GTGTGGGAGCACGCTTACTA-3′ and reverse: 5′-AGAGCTTAACATACTCAGCATAACG-3′; human catalase (CAT), forward: 5′-GATAGCCTTCGACCCAAGCAAC-3′ and reverse: 5′-TGATTGTCCTGCATGCACATCG-3′; human NOX4, forward: 5′-AGGATCACAGAAGGTTCCAAGC-3′ and reverse: 5′-TCCTCATCTCGGTATCTTGCTG-3′; human P22, forward: 5′-GTGTTTGTGTGCCTGCTGGAGT-3′ and reverse: 5′-CTGGGCGGCTGCTTGATGGT-3′; human CD36, forward: 5′-GCAACAAACCACACACTGGG-3′ and reverse: 5′-AGTCCTACACTGCAGTCCTCA-3′; 18 S, forward: 5′-ACACGGACAGGATTG ACAGA-3′ and reverse: 5′-GGACATCTAAGGGCATCACAG-3′. For all real-time PCR analysis, 18 S mRNA was used to normalize RNA inputs. Western blotting analysis The kidney and HK-2 cells were lysed in lysis buffer and centrifuged at 14 000 g for 20 min at 4°C and the supernatant protein was collected. The nuclear and cytosol protein of kidney tissues and HK-2 cells was extracted using a nuclear protein extraction kit (Invitrogen, Carlsbad, CA, USA). The concentration of the samples was determined using a BCA protein assay kit. Protein (50 μg) was separated through a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a semidry transfer blotting apparatus. The membranes were incubated with different appropriate primary antibodies for 18 h at 4°C. The primary antibodies were as follow: anti-TGF-β1 (1:1000 dilution, Abcam, Cambridge, UK), anti-MnSOD (1:2000 dilution, Proteintech, Chicago, IL, USA), anti-CAT (1:1000 dilution, Proteintech), anti-NOX4 (1:1500 dilution, Abcam), anti-p22 (1:1500 dilution, Cell Signaling Technology, Beverly, MA, USA), anti-CD36 (1:1000 dilution, Abcam), anti-NF-κB (1:1000 dilution, Abcam), anti-Histone H3 (1:1000 dilution, Signalway, College Park, MD, USA) and anti-β-actin (1:2000 dilution, Cell Signaling Technology). After washing with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody: anti-rabbit (or mouse) IgG (GE Healthcare, Piscataway, NJ, USA) at a 1:10 000 dilution for 2 h at room temperature. Band densities on each membrane were measured using LabWorks 4.5 software (UVP, Upland, CA, USA). Mitochondrial ROS detection The mitochondrial formation of ROS in HK-2 cells was measured by flow cytometry (BD Immunocytometry Systems, Franklin Lakes, NJ, USA) using the mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, HK-2 cells were incubated with 5-µM MitoSOX reagent working solution for 10 min at 37°C in the dark. After washing with warm buffer three times, the cells were resuspended in warm buffer for flow cytometry analysis (excitation/emission, 510/580 nm). Immunofluorescence Cells were plated on cover slips, fixed with 4% formaldehyde for 20 min at 4°C, and blocked with 10% BSA for 30 min. To punch holes in the cytomembrane, the cells were incubated in 0.1% Triton X-100 for 20 min at room temperature. The cells were then incubated with the primary antibody (anti-CD36 1:250 dilution, anti-NF-κB 1:250 dilution) overnight at 4°C. The next day, after incubation with FITC-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), the slides were incubated with 4, 6-diamidino-2-phenylindole (DAPI, Sigma) to label the nuclei and were then analyzed by fluorescence microscopy. Oil Red O staining Oil Red O staining was performed according to the instructions of the manufacturer (Sigma). Cells were fixed in 4% paraformaldehyde for 15 min and then stained for 10 min in1% Oil Red O. After washing with 70% alcohol for 15 s, the cells were counter-stained with Harris hematoxylin for 5 min. The stained samples were imaged using an Olympus microscope (Olympus Corporation, Tokyo, Japan) Statistical analysis Data are expressed as the mean ± standard deviation (SD). The differences among groups were analyzed for statistical significance using one-way analysis of variance (ANOVA), followed by post hoc testing using the Tukey–Kramer method. All experiments were performed at least three times. A P-value of <0.05 was considered significant. RESULTS Effect of SS31 on the biochemical characteristics in db/db mice The ratios of kidney weight to body weight were elevated significantly in db/db mice compared with the db/m mice, and this elevation was inhibited by SS31 treatment (Figure 1A). Biochemical parameters such as 24-h UAER (Figure 1B) and serum creatinine (Figure 1C) were increased significantly in db/db mice compared with those in the db/m group. This increase was rescued by administration of SS31. However, there were no significant differences in the levels of plasma glucose (Figure 1D), blood pressure (Figure 1E), total cholesterol (Figure 1F) and triglycerides (Figure 1G) among the four groups. FIGURE 1 View largeDownload slide Effect of SS31 on clinical parameters, histological changes, urinary MDA levels and 8-OHdG excretion in experimental animals. (A) Kidney/body weight ratio (mg/g). (B) Twenty-four-hour UAER (μg/24 h). (C) Serum creatinine (mg/dL). (D) Blood glucose (mmol/L). (E) Blood pressure (mmHg). (F) Total cholesterol (mM). (G) Triglyceride (mM). (H) The levels of urinary MDA were detected by ELISA (n = 8). (I) The levels of urinary 8-OHdG were detected by ELISA (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 1 View largeDownload slide Effect of SS31 on clinical parameters, histological changes, urinary MDA levels and 8-OHdG excretion in experimental animals. (A) Kidney/body weight ratio (mg/g). (B) Twenty-four-hour UAER (μg/24 h). (C) Serum creatinine (mg/dL). (D) Blood glucose (mmol/L). (E) Blood pressure (mmHg). (F) Total cholesterol (mM). (G) Triglyceride (mM). (H) The levels of urinary MDA were detected by ELISA (n = 8). (I) The levels of urinary 8-OHdG were detected by ELISA (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effects of SS31 on glomerular hypertrophy and tubular injury PAS staining and Masson staining were used to assess glomerular hypertrophy and tubular injury in each group. At 12 weeks, the db/db mice displayed a larger glomerular volume (Figure 2B), greater mesangial matrix fraction (Figure 2D) and increased glomerular injury index (Figure 2C) compared with the db/m mice. SS31 treatment reversed these changes (Figure 2). In the db/db mice, the collagen IV and fibronectin content in the renal cortex also increased visibly, as indicated by immunohistochemistry (Figure 2A). SS31 treatment was found to reduce the level of collagen IV and fibronectin (Figure 2A). FIGURE 2 View largeDownload slide Effect of SS31 on glomerular hypertrophy and injury. (A) Representative photomicrographs of PAS staining, Masson staining and immunohistochemistry images of the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of the glomerular volume at 20 weeks (n = 8). (C) Semiquantitative analyses of the glomerular injury index at 20 weeks (n = 8). (D) Semiquantitative analyses of the mesangial matrix fraction at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 2 View largeDownload slide Effect of SS31 on glomerular hypertrophy and injury. (A) Representative photomicrographs of PAS staining, Masson staining and immunohistochemistry images of the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of the glomerular volume at 20 weeks (n = 8). (C) Semiquantitative analyses of the glomerular injury index at 20 weeks (n = 8). (D) Semiquantitative analyses of the mesangial matrix fraction at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. In the db/db mice, the proximal tubular area became larger than that in the db/m mice (Figure 3A and B). Tubulointerstitial damage was increased compared with the db/m control, and SS31 attenuated the tubular damage in the db/db kidney (Figure 3A and C). The expression of TGF-β1 in the db/db mice was also reduced after SS31 treatment (Figure 3D). FIGURE 3 View largeDownload slide Effect of SS31 on tubular injury. (A) Representative photomicrographs of PAS staining, Masson staining in the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of proximal tubular area according to the outer diameter at 20 weeks (n = 8). (C) Semiquantitative analyses of the tubulointerstitial damage index at 20 weeks (n = 8). (D) Representative western blotting images of TGF-β1 at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 3 View largeDownload slide Effect of SS31 on tubular injury. (A) Representative photomicrographs of PAS staining, Masson staining in the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of proximal tubular area according to the outer diameter at 20 weeks (n = 8). (C) Semiquantitative analyses of the tubulointerstitial damage index at 20 weeks (n = 8). (D) Representative western blotting images of TGF-β1 at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on urinary MDA levels and 8-OHdG excretion in db/db mice The results showed a significant increase in MDA levels for the db/db group compared with the control db/m group. Treatment with SS31 induced a significant decrease in the MDA level compared with that in the db/db group (Figure 1H). In the db/db group, the 8-OHdG level increased, but was reduced by SS31 (Figure 1I). Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in db/db mice As shown in Figure 4, in the db/db group, renal MnSOD and CAT levels were significantly reduced compared with those in the db/m group; however, the SS31 group showed significant recovery of antioxidant enzyme levels (Figure 4A–C, F and G). Renal NADPH oxidase subunits p22 and Nox4 levels were increased in the db/db group compared with the db/m group. SS31 administration inhibited the increased expressions of NADPH oxidase subunits p22 and Nox4 (Figure 4A, D, E, H and I). FIGURE 4 View largeDownload slide Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in experimental animals. (A–E) The levels of MnSOD, CAT, NOX4 and P22 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (F–I) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 4 View largeDownload slide Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in experimental animals. (A–E) The levels of MnSOD, CAT, NOX4 and P22 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (F–I) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on CD36 mRNA and protein expression in db/db mice As shown in Figure 5, the mRNA and protein levels of CD36 in the kidney increased significantly in the db/db mice compared with the control db/m mice. However, SS31 treatment reduced the diabetes-induced increase in CD36 mRNA and protein levels markedly in the db/db mice compared with the normal levels (Figure 5B and C). In addition, immunohistochemical analysis indicated that CD36 mainly existed in proximal tubule and was significantly higher in the db/db mice than in the controls; SS31 reduced the level of CD36 in the db/db mice (Figure 5A). FIGURE 5 View largeDownload slide Effect of SS31 on CD36 and NF-κB p56 expression in experimental animals. (A) Representative immunohistochemistry images of CD36 expression in the renal tissues of mice (magnification 400×). (B) The levels of CD36 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (C) The mRNA expression levels of Cd36 were analyzed by real-time RT-PCR (n = 8). (D, E) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 5 View largeDownload slide Effect of SS31 on CD36 and NF-κB p56 expression in experimental animals. (A) Representative immunohistochemistry images of CD36 expression in the renal tissues of mice (magnification 400×). (B) The levels of CD36 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (C) The mRNA expression levels of Cd36 were analyzed by real-time RT-PCR (n = 8). (D, E) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on NF-κB p56 subunit protein expression in db/db mice Enhanced nuclear and decreased cytoplasmic levels of NF-κB p65 protein were observed in db/db mice compared with the control db/m mice. The level of NF-κB p65 nuclear translocation was attenuated significantly compared with db/db mice after treatment with SS31 for 12 weeks (Figure 5D and E). Effect of SS31 on ROS production in HG-induced HK-2 cells As shown in Figure 6, the fluorescence intensity in HK-2 cells was enhanced significantly after stimulation with HG for 48 h compared with the control group. However, this increase in ROS was reduced markedly in cells co-incubated with SS31 (Figure 6A). FIGURE 6 View largeDownload slide Effect of SS31 on ROS production, Mn SOD/CAT inactivation and NADPH oxidase activity in HK-2 cells. (A) Mitochondrial ROS was detected by flow cytometry using MitoSOX Red staining (n = 6). (B–F) The levels of MnSOD, CAT, NOX4 and p22 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (G–J) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 6). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 6 View largeDownload slide Effect of SS31 on ROS production, Mn SOD/CAT inactivation and NADPH oxidase activity in HK-2 cells. (A) Mitochondrial ROS was detected by flow cytometry using MitoSOX Red staining (n = 6). (B–F) The levels of MnSOD, CAT, NOX4 and p22 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (G–J) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 6). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in HG-induced HK-2 cells In HG-induced HK-2 cells, significantly decreased levels of MnSOD and CAT were observed compared with those in the control group; however, treatment with SS31 caused an increase in their levels (Figure 6B–D, G and H). In addition, the mRNA levels of NADPH oxidase subunits p22 and Nox4 increased in HG-induced HK-2 cells. SS31 treatment showed a beneficial effect of reducing NADPH oxidase activity in HG-induced HK-2 cells (Figure 6A, E, F, I and J). Effect of SS31 on lipid accumulation and CD36 mRNA and protein expression in HG-induced HK-2 cells Oil Red O staining revealed that lipid droplets accumulation was significantly enhanced in HK-2 cells after treatment with HG for 48 h. In contrast, treatment with SS31 markedly ameliorated the HG-induced lipid levels in HK-2 cells (Figure 7E). FIGURE 7 View largeDownload slide Effect of SS31 on CD36 mRNA and protein levels in HG-induced HK-2 cells. (A, B) The levels of CD36 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (C) The mRNA expression levels of CD36 were analyzed by real-time RT-PCR (n = 6). (D) Representative images of CD36 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. (E) Lipid droplets were detected by Oil Red O staining (magnification 400×). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 7 View largeDownload slide Effect of SS31 on CD36 mRNA and protein levels in HG-induced HK-2 cells. (A, B) The levels of CD36 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (C) The mRNA expression levels of CD36 were analyzed by real-time RT-PCR (n = 6). (D) Representative images of CD36 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. (E) Lipid droplets were detected by Oil Red O staining (magnification 400×). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. The CD36 mRNA and protein expression were at a low levels in the control group, and their levels were increased dramatically in HK-2 cells after stimulation with HG for 48 h. Treatment with SS31 attenuated CD36 mRNA (Figure 7C) and protein (Figure 7A and B) levels significantly after HG stimulation. The microscopic observation of immunofluorescence labeling was consistent with the protein and mRNA results. The number of CD36-positive cells increased significantly in HG-induced HK-2 cells. Treatment with SS31 decreased the number of CD36-positive cells significantly (Figure 7D). Effect of SS31 on NF-κB p56 subunit protein expression in HG-induced HK-2 cells As shown in Figure 8, we were unable to detect any difference between the NG and M groups under normal culture conditions. Compared with the control groups, the nuclear levels of NF-κB p65 were increased significantly and cytoplasmic levels were decreased in HG-induced HK-2 cells. SS31 treatment resulted in a significant reduction in NF-κB p65 nuclear translocation (Figure 8A and B). Immunofluorescence labeling showed that NF-κB p56 was localized predominately in the cytoplasm in the NG and M groups. In HG-induced HK-2 cells, nuclear staining increased. Treatment with SS31 significantly reversed the activation of NF-κB p56 (Figure 8C). FIGURE 8 View largeDownload slide Effect of SS31 on NF-κB p56 subunit protein levels in HG-induced HK-2 cells. (A, B) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 6). (C) Representative images of NF-κB p56 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) and HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 8 View largeDownload slide Effect of SS31 on NF-κB p56 subunit protein levels in HG-induced HK-2 cells. (A, B) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 6). (C) Representative images of NF-κB p56 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) and HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. DISCUSSION In the present study, we demonstrated that SS31 inhibits the renal functional and pathological changes in db/db mice. In addition, SS31 prevented increased oxidative stress, NADPH oxidase activity, overexpression of CD36, NF-κB (P65) and upregulated MnSOD/CAT inactivation in db/db mice and HG-induced HK-2 cells. Elevated levels of plasma creatinine have been identified as waste products of metabolism following increased renal structural injury [23]. Albuminuria is also a hallmark of DN, and has a close link with kidney degeneration [24]. Increasing concentrations of these metabolites during DN are representative of deteriorating kidney function [25]. In the present study, we observed increases in creatinine and albuminuria in the db/db mice, indicating impaired metabolic control, which is consistent with that reported previously. By contrast, a significant decrease in these parameters was observed in the db/db mice of the SS31 treatment group, but no significant improvement was observed in the control group. Our study also showed that SS31 administration protected against renal pathological damage in db/db mice, reflecting the protective effects of SS31 on renal lesions. These results indicated that SS31 improves kidney function in DN. However, SS31 did not improve plasma glucose, blood pressure and blood lipids in db/db mice, indicating that the renoprotection may be independent of plasma glucose, blood pressure and blood lipids. SS31 is an innovative cell-permeable mitochondrion-targeted antioxidant peptide. Studies have suggested that in various diseases, SS31 has protective effects, including neuroprotective [13, 26], cardioprotective [27] and transplanted pancreatic islet cell-protective properties [15], renoprotective effects [17, 18, 28], and protects against HG-induced injury in human retinal endothelial cells [16]. Our previous study in uninephrectomy, STZ-induced diabetic mice and HG-induced mesangial cells showed that SS31 could protect