Abstract Background Hyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN), respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated. Methods The expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20) by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5) and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software. Results CD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO), the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol) inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid. Conclusions CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.

Citation: Hua W, Huang H-z, Tan L-t, Wan J-m, Gui H-b, Zhao L, et al. (2015) CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress. PLoS ONE 10(5): e0127507. https://doi.org/10.1371/journal.pone.0127507 Academic Editor: Stuart E. Dryer, University of Houston, UNITED STATES Received: December 13, 2014; Accepted: April 13, 2015; Published: May 22, 2015 Copyright: © 2015 Hua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All data are available solely within the paper. Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81370816), the Natural Science Foundation of Chongqing Science and Technology Commission of China (No. cstc2012jjA10136) and Chongqing Municipal Health Bureau of China (No. 2011-1-016) to X. Du, and the National Natural Science Foundation of China (No. 81202318) to X. Chen. Competing interests: The authors have declared that no competing interests exist.

Introduction Diabetes mellitus (DM) has become a worldwide epidemic affecting nearly all developing and developed countries. Approximately 20 to 40% of patients with diabetes types 1 and 2, will develop diabetic nephropathy (DN), the main cause of chronic kidney disease (CKD), which ultimately progresses to end-stage renal disease (ESRD) associated with high costs substituting for kidney function [1]. Diabetic nephropathy is characterized by glomerular hypertrophy, widening of the glomerular basement membrane (GBM), mesangial expansion and podocytopenia, leading to nodular (Kimmelstiel-Wilson) glomerulosclerosis, which is called diabetic glomerulosclerosis [2]. Podocytes are highly branched, terminally differentiated visceral epithelial cells of the renal glomerulus that cover the urinary side of the GBM and play a crucial role in the regulation of glomerular function. Podocyte injury triggers proteinuria and glomerular sclerosis in human DN, and in animal models [3–5]. Reduction in podocyte number mediated by apoptosis has been shown to predict progressive decline in renal function and proteinuria in type 2 DM patients [6]. DM is often accompanied by lipid abnormalities. Experimental and clinical evidence suggests that lipid abnormalities in renal disease, including DN, contribute to the process of glomerulosclerosis with progressive decline in renal function [7–11]. Fatty acid translocase CD36 is expressed in several cell types, including macrophages, microvascular endothelial cells, platelets, adipocytes, and podocytes. Studies showed that hyperlipidemia-induced apoptosis mediated by CD36 was associated with increased uptake of ox-LDL or fatty acid in macrophages leading to atherosclerotic lesions[12], in hepatocytes leading to liver damage and fibrosis in obese patients[13], and in proximal tubular epithelial cells leading to progressive diabetic nephropathy[14]. However, the specific role of CD36 in podocyte apoptosis of DN with hyperlipidemia remains poorly investigated. The aim of the present study was therefore, to investigate the expression of CD36 in kidney tissues from DN patients with hyperlipidemia, and the effect of fatty acid including the mechanism of action on CD36 expression and apoptosis of podocytes using an in vitro model.

