Proteomic analysis of kidney and protective effects of grape seed
procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the
development of diabetic nephropathy
Zhen Zhang
a, Bao-ying Li
a, Xiao-li Li
a, Mei Cheng
a, Fei Yu
a, Wei-da Lu
a, Qian Cai
a, Jun-fu Wang
b,
Rui-hai Zhou
c, Hai-qing Gao
a,⁎
, Lin Shen
aa
Department of Geriatrics, Qi-Lu Hospital of Shandong University, Key Laboratory of Cardiovascular Proteomics of Shandong Province, People's Republic of China
b
Institute of Basic Science, Medical Science Academy of Shandong, People's Republic of China
c
Division of Cardiology, University of North Carolina at Chapel Hill, Chapel Hill, USA
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 11 November 2012
Received in revised form 4 February 2013 Accepted 27 February 2013
Available online 6 March 2013 Keywords:
Grape seed procyanidin B2 Diabetic nephropathy MFG-E8
db/db mice iTRAQ
Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1⁄2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. Thesefindings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
According to recent estimations, diabetes mellitus (DM) is affecting about 5–10% of the world population and rising rapidly in younger population groups, including children and adolescents[1–3]. In the last decade, diabetic nephropathy (DN), one of the most relevant diabetic complications, has become the leading cause of end-stage renal disease (ESRD), with data indicating that type 2 diabetes contributes to a large proportion of patients under renal dialysis treatment[4]. Although hy-perglycemia has been accepted to mediate the pathogenesis of DN, the
underlying mechanism remains unclear[5–7]. Moreover, the current therapies for DN including intensive insulin treatment, strict lipid control, reduction of blood hypertension and smoking cessation are not sufficient to prevent renal deteriorative progression to ERSD[2,8]. Hence, clarification of the molecular pathogenesis of DN and develop-ment of novel effective therapeutic strategies are high priorities.
Increasing evidence indicates that hyperglycemia-mediated oxida-tive stress is an early event and common mediator in the development of diabetic complications [9–11]. Excessive oxidative stress results in 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[12,13]. In diabetic rats, antioxidant treatments with tocotrienol, probucol,α-lipoic acid, taurine, or resveratrol improved renal complications such as albuminuria and glomerular hypertrophy by reversing the increased levels of reactive oxygen species (ROS) and activating antioxidant enzymes[10,14–16]. Therefore, targeting to alleviate oxidative stress may offer new prospec-tive approaches for DN.
Abbreviations: AGEs, advanced glycation end products; BUN, blood urea nitrogen; Cr, plasma creatinine concentration; DM, diabetes mellitus; DN, diabetic nephropathy; ERK1/2, extracellular signal-regulated kinase1/2; ESRD, end-stage renal disease; FBG, fasting blood glucose; GSK-3β, glycogen synthase kinase-3beta; GSPB2, grape seed procyanidin B2; GSPE, grape seed proanthocyanidin extracts; iTRAQ, isobaric tags for relative and absolute quantification; MFG-E8, milk fat globule EGF-8
⁎ Corresponding author at: 107 Wenhuaxi Road, Jinan, Shandong Province 250012, People's Republic of China. Tel.: + 86 531 82169371; fax: + 86 531 86927544.
E-mail address:[email protected](H. Gao).
0925-4439/$– see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbadis.2013.02.022
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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b b a d i sGrape 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[17–19]. Our previous studies have convincingly demonstrated the protective effects of GSPE on diabetic vas-cular complications, especially on diabetic kidney[20–22]. Grape seed procyanidin B2 (GSPB2) is a dimeric form of GSPE, more powerful than other water-soluble polyphenos in biological activities. GSPB2 has been reported to possess pharmacologic effects including anti-inflammatory, anti-apoptosis and anti-atherosclerosis [23–25], but little is known about its effects on DN.