against renal injury, which was linked to decreased renal cell apoptosis and alteration of mitochondrial potential and ATP [18]. In this study, we found that SS31 could attenuate renal levels of 8-OHdG, a marker for generalized oxidative DNA damage [29], and MDA, an end product of lipid peroxidation associated with ROS [30], which suggested that mechanism underlying the renoprotective effects of SS31 involves inhibiting oxidative stress. The chief source of oxidative stress in DN is the overproduction of ROS. ROS are generated by cellular respiration and the arachidonic acid cycle; however, in experimental models of DN, ROS production is dependent mainly on NADPH oxidase activation [31]. Among the seven isoforms (Nox1–5, Duox1 and Duox2) of NADPH oxidase, Nox4 is localized mainly in renal mitochondria and is a key player in glucose-mediated ROS production. Moreover, correlative studies support the view that p22phox acts as activator of Nox4 [32] and was able to stimulate ROS production. Similar to previous observations, we found that NOX4 and p22phox mRNA and protein expression were increased remarkably in both db/db mice and in HG-induced HK-2 cells, and these increases were ameliorated by SS31 treatment. These observations further reinforce the conclusion that SS31 reduces oxidative damage in the diabetic kidney by inhibiting NADPH oxidase-mediated ROS production. In addition to NADPH oxidase, evidence revealed that increased ROS generation under diabetic conditions is also caused by alterations in endogenous antioxidant systems, including enzymes such as SOD and CAT. In the diabetic kidney, overexpression of MnSOD or CAT is associated with protection against mitochondrial oxidative damage [33]. In the present study, both db/db mice and HG-induced HK-2 cells showed decreased expressions of MnSOD and CAT, compared with those in the normal group. Treatment with SS31 could upregulate the expressions of MnSOD and CAT effectively in the kidney of db/db mice and HG-induced HK-2 cells. These results indicated clearly that maintaining the balance between the production of ROS and the antioxidant system is one of the mechanisms by which SS31 exerts its renoprotective effect. CD36, a transmembrane glycoprotein, was reported recently to mediate the production of ROS in chronic kidney disease [9]. Recent evidence demonstrated that CD36 expression was increased markedly in diabetic kidneys and is involved in the mechanisms of apoptosis [10]. Our previous investigation showed that inhibition of CD36 overexpression could attenuate HG-induced ROS generation [11]. These findings suggested that reducing CD36 expression and function using antioxidant agents might be an approach to protect renal tubular cells from oxidative stress in DN. Indeed, Cho et al. demonstrated that SS31 could downregulate CD36, attenuate ROS production, and reverse the degree of ischemia in the ischemic area [14]. In the current study, we found that increased CD36 expression in db/db mice and HG-cultured HK-2 cells was decreased drastically by SS31 treatment, providing evidence that SS31 acts via inhibition of CD36 expression. CD36 is also known as a fatty acid translocase, and is responsible for lipid deposition in several tissues [34]. Previous studies have demonstrated that HG upregulates CD36 expression in renal cells [10, 11]. Increased expression of CD36 increases the cellular uptake of free fatty acids and aggravates HG-induced lipid accumulation in diabetic kidneys [35]. The present study indicated that SS31 attenuates CD36 expression in HG-cultured HK-2 cells. We further investigated the role of SS31 in HK-2 cells lipid accumulation induced by HG. Using Oil Red O staining, we confirmed that SS31 improves HG-induced lipid deposition. The renal protective effects of SS31 are partly caused by inhibiting CD36-triggered lipid accumulation. Activation of the NF-κB signaling pathway is related to increased inflammation in DN [36]. Furthermore, NF-κB is a redox-sensitive transcription factor, whose activation can attributed to overproduction of ROS [37]. Thus, it is conceivable that scavenging ROS using SS31 could suppress the increase in NF-κB transcription in DN. SS31 treatment inhibited ROS-induced NF-κB activation in several animal models [23, 24, 38]. In the present, we confirmed that SS31 inhibited NF-κB signaling activation significantly in DN. CONCLUSION In conclusion, the results presented here suggested that treatment with SS31 significantly alleviated renal hypertrophy, UAER and creatinine in db/db mice. SS31 also inhibited oxidative stress, NADPH oxidase activation, expression of CD36 and NF-κB p65, and promoted the activity of MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Therefore, SS31 might have a potential therapeutic relevance in DN, possibly by inhibiting oxidative stress and downregulating CD36 expression. FUNDING This work was supported by the Scientific Research Foundation for Doctors, the Second Hospital of Shanxi Medical University [grant number 201601-5]. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Thomas MC , Cooper ME , Zimmet P. Changing epidemiology of type 2 diabetes mellitus and associated chronic kidney disease . Nat Rev Nephrol 2016 ; 12 : 73 – 81 Google Scholar Crossref Search ADS PubMed 2 Zelmanovitz T , Gerchman F , Balthazar AP et al. Diabetic nephropathy . Diabetol Metab Syndr 2009 ; 1 : 10 Google Scholar Crossref Search ADS PubMed 3 Gnudi L , Coward RJ , Long DA. Diabetic nephropathy: perspective on novel molecular mechanisms . Trends Endocrinol Metab 2016 ; 27 : 820 – 830 Google Scholar Crossref Search ADS PubMed 4 Pedraza-Chaverri J , Sanchez-Lozada LG , Osorio-Alonso H et al. New pathogenic concepts and therapeutic approaches to oxidative stress in chronic kidney disease . Oxid Med Cell Longev 2016 ; 2016 : 6043601 Google Scholar Crossref Search ADS PubMed 5 Bondeva T , Wolf G. Reactive oxygen species in diabetic nephropathy: friend or foe? Nephrol Dial Transplant 2014 ; 29 : 1998 – 2003 Google Scholar Crossref Search ADS PubMed 6 Tavafi M. Diabetic nephropathy and antioxidants . J Nephropathol 2013 ; 2 : 20 – 27 Google Scholar Crossref Search ADS PubMed 7 Silverstein RL , Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior . Sci Signal 2009 ; 26 : 3 8 Liani R , Halvorsen B , Sestili S et al. Plasma levels of soluble CD36, platelet activation, inflammation, and oxidative stress are increased in type 2 diabetic patients . Free Radic Biol Med 2012 ; 52 : 1318 – 1324 Google Scholar Crossref Search ADS PubMed 9 Okamura DM , Pennathur S , Pasichnyk K et al. CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD . J Am Soc Nephrol 2009 ; 20 : 495 – 505 Google Scholar Crossref Search ADS PubMed 10 Susztak K , Ciccone E , McCue P et al. Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy . PLoS Med 2005 ; 2 : 45 Google Scholar Crossref Search ADS 11 Hou YJ , Wu M , Wei JY et al. CD36 is involved in high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells . Biochem Biophys Res Commun 2015 ; 468 : 281 – 286 Google Scholar Crossref Search ADS PubMed 12 Moon JS , Karunakaran U , Elumalai S et al. Metformin prevents glucotoxicity by alleviating oxidative and ER stress-induced CD36 expression in pancreatic beta cells . J Diabet Compl 2017 ; 31 : 21 – 30 Google Scholar Crossref Search ADS 13 Reddy PH , Manczak M , Kandimalla R. Mitochondria-targeted small molecule SS31, a potential candidate for the treatment of Alzheimer's disease . Hum Mol Genet 2017 ; 26 : 1483 – 1496 Google Scholar Crossref Search ADS PubMed 14 Cho S , Szeto HH , Kim E et al. A novel cell-permeable antioxidant peptide, SS31, attenuates ischemic brain injury by down-regulating CD36 . J Biol Chem 2007 ; 282 : 4634 – 4642 Google Scholar Crossref Search ADS PubMed 15 Thomas DA , Stauffer C , Zhao K et al. Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization, reduces islet cell apoptosis, increases islet cell yield, and improves posttransplantation function . J Am Soc Nephrol 2007 ; 18 : 213 – 222 Google Scholar Crossref Search ADS PubMed 16 Huang J , Li X , Li M et al. Mitochondria-targeted antioxidant peptide SS31 protects the retinas of diabetic rats . Curr Mol Med 2013 ; 13 : 935 – 945 Google Scholar Crossref Search ADS PubMed 17 Szeto HH , Liu S , Soong Y et al. Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury . J Am Soc Nephrol 2011 ; 22 : 1041 – 1052 Google Scholar Crossref Search ADS PubMed 18 Hou YJ , Li SC , Wu M et al. Mitochondria-targeted peptide SS-31 attenuates renal injury via an antioxidant effect in diabetic nephropathy . Am J Physiol Renal Physiol 2016 ; 310 : 547 – 559 Google Scholar Crossref Search ADS 19 Zhao K , Zhao GM , Wu D et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury . J Biol Chem 2004 ; 279 : 34682 – 34690 Google Scholar Crossref Search ADS PubMed 20 Zhao WY , Han S , Zhang L et al. Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells . Cell Physiol Biochem 2013 ; 32 : 591 – 600 Google Scholar Crossref Search ADS PubMed 21 Maric C , Sandberg K , Hinojosa-Laborde C. Glomerulosclerosis and tubulointerstitial fibrosis are attenuated with 17beta-estradiol in the aging Dahl salt sensitive rat . J Am Soc Nephrol 2004 ; 15 : 1546 – 1556 Google Scholar Crossref Search ADS PubMed 22 Tervaert TWC , Mooyaart AL , Amann K et al. Pathologic classification of diabetic nephropathy . J Am Soc Nephrol 2010 ; 21 : 556 – 563 Google Scholar Crossref Search ADS PubMed 23 Steubl D , Block M , Herbst V et al. Plasma uromodulin correlates with kidney function and identifies early stages in chronic kidney disease patients . Medicine (Baltimore) 2016 ; 95 : e3011 Google Scholar Crossref Search ADS PubMed 24 Lemley KV. An introduction to biomarkers: applications to chronic kidney disease . Pediatr Nephrol 2007 ; 22 : 1849 – 1859 Google Scholar Crossref Search ADS PubMed 25 Currie G , McKay G , Delles C. Biomarkers in diabetic nephropathy: present and future . World J Diabetes 2014 ; 5 : 763 – 776 Google Scholar Crossref Search ADS PubMed 26 Yang L , Zhao K , Calingasan NY et al. Mitochondria targeted peptides protect against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine neurotoxicity . Antioxid Redox Signal 2009 ; 11 : 2095 – 2104 Google Scholar Crossref Search ADS PubMed 27 Cho J , Won K , Wu D et al. Potent mitochondria-targeted peptides reduce myocardial infarction in rats . Coron Artery Dis 2007 ; 18 : 215 – 220 Google Scholar Crossref Search ADS PubMed 28 Mizuguchi Y , Chen J , Seshan SV et al. A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction . Am J Physiol Renal Physiol 2008 ; 295 : 1545 – 1553 Google Scholar Crossref Search ADS 29 Shigenaga MK , Gimeno CJ , Ames BN. Urinary 8-hydroxy-2-deoxyguanosine as a biological marker of in vivo oxidative DNA damage . Proc Natl Acad Sci USA 1989 ; 86 : 9697 – 9701 Google Scholar Crossref Search ADS PubMed 30 Rajasekaran S , Ravi K , Sivagnanam K et al. Beneficial effects of aloe vera leaf gel extract on lipid profile status in rats with streptozotocin diabetes . Clin Exp Pharmacol Physiol 2006 ; 33 : 232 – 237 Google Scholar Crossref Search ADS PubMed 31 Sedeek M , Nasrallah R , Touyz RM et al. NADPH oxidases, reactive oxygen species, and the kidney: friend and foe . J Am Soc Nephrol 2013 ; 24 : 1512 – 1518 Google Scholar Crossref Search ADS PubMed 32 Kawahara T , Quinn MT , Lambeth JD. Molecular evolution of the reactive oxygengenerating NADPH oxidase (Nox/Duox) family of enzymes . BMC Evol Biol 2007 ; 7 : 109 Google Scholar Crossref Search ADS PubMed 33 Flekac M , Skrha J , Hilgertova J et al. Gene polymorphisms of superoxide dismutases and catalase in diabetes mellitus . BMC Med Genet 2008 ; 9 : 30 Google Scholar Crossref Search ADS PubMed 34 Nath A , Li I , Roberts L et al. Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma . Sci Rep 2015 ; 5 : 14752 Google Scholar Crossref Search ADS PubMed 35 Feng L , Gu C , Li Y et al. High glucose promotes CD36 expression by upregulating peroxisome proliferator-activated receptor γ levels to exacerbate lipid deposition in renal tubular cells . Biomed Res Int 2017 ; 2017 : 1414070 Google Scholar PubMed 36 Sun L , Li W , Li W et al. Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH xidase/ROS/Akt/NF-κB pathway under high glucose conditions . Int J Mol Methods 2014 ; 34 : 167 – 176 Google Scholar Crossref Search ADS 37 Hayden MS , Ghosh S. Signaling to NF-kappaB . Genes Dev 2004 ; 18 : 2195 – 2224 Google Scholar Crossref Search ADS PubMed 38 Wu J , Li H , Sun X et al. A mitochondrion-targeted tioxidant ameliorates isoflurane-induced cognitive deficits in aging mice . PLoS One 2015 ; 10 : 0138256 © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nephrology Dialysis Transplantation Oxford University Press

The antioxidant peptide SS31 prevents oxidative stress, downregulates CD36 and improves renal function in diabetic nephropathy

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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0931-0509
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1460-2385
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10.1093/ndt/gfy021
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Abstract

Abstract Background Oxidative stress plays an independent role in the pathogenesis of diabetic nephropathy (DN). CD36, a class B scavenger receptor, mediates reactive oxygen species (ROS) production in DN. SS31 is a mitochondria-targeted antioxidant peptide that can scavenge mitochondrial ROS. The antioxidative effects of SS31 on DN and the interaction between SS31 and CD36 remain poorly understood. Herein, we examined the effects of SS31 and investigated whether SS31 treatment attenuates CD36 expression in db/db diabetic mice and high glucose (HG)-induced HK-2 cells. Methods Eight-week-old db/m mice and db/db mice were administered with SS31 (3 mg/kg/day) for 12 weeks by intraperitoneal injection. For the in vitro studies, HG-cultured HK-2 cells were used. Biochemical parameters, body weight and histological changes in the mice were measured. The levels of oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Mn superoxide dismutase (MnSOD) and catalase (CAT), and the expression of CD36, nuclear factor-κB (NF-κB) p65 in mice and HK-2 cells were also analyzed. Results The results showed that SS31 alleviated proteinuria, glomerular hypertrophy and tubular injury, and affected creatinine level in db/db mice. SS31 suppressed the levels of oxidative stress, NADPH oxidase subunits, CD36 and NF-κB p65, and activated MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Conclusion Taken together, these data demonstrated a renoprotective role of SS31 in DN by suppression of enhanced oxidative stress and CD36 expression. CD36, diabetic nephropathy, NADPH, ROS, SS31 INTRODUCTION Diabetic nephropathy (DN) is the most severe microvascular complication of diabetes mellitus worldwide, and has become the largest single cause of end-stage renal failure [1]. The specific renal morphology of DN is an increase in kidney size, glomerular and tubular hypertrophy, glomerular basement membrane thickening and mesangial expansion, followed by the accumulation of glomerular extracellular matrix [2]. Alterations of diabetic renal function include excessive urinary albumin excretion, and reduced creatinine clearance or glomerular filtration rate [3]. Many studies have shown that oxidative stress plays an independent role in the progression and severity of DN [4]. Prolonged hyperglycemia, activated transforming growth factor (TGF)-β1 and accumulated advanced glycation end products in the glomerular and tubular epithelial cells of the kidney all cause the production of reactive oxygen species (ROS), which contribute to oxidative stress [3]. ROS can damage renal cells by oxidizing membrane phospholipids, proteins, carbohydrates and nucleic acids. In addition, ROS are also secondary messengers that activate many signaling cascade events, ultimately leading to cell damage and deterioration of kidney functions in the diabetic kidney [5, 6]. Thus, it is believed that protecting renal cells by suppressing oxidative stress is a potential therapeutic strategy for DN. CD36, which belongs to a class B scavenger receptor family, is a glycosylated surface receptor that is present in the plasma membrane and mitochondria of renal tubular cells, macrophages, endothelial cells, skeletal muscle, adipocytes and platelets [7]. CD36 has a role in mediating oxidative stress injury in type 2 diabetes [8]. CD36 deficiency prevents high glucose (HG)-induced ROS production in chronic kidney disease [9]. Furthermore, Susztak et al. [10] reported that increased CD36 protein expression was induced by d-glucose in proximal tubular epithelial cells and mediates apoptosis, which might contribute to the development of DN. Previously, we showed that the CD36 level is increased in HG-induced HK-2 cells and is associated with oxidative stress [11]. In addition, metformin can downregulate the oxidative stress-induced increase in the CD36 level in pancreatic beta cells [12]. These findings suggested that CD36 might be a therapeutic target against oxidative stress in DN. SS31 is a cell-permeable, mitochondrion-targeted antioxidant peptide. Several studies have revealed that SS31 can partition readily to the mitochondrial inner membrane, and can protect mitochondria against ROS production, mitochondrial permeability transition, swelling and cytochrome c release, in a wide variety of cell types [13–18]. Our recent study demonstrated that SS31 could alleviate renal morphological and functional alterations, inhibit renal cell apoptosis and alleviate the alteration of mitochondrial potential and ATP in uninephrectomy, streptozotocin (STZ)-induced diabetic mice and HG-induced mesangial cells [18]. Previous studies have demonstrated that the ROS scavenging activity of SS31 is mediated by its dimethyltyrosine residue [19]. Furthermore, SS31 could attenuate ischemic injury by downregulating CD36 [14]. In the hypoxia/reoxygenation-stressed human renal tubular cell line NRK52E, the protective role of SS31 was p66Shc-dependent [20]. However, the mechanisms underlying the renoprotective effects of SS31 remain unclear. In the present study, we investigated the therapeutic potential of SS31 against oxidative stress and examined whether SS31-induced renoprotection is CD36-dependent in db/db mice and HG-induced HK-2 cells. MATERIALS AND METHODS Experimental animals Thirty-two male 8-week-old C57BLKS/J db/db diabetic and db/m normal male mice were purchased from the Model Animal Research Center of Nanjing University and housed in a temperature-controlled room in the animal center of the Shanxi Medical University. SS31 was provided