| BackgroundDiabetic nephropathy (DN), as one of the most relevant microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. DN results in high morbidity and mortality thus increased large medical burden in patients with DN. Hyperglycemia in diabetic patients has been regarded to the main reason of DN, but the underlying mechanism remains unknown. Moreover, the current therapies for DN in clinic are still limited, such as intensive insulin treatment, strict lipid control, reduction of blood hypertension and smoking cessation. They are not sufficient to prevent renal deteriorative progression to end-stage renal diseases. Hence, it is urgent to uncover the molecular pathogenesis of DN and develop some new effective therapeutic drugs.More and more evidence suggested that hyperglycemia-related oxidative stress is an early event and main mediator in the development of diabetic complications. Excessive oxidative stress leads to the activation of major biochemical harmful pathways, including advanced glycation end-products (AGEs) formation, protein kinase C(PKC)-extracellular-regulated protein kinase (ERK) pathway, polyol pathway, and growth factors and cytokines. In diabetic rats, antioxidant treatments with tocotrienol, probucol, a-lipoic acid, taurine, or resveratrol improved the renal complications such as albuminuria and glomerular hypertrophy. These antioxidant drugs reverse the increased levels of reactive oxygen species (ROS) and activating antioxidant enzymes. Therefore, targeting to alleviate oxidative stress may offer new prospective approaches for DN.Grape seed proanthocyanidin extracts (GSPE) is extracted from grape seeds, possessing a variety of potent pharmacological activities including anti-oxidative stress, free-radical scavenging, anti-inflammation, anti-AGEs, anti-atherosclerosis and so on. In our previous studies, we have convincingly demonstrated the protective effects of GSPE on diabetic vascular complications, especially on diabetic kidney. Grape seed procyanidin B2 (GSPB2) is dimeric form of GSPE, with the most powerful biological activities. GSPB2 has been reported to possess pharmacologic effects including anti-inflammatory, anti-apoptosis and anti-atherosclerosis. But little is known about its effects and molecular mechanism on DN.With the development of proteomics, mass spectrometry (MS)-based isobaric tags for relative and absolute quantification (iTRAQ) has become the main method in proteomic analysis. iTRAQ is applied to quantitively detect the differentially expressed proteins. It has been widely used to study molecular marks and pathogenesis of disease, and find the differentially expressed proteins between normal and abnormal state. Thus, our present study aims to evaluate the renopreventive effects of GSPB2 and explore its potential target proteins in diabetic nephropathy. We apply iTRAQ to get comprehensive differential protein profiles in kidneys of db/db mice. Furthermore, we investigated the molecular pathway of the identified proteins in diabetic kidney identified by iTRAQ, through knocking down MFG-E8 and injecting exogenous recombinant MFG-E8 in db/db mice.Objective1. To evaluate the effects of GSPB2 on renal functional and morphological changes.2. Applying iTRAQ to establish comprehensive differential protein profiles in kidneys of db/db mice with or without GSPB2 treatment.3. By knocking down MFG-E8 and injecting exogenous recombinant MFG-E8 in db/db mice to explore the molecular function of one identified protein in diabetic kidney.MethodsMale C57BLKS/J db/db mice (n= 16,7 weeks old) and db/m mice (n= 8,7 weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). After adaptation for a week, sixteen db/db mice were divided into 2 groups:an untreated diabetic group (DM, n= 8) administrated with normal saline solution by intragastric administration and GSPB2-treated group with a dosage of 30mg/kg/d (DMT, n= 8) for 10 weeks. C57BLKS/J db/m mice were selected as control group (CC, n= 8). Animals were weighed every week. Before the end of intervention,24h urine samples were collected from mice with metabolic cages to measure urinary albumin excretion. All mice were then fasted for 12hrs before scarification. Fasting blood was collected for FBG, AGEs, BUN and Cr. Perfused kidneys were dissected for histological examination (HE and PAS staining) under light microscopy and ultrastructural evaluation under electron microscope. Some kidneys were kept at-80℃ for iTRAQ proteomic analysis and western blot analysis.In this