SIRT4 Overexpression Protects Against Diabetic Nephropathy by Inhibiting Podocyte Apoptosis
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342 EXPERIMENTAL AND THERAPEUTIC MEDICINE 13: 342-348, 2017 SIRT4 overexpression protects against diabetic nephropathy by inhibiting podocyte apoptosis JIAN-XIA SHI, QI-JIN WANG, HUI LI and QIN HUANG Department of Endocrinology, Changhai Hospital, The Second Military Medical University, Shanghai 200433, P.R. China Received June 29, 2015; Accepted September 22, 2016 DOI: 10.3892/etm.2016.3938 Abstract. Diabetic nephropathy is a diabetic complication contribute to the progression of diabetic nephropathy; hyper- associated with capillary damage and increased mortality. glycemia, hypertension, obesity and advancing age have been Sirtuin 4 (SIRT4) plays an important role in mitochondrial extensively characterized (2,3). However, the precise mecha- function and the pathogenesis of metabolic diseases, including nism for this condition remains unclear. Diabetic nephropathy aging kidneys. The aim of the present study was to investigate is characterized by changes in kidney morphology and ultra- the association between SIRT4 and diabetic nephropathy in structure, resulting in an increased glomerular filtration rate, a glucose-induced mouse podocyte model. A CCK-8 assay increased glucose level and blockade of the renin-angiotensin showed that glucose simulation significantly inhibited podo- system (4-6). cyte proliferation in a time- and concentration-dependent Although maintaining control of the glycemic index is chal- manner. Reverse transcription-quantitative polymerase lenging, it lightens the symptoms of diabetic complications, chain reaction and western blot analysis showed that the suggesting that hyperglycemia is the critical induction factor mRNA and protein levels of SIRT4 were notably decreased in the development and progression of diabetic complications, in a concentration-dependent manner in glucose-simulated including diabetic nephropathy (7,8). Treatment strategies for podocytes. However, SIRT4 overexpression increased prolif- diabetic nephropathy, such as glycemic and blood pressure eration and suppressed apoptosis, which was accompanied by control, target various pathways contributing to the develop- increases in mitochondrial membrane potential and reduced ment of diabetic nephropathy (9,10). However, numerous production of reactive oxygen species (ROS). Notably, patients continue to experience progressive renal injury. SIRT4 overexpression downregulated the expression of Thus, investigations of additional pathogenic pathways and apoptosis-related proteins NOX1, Bax and phosphorylated p38 relevant therapeutic strategies involving candidate targets with and upregulated the expression of Bcl-2 in glucose-simulated a potential impact on diabetic nephropathy are worthwhile. podocytes. In addition, SIRT4 overexpression significantly Importantly, reactive oxygen species (ROS), apoptosis and attenuated the inflammatory response, indicated by reduc- inflammatory response in the kidney are associated with the tions in the levels of TNF-α, IL-1β and IL-6. These results development and progression of diabetic nephropathy (11,12). demonstrate for the first time that the overexpression of The sirtuin family has seven members, SIRT1-SIRT7, SIRT4 prevents glucose-induced podocyte apoptosis and ROS which have functions in lifespan regulation. In mammals, production and suggest that podocyte apoptosis represents an SIRT1, SIRT6 and SIRT7 are located in the nucleus, SIRT3, early pathological mechanism leading to diabetic nephropathy. SIRT4 and SIRT5 are located in the mitochondria and SIRT2 is located in the cytoplasm (13). SIRT1 has been shown to be Introduction associated with the regulation of apoptosis, inflammation, metabolism and mitochondrial biogenesis, and play a pivotal Diabetic nephropathy is a serious and common microvascular role in neural development and age-related diseases, including complication of diabetes and a major cause of end-stage renal type 2 diabetes (14). SIRT3 enhances lipid catabolism, regu- disease worldwide (1). Several factors have been shown to lates the tricarboxylic acid cycle and reduces the levels of ROS (15). The SIRT4 protein, which is localized to the mito- chondrial cellular compartment, uses nicotinamide adenine dinucleotide to adenosine diphosphate (ADP)-ribosylate gluta- mate dehydrogenase (GDH) and thereby repress GDH activity Correspondence to: Dr Qin Huang, Department of Endocrinology, and limit the generation of adenosine triphosphate (16,17). Changhai Hospital, The Second Military Medical University, 168 Changhai Road, Shanghai 200433, P.R. China The expression of sirtuins has been observed in the kidneys, E-mail: [email protected] and shown to be modulated by calorie restriction to protect against the