Materials and Methods Human kidney tissues Paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20) were collected from Pathology Department of the First Affiliated Hospital of Chongqing Medical University from January 2011 to December 2013, and the expression of CD36 was measured by immunohistochemistry and immunofluorescence. For control tissue samples, renal tissue was collected from 18 patients undergoing nephrectomy for renal trauma with no other evidence of renal disease. For DN tissue samples, renal tissue was collected from 20 DN patients with hyperlipidemia undergoing renal biopsies. The study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University and written informed consent was obtained from all the patients. Podocyte culture studies Conditionally immortalized mouse podocyte cell line (MPC5) was kindly shared by Dr Ruan of the Centre for Nephrology, Royal Free and University College Medical School, London, United Kingdom. Cells were cultured and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1000 U/L penicillin, 1 mg/L streptomycin and 10U/ml interferon-γ on type I collagen at “permissive” temperature of 33°C and 5% carbon dioxide. Podocyte differentiation was induced by culturing them under “nonpermissive” temperature of 37°C without interferon-γ. All experiments were performed on podocyte cell line after differentiation for 14 days. Podocytes were treated with palmitic acid (Sigma-Aldrich) after pre-treatment with or without Sulfo-N-succinimidyloleate (SSO, Toronto Research Chemicals, North York, ON, Canada), a specific inhibitor of the fatty acid binding site on CD36, antioxidant4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol, Sigma-Aldrich), or vehicle (Dimethyl sulfoxide, DMSO). The CD36 expression, lipid uptake, reactive oxygen species (ROS) production and cell apoptosis were measured. Immunohistochemistry staining Paraffin-embedded kidney tissue samples were sliced into 5-μm-thick sections, deparaffinized in xylene, and rehydrated in graded ethanol. Antigen recovery was performed in 10 mmol/L boiling sodium citrate buffer at pH 6.0 for 10 minutes at 92–98°C, and the specimens were incubated with 0.3% H 2 O 2 for 15 minutes. Non-specific binding was blocked with normal goat serum for 10 minutes at room temperature. The sections were incubated with mouse monoclonal anti-CD36 antibody (diluted 1:100, Abcam, ab23680, UK) at 4°C overnight, washed three times with phosphate-buffered saline (PBS) and incubated with biotinylated secondary antibody (Zhongshan Golden Bridge Inc., China) for 10 minutes. After washing with PBS for three times, sections were incubated with DAB (3, 3’-diaminobenzidine) for 5 minutes, counterstained with hematoxylin for 5 seconds, visualized using Nikon Eclipse 80i microscope. Immunofluorescence staining of CD36 Tissue staining was accomplished by first slicing the paraffin-embedded kidney tissue samples into 5-μm-thick sections. After deparaffinization, rehydration, antigen recovery and blocking, the sections were incubated with mouse monoclonal anti-CD36 antibody (1:100) at 4°C overnight, and washed three times with PBS and incubated with secondary antibody (goat anti-mouse-IgG AlexaFluor 488, Invitrogen, A11001, USA) for 1.5 hours in light-shielded conditions. After three washes with PBS, sections were mounted with antifade reagents, coverslipped, and visualized microscopically. Cultured cells were stained by plating the podocytes on glass coverslips. After treatment, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were washed with PBS, blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature, and incubated with mouse anti-CD36 antibody (1:100) at 4°C overnight. The cells were washed three times with PBS and incubated with goat anti-mouse-IgG for 1.5 hours in light-shielded conditions. Nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI; Invitrogen, USA) for 2 minutes. After three washes with PBS, coverslips were mounted with antifade reagents. The cells were visualized microscopically. Real-time PCR Total RNA was extracted by Bioteke Corporation Kit. First strand cDNAs synthesized from total RNA using PrimeScript RT reagent Kit (Takara, Japan) were used as templates. Specific primers were purchased from GeneCopoeia, USA. The levels of mRNA were quantified using All-in-One qPCR Mix Kit (GeneCopoeia, USA). Fold change for each group was determined using the delta-delta Ct method. Quantified mRNA levels were normalized to β-actin and presented relative to control group (podocytes were treated with 0 μmol/L palmitic acid for 0 hour). Western blot analysis Cells were rinsed twice with PBS, sonicated for 15 seconds in 500 μl of RIPA lysis buffer (Beyotime, China), and centrifuged at 14,000g for 5 