experiment, kidneys of four mice per group were chosed for proteomic analysis. In brief, renal proteins were digested by trypsin, and labeled with iTRAQ reagents,114 for the peptides of group CC,117 for group DM, and 115 for group DMT. All the peptides were fractionated with Strong Cation Exchange (SCX) chromatography and analyzed by a LTQ-Velos ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). The MS/MS spectra were extracted and automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version 3.72) using the Turbo SEQUEST program in the BioWorksTM 3.1 software suite. Data were further analyzed for protein subcellular location and functional cluster according to the Gene Ontology (GO) classes "cellular component", "molecular functions", and "biological process" in UniProt protein knowledge database (http://www.uniprot.org). Ingenuity pathway analysis (IPA) program (http://www.ingenuity.com) was applied to analyze the pathway of differentiall y expressed proteins identified by iTRAQ.Lentiviral vectors with short hairpin RNAs (shRNAs) for mouse MFG-E8 gene were constructed and chemically synthesized. The lentiviral vector with green fluorescence protein (GFP) was the RNAi control. Male C57BLKS/J db/db and db/m mice (n= 40,7 weeks old) were injected with MFG-E8 RNAi and exogenous recombinant MFG-E8 in this study. C57BLKS/J db/m mice were selected as control group (CC, n= 8). All db/db mice were divided into four groups:an untreated diabetic group (DM, n= 8), LV-GFP treated db/db group (LV-GFP, n=8), MFG-E8 RNAi treated db/db mice group (MRNAi, n= 8) and recombinant MFG-E8 treated db/db mice group (reMFG-E8, n= 8). At the age of 14 week, sixteen db/db mice were transinfected with MFG-E8 shRNAs or LV-GFP by tail vein injection, respectively. Another eight db/db mice were injected by 300ul of recombinant mouse MFG-E8 prediluted in PBS through tail vein at a dosage of 20ug/kg, twice a week for 4 weeks. At the end of the intervention, all mice were fasted overnight and then sacrificed. The perfused kidneys were dissected and kept at-80℃ until further analysis. Western blotting was applied to measure protein expression of MFG-E8, p-ERK1/2, p-Akt and p-GSK-3β in kidneys of db/db mouse.Results1. General observationThroughout the experimental periods, db/m mice in CC group were in good condition, without any diabetic symptoms, db/db mice in DM group were polydipsia, polyphagia and hyperdiuresis, with dirty furs and obesity. GSPB2 treated db/db mice looked abnormal, but better than the db/db mice. Body weights were consistently greater in db/db mice than in db/m mice (P< 0.05). However, the increase of body weight in db/db mice was significantly attenuated from the second week after GSPB2 treatment (P< 0.05).2. Effects of GSPB2 on Body Weight, FBG, AGEs, BUN, Cr and urinary albumin excretion rate.At the end of experiment, levels of FBG, AGEs, BUN, Cr and 24h urinary albumin excretion were remarkably elevated in db/db mice when compared to db/m mice. Treatment with GSPB2 to db/db mice induced a significant reduction in AGEs, BUN, Cr and 24h urinary albumin excretion. However, GSPB2 treatment did not affect blood glucose levels in db/db mice.3. Effects of GSPB2 on renal histology of db/db miceHE and PAS staining:The renal section of db/m mice demonstrating normal architecture with normal glomeruli under light microscopy. However, db/db mice exhibited glomerular hypertrophy, mesangial expansion related to mesangial cell proliferation and matrix accumulation, along with visceral epithelial cells proliferation. These alterations in diabetic kidneys were ameliorated to near normalcy after treatments with GSPB2.Ultrastructure examination under electron microscopy showed irregular thickened glomerular basement membranes (GBMs) with segmental nail-like prominencies, deformed podocytes with less glogi complexes and mitochondria, extensively fused foot processes and slit pores were striking in db/db mice, as compared to db/m mice. Treatment with GSPB2 markedly reversed the ultrasructural changes of glomeruli in db/db mice.4. Quantification and identification of the renal proteome by iTRAQ AnalysisIn this study, 2842 renal proteins were identified by iTRAQ analysis, of which 671 proteins with different abundance were identified between db/db mice and db/m mice (± 1.5-fold). Fifty-three proteins were down-regulated and sixty proteins were up-regulated in db/db mice after GSPB2 treatment. Strikingly, a novel molecule MFG-E8 drew our particular attention, with a 2.23-fold increase