The present study aims to evaluate the renopreventive effects of GSPB2 and explore its potential target proteins in diabetic nephropathy. We resorted to mass spectrometry (MS)-based isobaric tags for relative and absolute quantification (iTRAQ) to get comprehensive differential Fig. 1. Effect of GSPB2 on body weight in db/db mice. Values are expressed as mean ± SD.
Asterisk (*) indicating a pb 0.05 versus group CC; pound sign (#) indicating a p b 0.05 versus group DM. CC, control db/m mice; DM, untreated db/db mice; DMT, db/db mice treated with GSPB2 (30 mg/kg per day for 10 weeks).
Fig. 2. Effect of GSPB2 on the level of FBG(A), AGEs(B), BUN(C), Cr(D) and urinary albumin(E) in db/db mice. Values are expressed as mean ± SD. Asterisk (*) indicating a pb 0.05 versus group CC; pound sign (#) indicating a pb 0.05 versus group DM.
protein profiles in kidneys of db/db mice. Furthermore, we investigated the molecular pathway of milk fat globule EGF-8 (MFG-E8), one of the identified up-regulated proteins in diabetic kidney identified by iTRAQ, through knocking down MFG-E8 and injecting exogenous recombinant MFG-E8 in db/db mice.
2. Materials and methods 2.1. Materials
The detailed information of the main materials is available in the Supplemental material.
2.2. Animals
Male C57BLKS/J db/db and their nondiabetic db/m littermates, (n = 24, 7 weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). All mice were kept at con-trolled temperature (23 ± 2 °C) and humidity (55 ± 5%) levels under an artificial light cycle, with free access to laboratory pellet chow and tap water. All experimental procedures were approved by the animal ethics committee of Shandong University. After adaptation for a week, db/m mice were selected as control group (CC, n = 8). Sixteen db/db mice were randomly divided into two groups: the vehicle-treated diabetic group (DM, n = 8) administered normal saline solution and the other diabetic group (DMT, n = 8) treated with GSPB2 (30 mg/kg body weight/day) in normal saline solution orally for 10 weeks. Animals
were weighed every week. At the end of the intervention, 24-h urine samples were collected from mice with metabolic cages to measure urinary albumin excretion. All mice were then sacrificed. Fasting blood was collected and perfused kidneys were dissected. The sera and tissues were kept at−80 °C until further analysis.
2.3. Estimation of fasting blood glucose (FBG), AGEs, blood urea nitrogen (BUN), plasma creatinine concentration (Cr) and urinary albumin excretion rate
The FBG, BUN, Cr were determined by DVI-1650 Automatic Biochemistry and Analysis Instrument (Bayer, Germany). AGE spe-cific fluorescence determinations were performed by measuring emission at 440 nm on excitation at 370 nm using afluorescence spectrophotometer (HITACHI F-2500, Japan). Urinary albumin levels were measured by a competitive ELISA, according to the manufacturer's instructions.
2.4. Proteomic analysis
In the experiment, kidneys of four mice per group were analyzed. Detailed experimental procedures were described in the Supplemen-tal material. In brief, renal proteins were digested, labeled with iTRAQ reagents (114 for the peptides of group CC, 117 for group DM, and 115 for group DMT), fractionated with Strong Cation Exchange (SCX) chromatography and analyzed on mass spectrometers. Fig. 3. Bioinformatic analysis of differentially expressed renal protein after treatment with GSPB2 in db/db mice. GO classification of proteins based on (A) cellular component and (B) molecular functions. (C) Interplaying network of proteins with abundance change generated by Ingenuity pathway analysis (IPA). The red and green nodes represent up-regulated and down-regulated genes or gene products in db/db mice kidney respectively. The edge represents the biological relationship between two nodes. The intensity of the node color indicates the abundance and the various shapes of the nodes represent the molecular class of the gene product.