minutes. Protein concentration was then determined by bicinchoninic acid protein assay (Beyotime, Beijing, China). The sample loading buffer was added to the protein sample and heated at 100°C for 10 minutes. The proteins were separated by electrophoresis in 10% tris-glycine polyacrylamide gradient gels. The separated proteins were then transferred onto PVDF membrane (Millipore), blocked with 5% skimmed milk for 2 hours, and incubated with rabbit monoclonal anti-CD36 antibody (1:800, Abcam, ab133625, UK), or rabbit monoclonal cleaved-caspase3 antibody (1:1000, Cell Signaling Technology, 9664S, USA) overnight at 4°C. After washing and incubation with horseradish peroxidase (HRP)-labeled Goat anti-rabbit (1:8,000, MultiSciences, GAR007, China) or Goat anti-mouse IgG (1:8,000, MultiSciences, GAM007, China) at room temperature for 1.5 hours, the membranes were probed with chemiluminescence reagents using a commercially available kit (Pierce Biotechnology, USA). Protein expression was detected using a chemiluminescent staining reagent kit to visualize the signals, followed by exposure to x-ray films. Band intensities were quantified with Quantity One software, and calculated as the optical density×area of the band. Measurement of lipid uptake Lipid uptake was measured using Oil Red O staining and BODIPY lipid probes. Oil Red O staining was carried out for 1 hour followed by 3 washes with distilled water. Lipid droplets were then visualized by microscope. To measure the uptake of fatty acid by BODIPY lipid probes, we incubated podocytes with 4, 4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (10 μg/ml, BODIPY500/510 C1, C12, Invitrogen, USA) at 37°C for 2 hours. Then the podocytes were washed with PBS to remove any extracellular fatty acid. Images were visualized under Olympus FluoView laser scanning confocal microscope (Olympus, Tokyo, Japan). Detection of ROS Intracellular ROS level was measured using the 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Pretreated cells were reacted with DCFH-DA (10 μmol/L) at 37.0°C for 1 hour. Cell images were captured with fluorescence microscope (excitation wavelength was 488 nm and emission wavelength was 520 nm). Flow cytometry analysis An apoptosis assay kit (Sungene Biotech, China) was used to determine apoptosis in confluent podocytes. Flow cytometry with BD FACSVantage SE cytometer was used to count cells that were annexin V-positive and propidium iodide-negative for early stages of apoptosis. Apoptosis detection by TUNEL assay In situ detection of DNA fragmentation was performed using the ApoTag TUNEL assay following the manufacturer's protocol (Intergen, Purchase, New York, United States). Apoptotic nuclei were detected using PI staining (1 μg/ml) in cell cultures fixed with 4% paraformaldehyde, and the TUNEL-positive cells (Green) were analyzed via fluorescence microscopy. To quantify apoptosis, 400 nuclei from random microscopic fields were analyzed by an observer blinded to the treatment groups. The total number of apoptotic cells in each section was summed and expressed as the percentage of the total cell number. At least 10 individual sections were evaluated per slide. Each observer was blinded to other data concerning the cells, as well as to the results of the other observer. Statistical analysis All statistical analyses were performed using SPSS 21.0 statistical software. All samples were analyzed in triplicate. Numeric data were shown as means ± SD. Statistical significance between groups was analyzed by Student's t-test and one-way ANOVA. P < 0.05 was considered statistically significant.

Discussion DN, one of the microvascular complications of diabetes, is the most common cause of CKD worldwide, with a prevalence of 40% in patients with ESRD[20]. Hyperglycemia-associated increased formation of intracellular advanced glycation end-products (AGEs) and activation of protein kinase C isoforms [21, 22], systemic and glomerular hypertension, and activation of renin-angiotensin system (RAS) are major factors in the pathogenesis of DN. Current therapies for DN focus on controlling blood sugar and blood pressure, and inhibiting the renin-angiotensin system (RAS) to reduce proteinuria, in an effort to delay the progression of DN [23, 24]. However, with increasing incidence of DN and deteriorating kidney function resulting in ESRD, it is imperative to understand the pathogenesis of DN comprehensively and identify effective therapeutic interventions and strategies. It has been found that DM is often complicated with dyslipidaemia associated with elevated triglycerides (TG) [25]. Hyperlipidemia is associated with DN progression, and lipid-lowering agents might protect renal function [7]. CD36 is a transmembrane protein of the class