by ChinaPeptides (Shanghai, China). Half of the db/db and db/m mice were injected with saline intraperitoneally and used as controls; the other half was injected with 3 mg/kg/day SS31 intraperitoneally for 12 weeks. The dosage (3 mg/kg/day) was based on related studies showing the efficacy of SS31 without adverse effect [16, 18]. All mice were given free access to food and water, and sacrificed at 20 weeks of age. Serum samples, 24-h urine samples and kidney tissues were collected from each mouse for further study. All experimental protocols were conducted according to the Ethics Review Committee for Animal Experimentation of Shanxi Medical University. Cell culture HK-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in an atmosphere of 5% CO2. d-glucose and mannitol were purchased from Sigma (St Louis, MO, USA). After fasting for 24 h, the HK-2 cells were stimulated with 5.6 mmol/L glucose (normal glucose, NG), NG plus 24.4 mM mannitol (M), 30 mmol/L glucose (high glucose, HG) and HG plus 100 nM SS31 (SS31) for 48 h. Metabolic data Urine volume, body weight, blood pressure, blood glucose and albumin concentrations were measured at 20 weeks of age. Urine was collected over a 24-h period, with each mouse placed individually in a metabolic cage. Urinary albumin, urinary creatinine, serum creatinine, serum total cholesterol and serum triglycerides were measured using reagent kits (BioSino Bio-technology and Science Inc., Beijing, China), according to the respective manufacturer's instructions. The 24-h urinary albumin excretion rate (UAER) = urinary albumin (μg/mL) × 24-h urine volume (mL). Renal pathology Renal tissues were fixed with 4% paraformaldehyde overnight at 4°C, dehydrated and embedded in paraffin. Sections (2-μm-thick) were prepared for periodic acid-Schiff (PAS) and Masson trichrome staining. Thirty glomeruli and approximately 80 ± 100 proximal tubules in each mouse (eight mice in each group) were measured for mesangial matrix fraction and tubular area using the image processing and analysis system, ImageJ. We analyzed the degree of glomerular and tubular injury semiquantitatively. The mean glomerular tuft volume (GV) was determined from the mean glomerular cross-sectional tuft area (GA), as described previously [18]. GV was calculated as GV = β/k × (GA)3/2, with β = 1.38, the shape coefficient for spheres, and k = 1.1, a size distribution coefficient. The fraction of the mesangial matrix was expressed as the ratio of the PAS-positive material in the mesangium to the glomerular tuft area. The glomerular and tubulointerstitial injury index was conducted by a pathologist in a blinded fashion, as described previously [21, 22]. The glomerular injury index was graded from 0 to 4 on the basis of the degree of glomerulosclerosis and mesangial matrix expansion: grade 0 represented normal glomeruli; grade 1 represented a mesangial matrix expansion area up to 25%; grade 2 represented mesangial matrix expansion of >25–50%; grade 3 represented mesangial matrix expansion of >50–75%; and grade 4 represented >75% mesangial matrix expansion. The percentage of damaged tubules (interstitial inflammation and fibrosis, tubular dilation and cast formation) was graded from 0 to 3 as follows: 0, normal; 1, tubular lesion <25%; 2, 25–50% lesion and 3, lesion >50%. Immunohistochemistry The kidney sections (4 µm) were dewaxed and rehydrated in graded ethanol. After 15 min of antigen retrieval by microwave treatment in 10 mM citrate (pH 6.0) buffer, nonspecific binding was blocked with phosphate-buffered saline containing 10% goat serum at room temperature for 30 min. The sections were stained overnight with antibodies against collagen IV (1:200), TGF-β1 (1:150), fibronectin (1:200) and CD36 (1:150), respectively, at 4°C. The sections were further incubated with biotinylated secondary antibody for 1 h. Labeling was visualized with 3,3-diaminobenzidine to produce a brown color. Measurement of urine malondialdehyde and 8-hydroxydeoxyguanosine levels by an enzyme-linked immunosorbent assay Urine measurement of urine malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from the samples using an mRNA extraction kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction. cDNA was prepared using PrimeScript RT Master Mix Kit (Takara Bio Inc., Shiga, Japan). Real-time PCR was performed with a SYBR Premix ExTaq™ (Takara Bio Inc.) in the Agilent Mx3000P QPCR Systems (Agilent, Palo Alto, CA, USA). The sequences of the primers used were as follows: mouse Mnsod, forward: 5′-CAGGATGCCGCTCCGTTAT-3′ and reverse: 5′-TGAGGTTTACACGACCGCTG-3′; mouse Cat, forward: 5′-CAGCGACCAGATGAAGCAGTG-3′ and reverse: 5′-GTACCACTCTCTCAGGAATCCG-3′; mouse Nox4, forward: 5′-GCATCTGCATCTGTCCTG AA-3′ and reverse: 5′-TGGAACTTGGGTTCT TCCAG-3′; mouse P22, forward: 5′-TGG ACGTTTCACACAGTGGT-3′ and reverse: 5′-TAGGCTCAATGGGAGTCCAC-3; mouse Cd36, forward: 5′-CTCCTAGTAGGCGTGGGTCT-3′ and reverse: 5′-CACGGGGTCTCAACCATTCA-3′; human Mn superoxide dismutase (MnSOD), forward: 5′-GTGTGGGAGCACGCTTACTA-3′ and reverse: 5′-AGAGCTTAACATACTCAGCATAACG-3′; human catalase (CAT), forward: 5′-GATAGCCTTCGACCCAAGCAAC-3′ and reverse: 5′-TGATTGTCCTGCATGCACATCG-3′; human NOX4, forward: 5′-AGGATCACAGAAGGTTCCAAGC-3′ and reverse: 5′-TCCTCATCTCGGTATCTTGCTG-3′; human P22, forward: 5′-GTGTTTGTGTGCCTGCTGGAGT-3′ and reverse: 5′-CTGGGCGGCTGCTTGATGGT-3′; human CD36, forward: 5′-GCAACAAACCACACACTGGG-3′ and reverse: 5′-AGTCCTACACTGCAGTCCTCA-3′; 18 S, forward: 5′-ACACGGACAGGATTG ACAGA-3′ and reverse: 5′-GGACATCTAAGGGCATCACAG-3′. For all real-time PCR analysis, 18 S mRNA was used to normalize RNA inputs. Western blotting analysis The kidney and HK-2 cells were lysed in lysis buffer and centrifuged at 14 000 g for 20 min at 4°C and the supernatant protein was collected. The nuclear and cytosol protein of kidney tissues and HK-2 cells was extracted using a nuclear protein extraction kit (Invitrogen, Carlsbad, CA, USA). The concentration of the samples was determined using a BCA protein assay kit. Protein (50 μg) was separated through a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a semidry transfer blotting apparatus. The membranes were incubated with different appropriate primary antibodies for 18 h at 4°C. The primary antibodies were as follow: anti-TGF-β1 (1:1000 dilution, Abcam, Cambridge, UK), anti-MnSOD (1:2000 dilution, Proteintech, Chicago, IL, USA), anti-CAT (1:1000 dilution, Proteintech), anti-NOX4 (1:1500 dilution, Abcam), anti-p22 (1:1500 dilution, Cell Signaling Technology, Beverly, MA, USA), anti-CD36 (1:1000 dilution, Abcam), anti-NF-κB (1:1000 dilution, Abcam), anti-Histone H3 (1:1000 dilution, Signalway, College Park, MD, USA) and anti-β-actin (1:2000 dilution, Cell Signaling Technology). After washing with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody: anti-rabbit (or mouse) IgG (GE Healthcare, Piscataway, NJ, USA) at a 1:10 000 dilution for 2 h at room temperature. Band densities on each membrane were measured using LabWorks 4.5 software (UVP, Upland, CA, USA). Mitochondrial ROS detection The mitochondrial formation of ROS in HK-2 cells was measured by flow cytometry (BD Immunocytometry Systems, Franklin Lakes, NJ, USA) using the mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, HK-2 cells were incubated with 5-µM MitoSOX reagent working solution for 10 min at 37°C in the dark. After washing with warm buffer three times, the cells were resuspended in warm buffer for flow cytometry analysis (excitation/emission, 510/580 nm). Immunofluorescence Cells were plated on cover slips, fixed with 4% formaldehyde for 20 min at 4°C, and blocked with 10% BSA for 30 min. To punch holes in the cytomembrane, the cells were incubated in 0.1% Triton X-100 for 20 min at room temperature. The cells were then incubated with the primary antibody (anti-CD36 1:250 dilution, anti-NF-κB 1:250 dilution) overnight at 4°C. The next day, after incubation with FITC-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), the slides were incubated with 4, 6-diamidino-2-phenylindole (DAPI, Sigma) to label the nuclei and were then analyzed by fluorescence microscopy. Oil Red O staining Oil Red O staining was performed according to the instructions of the manufacturer (Sigma). Cells were fixed in 4% paraformaldehyde for 15 min and then stained for 10 min in1% Oil Red O. After washing with 70% alcohol for 15 s, the cells were counter-stained with Harris hematoxylin for 5 min. The stained samples were imaged using an Olympus microscope (Olympus Corporation, Tokyo, Japan) Statistical analysis Data are expressed as the mean ± standard deviation (SD). The differences among groups were analyzed for statistical significance using one-way analysis of variance (ANOVA), followed by post hoc testing using the Tukey–Kramer method. All experiments were performed at least three times. A P-value of <0.05 was considered significant. RESULTS Effect of SS31 on the biochemical characteristics in db/db mice The ratios of kidney weight to body weight were elevated significantly in db/db mice compared with the db/m mice, and this elevation was inhibited by SS31 treatment (Figure 1A). Biochemical parameters such as 24-h UAER (Figure 1B) and serum creatinine (Figure 1C) were increased significantly in db/db mice compared with those in the db/m group. This increase was rescued by administration of SS31. However, there