development and progression of damage in the Key words: diabetic nephropathy, podocytes, apoptosis, glucose, aging kidney (18), suggesting that sirtuins may be involved in sirtuin 4 evoking susceptibility to diabetic nephropathy. In the present study, the first comprehensive characteriza- tion of SIRT4 as a candidate gene for diabetic nephropathy is SHI et al: SIRT4 INHIBITS GLUCOSE-INDUCED PODOCYTE APOPTOSIS 343 provided, and the association between SIRT4 overexpression saline (PBS) and the density was adjusted to 1x105 cells/ml. and diabetic nephropathy investigated in an experimental The podocytes were then incubated with 0.5 ml JC-1 in an glucose-induced mouse podocyte model. incubator (37˚C, 100% humidity and 5% CO2) for 20 min and subsequently subjected to flow cytometric analysis. Materials and methods ROS detection. A dichlorodihydrofluorescein diacetate Cell culture and glucose treatment. Mouse podocytes were (DCFH-DA) fluorescent probe combined with flow cytometric obtained from Shanghai Cell Bank, Chinese Academy of analysis was used to detect the changes of ROS levels, as Sciences (Shanghai, China) and cultured in RPMI-1640 described previously (19). Briefly, podocytes were resuspended (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, in PBS and the density was adjusted to 5x105 cells/ml. The USA) supplemented with 10% fetal bovine serum (Invitrogen; podocytes were then incubated with 10 µM DCFH-DA for Thermo Fisher Scientific, Inc.), 100X penicillin-streptomycin 20 min in the dark at 37˚C and subsequently subjected to flow solution and 10 U/ml interferon (IFN)-γ (ProSpec-Tany cytometric analysis. TechnoGene Ltd., East Brunswick, NJ, USA), and incubated in a humidified atmosphere at 33˚C with 5% CO2. After prolif- Reverse transcription‑quantitative polymerase chain reaction eration to 70%-80% confluence, podocytes were cultured in (RT‑qPCR). Total RNA was extracted from the podocytes the same medium without 10 U/ml IFN-γ and incubated in a using TRIzol reagent (Gibco-BRL; Thermo Fisher Scientific, humidified atmosphere at 37˚C with 5% CO2 for 10-14 days. Inc.) according to the manufacturer's instructions. Briefly, Podocytes were exposed to normal glucose (5.5 mM) and high 1 µg RNA was reverse transcribed to cDNA using a cDNA glucose (10, 20, 30 and 40 mM), respectively. The normal synthesis kit (Thermo Fisher Scientific, Inc.) according to glucose (5.5 mM) treatment was used as control. the manufacturer's protocol. qPCR was then performed to measure the mRNA levels of SIRT4 using an ABI-7300 Lentiviral production and transduction. The SIRT4 coding Real-Time PCR system (Applied Biosystems; Thermo Fisher sequence was cloned into a pLVX-AcGFP-C1 lentiviral vector Scientific, Inc.). Maxima SYBR Green/ROX qPCR Master (Sangon Biotech, Shanghai, China). A blank vector was used Mix (K0223; Finnzymes; Thermo Fisher Scientific, Inc.) was as negative control. The constructs were then transduced into used, according to the manufacturer's protocol. The PCR HEK293T cells (Shanghai Cell Bank, Chinese Academy cycling conditions were as follows: 95˚C for 10 min, followed of Sciences, Shanghai, China) with psPAX2 and pMD2G by 40 cycles at 95˚C for 15 sec and 60˚C for 45 sec, and a lentiviral packaging vectors (Addgene, Cambridge, MA, final extension step of 95˚C for 15 sec, 60˚C for 1 min, 95˚C USA) using Lipofectamine 2000 (Invitrogen; Thermo Fisher for 15 sec and 60˚C for 15 sec. Gene expression was calcu- Scientific, Inc.) according to the manufacturer's instructions. lated using the ΔΔCq method (20). The primers used were as After 48 h of transduction, the lentivirus was collected and follows: SIRT4, 5'-TTG TGG GCT GGC CTC AATTC-3' and used to infect mouse podocytes. Mouse podocytes were 5'-AGT GCA AAG CGT CCA CGTTC-3'; and GAPDH, 5'-ATC infected with the lentivirus at an multiplicity of infection of 20 ACT GCC ACC CAG AAG-3' and 5'-TCC ACG ACG GAC ACA in the presence of 8 µg/ml Polybrene (Sigma-Aldrich). TTG-3'. The experiment was repeated three times. Cell proliferation assay. Podocytes were washed, trypsinized Protein extraction and western blotting. Podocytes were and adjusted to 3x103 cells/well in 96-well plates, and cultured harvested and lysed on ice for 30 min in radioimmunoprecipita- for 0, 12, 24, 36, 48, 60 and 72 h after transduction. Cell prolif- tion assay buffer (Beyotime Institute of Biotechnology, Haimen, eration was then determined using a Cell Counting kit (CCK)-8 China) containing 1 mM phenylmethylsulfonyl fluoride. The assay (Dojindo Laboratories, Kumamoto, Japan) according to protein concentration was assessed using a bicinchoninic acid the manufacturer's protocol. Briefly, CCK-8 solution (10 µl protein assay kit (cat. no. PICPI23223; Thermo Fisher Scientific, in 100 µl RMPI-160 medium) was added to each well and Inc.). Equal amounts of cell lysates (35