2.5. Protein pathway analysis
Ingenuity pathway analysis (IPA) program (http://www.ingenuity. com) was applied to analyze the pathway of differentially expressed pro-teins identified by iTRAQ. The proteins with their ID in IPI database and corresponding abundance changes were uploaded as an Excel spread-sheetfile into the Ingenuity software. Each identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Ingenuity software made use of the data to extract interactive networks between the proteins. A score better than 2 is usually considered as a valid network.
2.6. Transfection of MFG-E8 shRNA and injection of recombinant MFG-E8 in db/db mice
Lentiviral vectors with short hairpin RNAs (LV–shRNAs) against MFG-E8 was chosen for silencing MFG-E8 gene expression, and lentiviral
vector with green fluorescence protein (LV–GFP) was used as the MFG-E8 RNAi control. Both MFG-E8 LV–shRNAs and LV–GFP were dilut-ed to a total volume of 300μl containing 4 × 107TU. Male C57BLKS/J
db/db and db/m mice (n = 40, 7 weeks old) were used in this study. C57BLKS/J db/m mice were selected as control group (CC, n = 8). Thirty-two db/db mice were randomly divided into four groups: an untreated diabetic group (DM, n = 8), LV–GFP transinfected db/db group (LV–GFP, n = 8), MFG-E8 shRNA transinfected db/db group (MRNAi, n = 8) and recombinant mouse MFG-E8 treated db/db group (reMFG-E8, n = 8). At the age of 14 weeks, sixteen db/db mice were transinfected with MFG-E8 shRNAs or LV–GFP by tail vein injection, respectively. Another 8 db/db mice were injected by 300μl of recombinant mouse MFG-E8 prediluted in PBS through tail vein at a dos-age of 20μg/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 °C until further analysis. Western blot-ting was applied to measure transfection efficiency of MFG-E8 shRNAs. Fig. 4. Effect of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the protein expression of MFG-E8 in db/db mice. A–C, representative Western blot picture showing protein expressions of MFG-E8; D–F, densitometric quantification of MFG-E8 by the NIH software ImageJ, values are expressed as mean ± SD. Asterisk (*) indicating a p b 0.05 versus group CC; pound sign (#) indicating a pb 0.05 versus group DM. CC, control db/m mice; DM, untreated db/db mice; DMT, db/db mice treated with GSPB2 (30 mg/kg per day for 10 weeks); LV–GFP, db/db mice transinfected with lentiviral vector with greenfluorescence protein; MRNAi, db/db mice transinfected with MFG-E8 shRNA; reMFG-E8, db/db mice injected recombinant MFG-E8 through tail vein at a dosage of 20μg/kg, twice a week for 4 weeks.
2.7. Histological examination
4% paraformaldehyde-fixed kidneys were embedded in paraffin, sectioned at 5μm, and stained with hematoxylin–eosin for histological examination under light microscopy.
For morphometric analysis of the glomeruli, sections were stained with periodic acid-Sciff (PAS). To quantify the mesangial expansion, ten glomeruli, randomly selected in the two sections from each mouse, were evaluated by an investigator unaware of the sections' origins. Mesangial matrix expansion was determined by the presence of PAS-positive and nuclei-free area in the mesangium. The glomerular area was also traced along the outline of the capillary loop using computer-assisted color image analyzer LUZEX F (Nikon, Tokyo, Japan). Relative mesangial area was shown as the ratio of mesangial area/glomerular area.
For ultrastructural evaluation, parts of kidneys werefixed in 3% glutaraldehyde in cacodylate buffer at 4 °C for 4 h and subsequently postfixated in a 1% osmium–tetroxide phosphate buffer solution for 1 h. Next, the samples were dehydrated in a graded ethanol series with acetone, permeated, and embedded in epoxide resin. Ultrathin sections were stained with uranylacetate and lead citrate, and exam-ined under an H-800 electron microscope (TEM, Hitachi Electronic Instruments, Tokyo, Japan).