in db/db mice versus db/m mice, and back-regulated by GSPB2 treatment at a ratio of 0.47.Location analysis of the differentially expressed protein showed that the largest proportion was located in nucleus (22%). Proteins located in cytosol (19%) and plasma membranes (14%) were the next two highest parts. Besides, up to 12% of total 113 proteins located in the mitochondrion. The differentially expressed proteins were clustered into a number of cellular pathways. Notably, the majority of changed proteins after treatment with GSPB2 were associated with protein or amino acid transport and metabolism, nearly accounting for 19%. The proteins involved in apoptosis/cell cycle/cell differentiation/proliferation and signal transduction constituted of another two prominent functional groups, occupying about 14% and 11%, respectively. Moreover, proteins involved in oxidative stress, carbohydrate and lipid metabolism accounted for 7 %,6%, and 4%, respectively. The ingenuity pathway analysis of the differentially expressed proteins suggested that apoptosis, oxidative stress, and metabolism were involved in the development of DN. In particular, MFG-E8 is directly linked to extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathway in this network.5. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the protein expression of MFG-E8 in db/db miceWestern blot analysis showed that the protein expression of MFG-E8 was significantly increased in db/db mice compared to db/m mice, and nomalized by GSPB2 treatment. MFG-E8 shRNA successfully knocked down MFG-E8 protein level by～60% in the kidneys of db/db mice. Conversely, the injection of exogenous recombinant MFG-E8 remarkably elevated MFG-E8 protein expression. LV-GFP had no significant influence on the protein level of MFG-E8 (P>0.05).6. Effects of MFG-E8 RNAi and recombinant MFG-E8 on renal histology of db/db miceHE and PAS staining: LV-GFP transfected db/db mice showed the similar histological changes as db/db mice, with glomerular hypertrophy and mesangial expansion. Silencing MFG-E8 significantly suppressed glomerular hypertrophy and mesangial expansion in the kidneys of db/db mice. However, exogenously added reMFG-E8 to db/db mice remarkably exacerbated renal lesions, characterized by notable glomerular hypertrophy, mesangial expansion and inflammatory cell accumulation.Ultrastructure examination under electron microscopy:The db/db mice transfected by LV-GFP showed similar diabetic changes to those in diabetic mice, such as irregular thickened GBMs with segmental nail-like prominencies, deformed podocytes with less glogi complexes and mitochondria, extensively fused foot processes and slit pores. The diabetic alterations in kidneys of db/db mice were remarkably attenuated by MFG-E8 RNAi, but exacerbated by injection of reMFG-E8, consist with the observations under light microscope.7. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the protein expression of p-ERK1/2、p-Akt、p-GSK-3β in kidneys of db/db miceThe protein expression level of p-ERK1/2, p-Akt、p-GSK-3β were remarkably higher in the kidneys of db/db mice than in db/m mice, and were significantly suppressed by treatment with GSPB2. MFG-E8 silencing significantly decreased the phosphorylation level of p-ERK1/2、p-Akt、p-GSK-3β compared with db/db mice. Conversely, exogenous MFG-E8 resulted in a significantly increase level of p-ERK1/2、p-Akt、p-GSK-3β in kidneys of db/db mice.Conclusion1. GSPB2 30mg/kg/day treatment significantly inhibit the obesity, decrease the AGEs level, improve renal function and alleviate renal histology injury in db/db mice.2. iTRAQ identified 671 with significant different abundance between kidneys of db/m mice and db/db mice, of which the protein expression level of 113 proteins were normalized by GSPB2 treatment. Most of these differentially expressed proteins located in cytoplasm, and involved in cell apoptosis, oxidative, and lipid metabolism. GSPB2 alleviate diabetic nephropathy partly by regulating these protein expression levels in db/db mice.3. MFG-E8 contributed to the pathogenesis of diabetic nephropathy, by regulating ERK1/2, Akt, and GSK-3β signaling pathway. GSPB2 performed potent protective effect on DN by inhibition of MFG-E8, phosphorylation of ERK1/2, Akt, and GSK-3β signaling pathway. Our research provides a new insight into the pathogenesis of diabetic nephropathy and indicates that targeting MFG-E8 could be a novel strategy to retard the progression of DN. |
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