B scavenger receptor family, a broadly expressed membrane glycoprotein mediating the uptake of LCFA in adipocytes and fibroblasts, in arteriosclerosis, fatty liver, obesity and inflammatory responses, and so on[12, 13, 26–28]. In present study, using immunofluorescence and immunohistochemical staining we found an elevated expression of CD36 in the kidney of DN patients with hyperlipidemia, suggesting that CD36-mediated renal uptake of lipids might be associated with DN. The CD36 cDNA predicts a polypeptide of 53-kDa composed of 471 amino-acid residues. After variable post-translational N-linked glycosylations, which are necessary for trafficking to the plasma membrane, CD36 protein presents different molecular masses (78, 88 or 94 KDa) corresponding to different glycoforms [29]. LCFA uptake in adipocytes requires plasma membrane rafts, and FAT/CD36 which recycles from intracellular nonlipid raft domains to lipid raft regions of the plasma membrane might control LCFA uptake [16]. In the present study, treating podocytes with palmitic acid increased the CD36 expression in podocytes at both transcriptional and protein levels (53KDa, 78KDa and 88KDa), in the cell membrane and cytoplasm. Further research showed palmitate induced lipid accumulation in podocytes, and SSO, which specifically binds to and cross-links FAT/CD36 on the plasma membrane without permeating the plasma membrane, disrupted the lipid uptake. These results confirmed that palmitate not only upregulated CD36 expression but also promoted CD36 translocation from cytoplasm to plasma membrane in podocytes, leading to lipid accumulation in podocytes. DN is characterized by early-stage microalbuminuria and an increasing magnitude of proteinuria. Massive proteinuria is an adverse prognostic factor in chronic renal failure (CRF) irrespective of etiology, and interventions designed to minimize excessive glomerular protein filtration may halt or slow the loss of function at early and late stages of CRF [30, 31]. Podocyte apoptosis has been suggested as a potent mechanism of proteinuria in diabetic nephropathy [32]. Additional studies are required to investigate the detailed mechanisms of podocyte apoptosis. We treated podocytes with PA, and found increased apoptosis, which was inhibited by SSO, a specific inhibitor of lipid uptake mediated by FAT/CD36 on the plasma membrane, suggesting that CD36 might play a key role in podocyte apoptosis induced by FFA. Fatty acids and triglyceride-rich emulsions stimulate ROS production in leukocytes [33]. Oxidative stress plays a pathological role in the development of various diseases including DM and atherosclerosis [34], and an increase in podocyte ROS levels was a potential mediator of podocyte apoptosis in DM [6]. Here, we treated podocytes with PA, and found ROS production was considerably increased. Inhibition of ROS overproduction by tempol largely abrogated podocyte apoptosis stimulated by palmitic acid, suggesting that oxidative stress played an important role in fatty acid-induced podocyte apoptosis. We further confirmed that pretreatment of SSO decreased ROS production induced by PA in podocytes. Thus, oxidative stress might participate in CD36-mediated palmitic acid-induced podocyte apoptosis. The inhibition of ROS by modulating CD36 expression and translocation might be part of a protective mechanism against saturated FFAs that drives podocyte apoptosis. However, the exact mechanism of action and regulation of CD36 involved in fatty acid-inducing podocyte apoptosis is not well understood and is the subject of ongoing investigations. In summary, we have shown that CD36 expression was increased in kidney tissue of DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted CD36 translocation from cytoplasm to plasma membrane in podocytes. Increased lipid uptake, ROS production and apoptosis in podocytes induced by palmitic acid were mediated by CD36. All of these suggested CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN. Further studies are required to determine the exact mechanism of action and regulation of CD36 involved in fatty acid-induced podocyte apoptosis.

Acknowledgments This work was supported by grant from National Natural Science Foundation of China (No. 81370816) and grant from Natural Science Foundation of Chongqing Science and Technology Commission of China (No.cstc2012jjA10136) and Chongqing Municipal Health Bureau of China (No. 2011-1-016) to X. Du, National Natural Science Foundation of China (No. 81202318) to X. Chen.

Author Contributions Conceived and designed the experiments: XMC XGD. Performed the experiments: WH. Analyzed the data: WH XMC XGD. Contributed reagents/materials/analysis tools: HZH LTT JMW HBG LZ XZR. Wrote the paper: WH XMC XGD.