were no significant differences in the levels of plasma glucose (Figure 1D), blood pressure (Figure 1E), total cholesterol (Figure 1F) and triglycerides (Figure 1G) among the four groups. FIGURE 1 View largeDownload slide Effect of SS31 on clinical parameters, histological changes, urinary MDA levels and 8-OHdG excretion in experimental animals. (A) Kidney/body weight ratio (mg/g). (B) Twenty-four-hour UAER (μg/24 h). (C) Serum creatinine (mg/dL). (D) Blood glucose (mmol/L). (E) Blood pressure (mmHg). (F) Total cholesterol (mM). (G) Triglyceride (mM). (H) The levels of urinary MDA were detected by ELISA (n = 8). (I) The levels of urinary 8-OHdG were detected by ELISA (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 1 View largeDownload slide Effect of SS31 on clinical parameters, histological changes, urinary MDA levels and 8-OHdG excretion in experimental animals. (A) Kidney/body weight ratio (mg/g). (B) Twenty-four-hour UAER (μg/24 h). (C) Serum creatinine (mg/dL). (D) Blood glucose (mmol/L). (E) Blood pressure (mmHg). (F) Total cholesterol (mM). (G) Triglyceride (mM). (H) The levels of urinary MDA were detected by ELISA (n = 8). (I) The levels of urinary 8-OHdG were detected by ELISA (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effects of SS31 on glomerular hypertrophy and tubular injury PAS staining and Masson staining were used to assess glomerular hypertrophy and tubular injury in each group. At 12 weeks, the db/db mice displayed a larger glomerular volume (Figure 2B), greater mesangial matrix fraction (Figure 2D) and increased glomerular injury index (Figure 2C) compared with the db/m mice. SS31 treatment reversed these changes (Figure 2). In the db/db mice, the collagen IV and fibronectin content in the renal cortex also increased visibly, as indicated by immunohistochemistry (Figure 2A). SS31 treatment was found to reduce the level of collagen IV and fibronectin (Figure 2A). FIGURE 2 View largeDownload slide Effect of SS31 on glomerular hypertrophy and injury. (A) Representative photomicrographs of PAS staining, Masson staining and immunohistochemistry images of the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of the glomerular volume at 20 weeks (n = 8). (C) Semiquantitative analyses of the glomerular injury index at 20 weeks (n = 8). (D) Semiquantitative analyses of the mesangial matrix fraction at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 2 View largeDownload slide Effect of SS31 on glomerular hypertrophy and injury. (A) Representative photomicrographs of PAS staining, Masson staining and immunohistochemistry images of the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of the glomerular volume at 20 weeks (n = 8). (C) Semiquantitative analyses of the glomerular injury index at 20 weeks (n = 8). (D) Semiquantitative analyses of the mesangial matrix fraction at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. In the db/db mice, the proximal tubular area became larger than that in the db/m mice (Figure 3A and B). Tubulointerstitial damage was increased compared with the db/m control, and SS31 attenuated the tubular damage in the db/db kidney (Figure 3A and C). The expression of TGF-β1 in the db/db mice was also reduced after SS31 treatment (Figure 3D). FIGURE 3 View largeDownload slide Effect of SS31 on tubular injury. (A) Representative photomicrographs of PAS staining, Masson staining in the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of proximal tubular area according to the outer diameter at 20 weeks (n = 8). (C) Semiquantitative analyses of the tubulointerstitial damage index at 20 weeks (n = 8). (D) Representative western blotting images of TGF-β1 at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 3 View largeDownload slide Effect of SS31 on tubular injury. (A) Representative photomicrographs of PAS staining, Masson staining in the db/m, db/m+ SS31, db/db and db/db+ SS31 groups at 20 weeks (magnification 400×). (B) Semiquantitative analyses of proximal tubular area according to the outer diameter at 20 weeks (n = 8). (C) Semiquantitative analyses of the tubulointerstitial damage index at 20 weeks (n = 8). (D) Representative western blotting images of TGF-β1 at 20 weeks (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on urinary MDA levels and 8-OHdG excretion in db/db mice The results showed a significant increase in MDA levels for the db/db group compared with the control db/m group. Treatment with SS31 induced a significant decrease in the MDA level compared with that in the db/db group (Figure 1H). In the db/db group, the 8-OHdG level increased, but was reduced by SS31 (Figure 1I). Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in db/db mice As shown in Figure 4, in the db/db group, renal MnSOD and CAT levels were significantly reduced compared with those in the db/m group; however, the SS31 group showed significant recovery of antioxidant enzyme levels (Figure 4A–C, F and G). Renal NADPH oxidase subunits p22 and Nox4 levels were increased in the db/db group compared with the db/m group. SS31 administration inhibited the increased expressions of NADPH oxidase subunits p22 and Nox4 (Figure 4A, D, E, H and I). FIGURE 4 View largeDownload slide Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in experimental animals. (A–E) The levels of MnSOD, CAT, NOX4 and P22 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (F–I) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 4 View largeDownload slide Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in experimental animals. (A–E) The levels of MnSOD, CAT, NOX4 and P22 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (F–I) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on CD36 mRNA and protein expression in db/db mice As shown in Figure 5, the mRNA and protein levels of CD36 in the kidney increased significantly in the db/db mice compared with the control db/m mice. However, SS31 treatment reduced the diabetes-induced increase in CD36 mRNA and protein levels markedly in the db/db mice compared with the normal levels (Figure 5B and C). In addition, immunohistochemical analysis indicated that CD36 mainly existed in proximal tubule and was significantly higher in the db/db mice than in the controls; SS31 reduced the level of CD36 in the db/db mice (Figure 5A). FIGURE 5 View largeDownload slide Effect of SS31 on CD36 and NF-κB p56 expression in experimental animals. (A) Representative immunohistochemistry images of CD36 expression in the renal tissues of mice (magnification 400×). (B) The levels of CD36 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (C) The mRNA expression levels of Cd36 were analyzed by real-time RT-PCR (n = 8). (D, E) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. FIGURE 5 View largeDownload slide Effect of SS31 on CD36 and NF-κB p56 expression in experimental animals. (A) Representative immunohistochemistry images of CD36 expression in the renal tissues of mice (magnification 400×). (B) The levels of CD36 were analyzed by western blotting and normalized to the level of β-actin (n = 8). (C) The mRNA expression levels of Cd36 were analyzed by real-time RT-PCR (n = 8). (D, E) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 8). Data are expressed as means ± SD. **P < 0.01 versus the db/m group; #P < 0.05, compared with the db/db group by ANOVA. Effect of SS31 on NF-κB p56 subunit protein expression in db/db mice Enhanced nuclear and decreased cytoplasmic levels of NF-κB p65 protein were observed in db/db mice compared with the control db/m mice. The level of NF-κB p65 nuclear translocation was attenuated significantly compared with db/db mice after treatment with SS31 for 12 weeks (Figure 5D and E). Effect of SS31 on ROS production in HG-induced HK-2 cells As shown in Figure 6, the fluorescence intensity in HK-2 cells was enhanced significantly after stimulation with HG for 48 h compared with the control group. However, this increase in ROS was reduced markedly in cells co-incubated with SS31 (Figure 6A). FIGURE 6 View largeDownload slide Effect of SS31 on ROS production, Mn SOD/CAT inactivation and NADPH oxidase activity in HK-2 cells. (A) Mitochondrial ROS was detected by flow cytometry using MitoSOX Red staining (n = 6). (B–F) The levels of MnSOD, CAT, NOX4 and p22 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (G–J) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 6). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 6 View largeDownload slide Effect of SS31 on ROS production, Mn SOD/CAT inactivation and NADPH oxidase activity in HK-2 cells. (A) Mitochondrial ROS was detected by flow cytometry using MitoSOX Red staining (n = 6). (B–F) The levels of MnSOD, CAT, NOX4 and p22 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (G–J) The mRNA expression levels of Mnsod, Cat, Nox4 and P22 were analyzed by real-time RT-PCR (n = 6). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. Effect of SS31 on MnSOD/CAT inactivation and NADPH oxidase activity in HG-induced HK-2 cells In HG-induced HK-2 cells, significantly decreased levels of MnSOD and CAT were observed compared with those in the control group; however, treatment with SS31 caused an increase in their levels (Figure 6B–D, G and H). In addition, the mRNA levels of NADPH oxidase subunits p22 and Nox4 increased in HG-induced HK-2 cells. SS31 treatment showed a beneficial effect of reducing NADPH oxidase activity in HG-induced HK-2 cells (Figure 6A, E, F, I and J). Effect of SS31 on lipid accumulation and CD36 mRNA and protein expression in HG-induced HK-2 cells Oil Red O staining revealed that lipid droplets accumulation was significantly enhanced in HK-2 cells after treatment with HG for 48 h. In contrast, treatment with SS31 markedly ameliorated the HG-induced lipid levels in HK-2 cells (Figure 7E). FIGURE 7 View largeDownload slide Effect of SS31 on CD36 mRNA and protein levels in HG-induced HK-2 cells. (A, B) The levels of CD36 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (C) The mRNA expression levels of CD36 were analyzed by real-time RT-PCR (n = 6). (D) Representative images of CD36 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. (E) Lipid droplets were detected by Oil Red O staining (magnification 400×). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 7 View largeDownload slide Effect of SS31 on CD36 mRNA and protein levels in HG-induced HK-2 cells. (A, B) The levels of CD36 were analyzed by western blotting and normalized to the levels of β-actin (n = 6). (C) The mRNA expression levels of CD36 were analyzed by real-time RT-PCR (n = 6). (D) Representative images of CD36 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. (E) Lipid droplets were detected by Oil Red O staining (magnification 400×). HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) or HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. The CD36 mRNA and protein expression were at a low levels in the control group, and their levels were increased dramatically in HK-2 cells after stimulation with HG for 48 h. Treatment with SS31 attenuated CD36 mRNA (Figure 7C) and protein (Figure 7A and B) levels significantly after HG stimulation. The microscopic observation of immunofluorescence labeling was consistent with the protein and mRNA results. The number of CD36-positive cells increased significantly in HG-induced HK-2 cells. Treatment with SS31 decreased the number of CD36-positive cells significantly (Figure 7D). Effect of SS31 on NF-κB p56 subunit protein expression in HG-induced HK-2 cells As shown in Figure 8, we were unable to detect any difference between the NG and M groups under normal culture conditions. Compared with the control groups, the nuclear levels of NF-κB p65 were increased significantly and cytoplasmic levels were decreased in HG-induced HK-2 cells. SS31 treatment resulted in a significant reduction in NF-κB p65 nuclear translocation (Figure 8A and B). Immunofluorescence labeling showed that NF-κB p56 was localized predominately in the cytoplasm in the NG and M groups. In HG-induced HK-2 cells, nuclear staining increased. Treatment with SS31 significantly reversed the activation of NF-κB p56 (Figure 8C). FIGURE 8 View largeDownload slide Effect of SS31 on NF-κB p56 subunit protein levels in HG-induced HK-2 cells. (A, B) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 6). (C) Representative images of NF-κB p56 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) and HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. FIGURE 8 View largeDownload slide Effect of SS31 on NF-κB p56 subunit protein levels in HG-induced HK-2 cells. (A, B) The levels of nuclear and cytosolic NF-κB p56 were analyzed by western blotting and normalized to the levels of Histone H3 and β-actin, respectively (n = 6). (C) Representative images of NF-κB p56 immunofluorescence labeling in HK-2 cells (n = 6) (magnification 400×). Positive staining is green. HK-2 cells were treated with 5.6 mM glucose (NG), NG + 24.4 mM mannitol (M), 30 mM glucose (HG) and HG + 100 nM SS31 (HG + SS31) for 48 h. Data are expressed as means ± SD. **P < 0.01 versus the NG group; #P < 0.05, compared with the HG group by ANOVA. DISCUSSION In the present study, we demonstrated that SS31 inhibits the renal functional and pathological changes in db/db mice. In addition, SS31 prevented increased oxidative stress, NADPH oxidase activity, overexpression of CD36, NF-κB (P65) and upregulated MnSOD/CAT inactivation in db/db mice and HG-induced HK-2 cells. Elevated levels of plasma creatinine have been identified as waste products of metabolism following increased renal structural injury [23]. Albuminuria is also a hallmark of DN, and has a close link with kidney degeneration [24]. Increasing concentrations of these metabolites during DN are representative of deteriorating kidney function [25]. In the present study, we observed increases in creatinine and albuminuria in the db/db mice, indicating impaired metabolic control, which is consistent with that reported previously. By contrast, a significant decrease in these parameters was observed in the db/db mice of the SS31 treatment group, but no significant improvement was observed in the control group. Our study also showed that SS31 administration protected against renal pathological damage in db/db mice, reflecting the protective effects of SS31 on renal lesions. These results indicated that SS31 improves kidney function in DN. However, SS31 did not improve plasma glucose, blood pressure and blood lipids in db/db mice, indicating that the renoprotection may be independent of plasma glucose, blood pressure and blood lipids. SS31 is an innovative cell-permeable mitochondrion-targeted antioxidant peptide. Studies have suggested that in various diseases, SS31 has protective effects, including neuroprotective [13, 26], cardioprotective [27] and transplanted pancreatic islet cell-protective properties [15], renoprotective effects [17, 18, 28], and protects against HG-induced injury in human retinal endothelial cells [16]. Our previous study in uninephrectomy, STZ-induced diabetic mice and HG-induced mesangial cells showed that SS31 could protect against renal injury, which was linked to decreased renal cell apoptosis and alteration of mitochondrial potential and ATP [18]. In this study, we found that SS31 could attenuate renal levels of 8-OHdG, a marker for generalized oxidative DNA damage [29], and MDA, an end product of lipid peroxidation associated with ROS [30], which suggested that mechanism underlying the renoprotective effects of SS31 involves inhibiting oxidative stress. The chief source of oxidative stress in DN is the overproduction of ROS. ROS are generated by cellular respiration and the arachidonic acid cycle; however, in experimental models of DN, ROS production is dependent mainly on NADPH oxidase activation [31]. Among the seven isoforms (Nox1–5, Duox1 and Duox2) of NADPH oxidase, Nox4 is localized mainly in renal mitochondria and is a key player in glucose-mediated ROS production. Moreover, correlative studies support the view that p22phox acts as activator of Nox4 [32] and was able to stimulate ROS production. Similar to previous observations, we found that NOX4 and p22phox mRNA and protein expression were increased remarkably in both db/db mice and in HG-induced HK-2 cells, and these increases were ameliorated by SS31 treatment. These observations further reinforce the conclusion that SS31 reduces oxidative damage in the diabetic kidney by inhibiting NADPH oxidase-mediated ROS production. In addition to NADPH oxidase, evidence revealed that increased ROS generation under diabetic conditions is also caused by alterations in endogenous antioxidant systems, including enzymes such as SOD and CAT. In the diabetic kidney, overexpression of MnSOD or CAT is associated with protection against mitochondrial oxidative damage [33]. In the present study, both db/db mice and HG-induced HK-2 cells showed decreased expressions of MnSOD and CAT, compared with those in the normal group. Treatment with SS31 could upregulate the expressions of MnSOD and CAT effectively in the kidney of db/db mice and HG-induced HK-2 cells. These results indicated clearly that maintaining the balance between the production of ROS and the antioxidant system is one of the mechanisms by which SS31 exerts its renoprotective effect. CD36, a transmembrane glycoprotein, was reported recently to mediate the production of ROS in chronic kidney disease [9]. Recent evidence demonstrated that CD36 expression was increased markedly in diabetic kidneys and is involved in the mechanisms of apoptosis [10]. Our previous investigation showed that inhibition of CD36 overexpression could attenuate HG-induced ROS generation [11]. These findings suggested that reducing CD36 expression and function using antioxidant agents might be an approach to protect renal tubular cells from oxidative stress in DN. Indeed, Cho et al. demonstrated that SS31 could downregulate CD36, attenuate ROS production, and reverse the degree of ischemia in the ischemic area [14]. In the current study, we found that increased CD36 expression in db/db mice and HG-cultured HK-2 cells was decreased drastically by SS31 treatment, providing evidence that SS31 acts via inhibition of CD36 expression. CD36 is also known as a fatty acid translocase, and is responsible for lipid deposition in several tissues [34]. Previous studies have demonstrated that HG upregulates CD36 expression in renal cells [10, 11]. Increased expression of CD36 increases the cellular uptake of free fatty acids and aggravates HG-induced lipid accumulation in diabetic kidneys [35]. The present study indicated that SS31 attenuates CD36 expression in HG-cultured HK-2 cells. We further investigated the role of SS31 in HK-2 cells lipid accumulation induced by HG. Using Oil Red O staining, we confirmed that SS31 improves HG-induced lipid deposition. The renal protective effects of SS31 are partly caused by inhibiting CD36-triggered lipid accumulation. Activation of the NF-κB signaling pathway is related to increased inflammation in DN [36]. Furthermore, NF-κB is a redox-sensitive transcription factor, whose activation can attributed to overproduction of ROS [37]. Thus, it is conceivable that scavenging ROS using SS31 could suppress the increase in NF-κB transcription in DN. SS31 treatment inhibited ROS-induced NF-κB activation in several animal models [23, 24, 38]. In the present, we confirmed that SS31 inhibited NF-κB signaling activation significantly in DN. CONCLUSION In conclusion, the results presented here suggested that treatment with SS31 significantly alleviated renal hypertrophy, UAER and creatinine in db/db mice. SS31 also inhibited oxidative stress, NADPH oxidase activation, expression of CD36 and NF-κB p65, and promoted the activity of MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Therefore, SS31 might have a potential therapeutic relevance in DN, possibly by inhibiting oxidative stress and downregulating CD36 expression. FUNDING This work was supported by the Scientific Research Foundation for Doctors, the Second Hospital of Shanxi Medical University [grant number 201601-5]. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Thomas MC , Cooper ME , Zimmet P. Changing epidemiology of type 2 diabetes mellitus and associated chronic kidney disease . Nat Rev Nephrol 2016 ; 12 : 73 – 81 Google Scholar Crossref Search ADS PubMed 2 Zelmanovitz T , Gerchman F , Balthazar AP et al. Diabetic nephropathy . Diabetol Metab Syndr 2009 ; 1 : 10 Google Scholar Crossref Search ADS PubMed 3 Gnudi L , Coward RJ , Long DA. Diabetic nephropathy: perspective on novel molecular mechanisms . Trends Endocrinol Metab 2016 ; 27 : 820 – 830 Google Scholar Crossref Search ADS PubMed 4 Pedraza-Chaverri J , Sanchez-Lozada LG , Osorio-Alonso H et al. New pathogenic concepts and therapeutic approaches to oxidative stress in chronic kidney disease . Oxid Med Cell Longev 2016 ; 2016 : 6043601 Google Scholar Crossref Search ADS PubMed 5 Bondeva T , Wolf G. Reactive oxygen species in diabetic nephropathy: friend or foe? Nephrol Dial Transplant 2014 ; 29 : 1998 – 2003 Google Scholar Crossref Search ADS PubMed 6 Tavafi M. Diabetic nephropathy and antioxidants . J Nephropathol 2013 ; 2 : 20 – 27 Google Scholar Crossref Search ADS PubMed 7 Silverstein RL , Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior . Sci Signal 2009 ; 26 : 3 8 Liani R , Halvorsen B , Sestili S et al. Plasma levels of soluble CD36, platelet activation, inflammation, and oxidative stress are increased in type 2 diabetic patients . Free Radic Biol Med 2012 ; 52 : 1318 – 1324 Google Scholar Crossref Search ADS PubMed 9 Okamura DM , Pennathur S , Pasichnyk K et al. CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD . J Am Soc Nephrol 2009 ; 20 : 495 – 505 Google Scholar Crossref Search ADS PubMed 10 Susztak K , Ciccone E , McCue P et al. Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy . PLoS Med 2005 ; 2 : 45 Google Scholar Crossref Search ADS 11 Hou YJ , Wu M , Wei JY et al. CD36 is involved in high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells . Biochem Biophys Res Commun 2015 ; 468 : 281 – 286 Google Scholar Crossref Search ADS PubMed 12 Moon JS , Karunakaran U , Elumalai S et al. Metformin prevents glucotoxicity by alleviating oxidative and ER stress-induced CD36 expression in pancreatic beta cells . J Diabet Compl 2017 ; 31 : 21 – 30 Google Scholar Crossref Search ADS 13 Reddy PH , Manczak M , Kandimalla R. Mitochondria-targeted small molecule SS31, a potential candidate for the treatment of Alzheimer's disease . Hum Mol Genet 2017 ; 26 : 1483 – 1496 Google Scholar Crossref Search ADS PubMed 14 Cho S , Szeto HH , Kim E et al. A novel cell-permeable antioxidant peptide, SS31, attenuates ischemic brain injury by down-regulating CD36 . J Biol Chem 2007 ; 282 : 4634 – 4642 Google Scholar Crossref Search ADS PubMed 15 Thomas DA , Stauffer C , Zhao K et al. Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization, reduces islet cell apoptosis, increases islet cell yield, and improves posttransplantation function . J Am Soc Nephrol 2007 ; 18 : 213 – 222 Google Scholar Crossref Search ADS PubMed 16 Huang J , Li X , Li M et al. Mitochondria-targeted antioxidant peptide SS31 protects the retinas of diabetic rats . Curr Mol Med 2013 ; 13 : 935 – 945 Google Scholar Crossref Search ADS PubMed 17 Szeto HH , Liu S , Soong Y et al. Mitochondria-targeted peptide accelerates ATP recovery and reduces ischemic kidney injury . J Am Soc Nephrol 2011 ; 22 : 1041 – 1052 Google Scholar Crossref Search ADS PubMed 18 Hou YJ , Li SC , Wu M et al. Mitochondria-targeted peptide SS-31 attenuates renal injury via an antioxidant effect in diabetic nephropathy . Am J Physiol Renal Physiol 2016 ; 310 : 547 – 559 Google Scholar Crossref Search ADS 19 Zhao K , Zhao GM , Wu D et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury . J Biol Chem 2004 ; 279 : 34682 – 34690 Google Scholar Crossref Search ADS PubMed 20 Zhao WY , Han S , Zhang L et al. Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells . Cell Physiol Biochem 2013 ; 32 : 591 – 600 Google Scholar Crossref Search ADS PubMed 21 Maric C , Sandberg K , Hinojosa-Laborde C. Glomerulosclerosis and tubulointerstitial fibrosis are attenuated with 17beta-estradiol in the aging Dahl salt sensitive rat . J Am Soc Nephrol 2004 ; 15 : 1546 – 1556 Google Scholar Crossref Search ADS PubMed 22 Tervaert TWC , Mooyaart AL , Amann K et al. Pathologic classification of diabetic nephropathy . J Am Soc Nephrol 2010 ; 21 : 556 – 563 Google Scholar Crossref Search ADS PubMed 23 Steubl D , Block M , Herbst V et al. Plasma uromodulin correlates with kidney function and identifies early stages in chronic kidney disease patients . Medicine (Baltimore) 2016 ; 95 : e3011 Google Scholar Crossref Search ADS PubMed 24 Lemley KV. An introduction to biomarkers: applications to chronic kidney disease . Pediatr Nephrol 2007 ; 22 : 1849 – 1859 Google Scholar Crossref Search ADS PubMed 25 Currie G , McKay G , Delles C. Biomarkers in diabetic nephropathy: present and future . World J Diabetes 2014 ; 5 : 763 – 776 Google Scholar Crossref Search ADS PubMed 26 Yang L , Zhao K , Calingasan NY et al. Mitochondria targeted peptides protect against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine neurotoxicity . Antioxid Redox Signal 2009 ; 11 : 2095 – 2104 Google Scholar Crossref Search ADS PubMed 27 Cho J , Won K , Wu D et al. Potent mitochondria-targeted peptides reduce myocardial infarction in rats . Coron Artery Dis 2007 ; 18 : 215 – 220 Google Scholar Crossref Search ADS PubMed 28 Mizuguchi Y , Chen J , Seshan SV et al. A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction . Am J Physiol Renal Physiol 2008 ; 295 : 1545 – 1553 Google Scholar Crossref Search ADS 29 Shigenaga MK , Gimeno CJ , Ames BN. Urinary 8-hydroxy-2-deoxyguanosine as a biological marker of in vivo oxidative DNA damage . Proc Natl Acad Sci USA 1989 ; 86 : 9697 – 9701 Google Scholar Crossref Search ADS PubMed 30 Rajasekaran S , Ravi K , Sivagnanam K et al. Beneficial effects of aloe vera leaf gel extract on lipid profile status in rats with streptozotocin diabetes . Clin Exp Pharmacol Physiol 2006 ; 33 : 232 – 237 Google Scholar Crossref Search ADS PubMed 31 Sedeek M , Nasrallah R , Touyz RM et al. NADPH oxidases, reactive oxygen species, and the kidney: friend and foe . J Am Soc Nephrol 2013 ; 24 : 1512 – 1518 Google Scholar Crossref Search ADS PubMed 32 Kawahara T , Quinn MT , Lambeth JD. Molecular evolution of the reactive oxygengenerating NADPH oxidase (Nox/Duox) family of enzymes . BMC Evol Biol 2007 ; 7 : 109 Google Scholar Crossref Search ADS PubMed 33 Flekac M , Skrha J , Hilgertova J et al. Gene polymorphisms of superoxide dismutases and catalase in diabetes mellitus . BMC Med Genet 2008 ; 9 : 30 Google Scholar Crossref Search ADS PubMed 34 Nath A , Li I , Roberts L et al. Elevated free fatty acid uptake via CD36 promotes epithelial-mesenchymal transition in hepatocellular carcinoma . Sci Rep 2015 ; 5 : 14752 Google Scholar Crossref Search ADS PubMed 35 Feng L , Gu C , Li Y et al. High glucose promotes CD36 expression by upregulating peroxisome proliferator-activated receptor γ levels to exacerbate lipid deposition in renal tubular cells . Biomed Res Int 2017 ; 2017 : 1414070 Google Scholar PubMed 36 Sun L , Li W , Li W et al. Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH xidase/ROS/Akt/NF-κB pathway under high glucose conditions . Int J Mol Methods 2014 ; 34 : 167 – 176 Google Scholar Crossref Search ADS 37 Hayden MS , Ghosh S. Signaling to NF-kappaB . Genes Dev 2004 ; 18 : 2195 – 2224 Google Scholar Crossref Search ADS PubMed 38 Wu J , Li H , Sun X et al. A mitochondrion-targeted tioxidant ameliorates isoflurane-induced cognitive deficits in aging mice . PLoS One 2015 ; 10 : 0138256 © The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Nephrology Dialysis TransplantationOxford University Press

Published: Nov 1, 2018

References

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