2.8. Western blot analysis
Proteins from isolated kidneys of db/db mice were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The transferred membranes were incu-bated with blocking buffer (1 × PBS and 5% non-fat dry milk) for 1 h at room temperature and then probed with the primary antibody (described in Supplemental material) at 4 °C for 24 h. HRP-conjugated IgG were used as secondary antibodies and detected with enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The intensities were quantified with the NIH software ImageJ.β-Actin was used as loading control.
2.9. Statistical analysis
Statistic analysis was performed with SPSS 16.0 software. All exper-imental data were represented as mean ± SD and compared by using ANOVA followed by the post hoc test, least significant difference (LSD). pb 0.05 was accepted as statistically significant.
3. Results
3.1. Effect of GSPB2 on body weight, FBG, AGEs, BUN, Cr and urinary albumin excretion rate
Throughout the experimental periods, the db/db mice exhibited striking obesity compared with their nondiabetic db/m littermates. As shown inFig. 1, body weights were consistently greater in db/db mice than in db/m mice (pb 0.05). However, the increase of body weight in db/db mice was significantly attenuated from the second week after GSPB2 treatment (pb 0.05).
The effects of GSPB2 on the FBG, AGEs, BUN, Cr, and 24 h urinary albumin excretion rate were epitomized inFig. 2. At the end of experi-ment, levels of FBG, AGEs, BUN, Cr and urinary albumin excretion were remarkably elevated in db/db mice as compared to db/m mice. Treatment with GSPB2 to db/db mice induced a significant reduction in AGEs, BUN, Cr and urinary albumin excretion. However, GSPB2 treat-ment did not affect blood glucose levels.
3.2. Quantification and identification of the renal proteome by iTRAQ analysis
In total, 2842 proteins were identified to have different abundance between db/db mice and db/m mice. A strict cutoff value of a 1.5-fold change resulted in afinal set of 113 differentially expressed proteins, of which, 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 Fig. 5. Light microscopicfindings of renal morphological changes in db/db mice by hematoxylin–eosin (HE) staining at ×400 magnification. A, control db/m mice; B, untreated db/db mice; C, db/db mice treated with GSPB2 (30 mg/kg per day for 10 weeks); D, db/db mice transfected with LV–GFP; E, db/db mice transinfected with MFG-E8 shRNA; F, db/db mice injected with reMFG-E8.
treatment at a ratio of 0.47. The protein ID in IPI, name, molecular weight and main functions with abundance changes are epitomized in Supple-mental Table 1 and Table 2, respectively, Table 1 for down-regulated pro-teins and Table 2 for up-regulated propro-teins by GSPB2.
3.3. Bioinformatic functional analysis of differentially expressed renal proteins after GSPB2 treatment
To gain insights into the biological roles of the differential proteins in the effect of GSPB2 on diabetic nephropathy, the differentially expressed proteins were categorized into various location and function classes based on GO analysis according to UniProt protein knowledge database and literature search. In the cellular component of GO analysis, the larg-est proportion of differentially expressed proteins 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 a total of 113 proteins were located in the mitochondrion (Fig. 3A). The differentially expressed proteins were clustered into a number of cellular path-ways. Notably, the majority of changed proteins after treatment with GSPB2 were associated with protein or amino acid transport and metabolism, nearly accounting for 19% (Fig. 3B). The proteins involved in apoptosis/cell cycle/cell differentiation/proliferation and signal trans-duction constituted of another two prominent functional groups, occu-pying about 14% and 11%, respectively. Moreover, proteins involved in
oxidative stress, carbohydrate and lipid metabolism accounted for 7%, 6%, and 4%, respectively.
Fig. 3C depicts the primary protein network generated by the
ingenuity pathway analysis of the differentially expressed proteins. This network scored 50 and consisted of 35 proteins involved in apopto-sis, oxidative stress, and metabolism. Among these, twenty-two proteins were present in our iTRAQ list. In particular, MFG-E8, the protein mark-edly over-expressed in diabetic kidneys, is directly linked to extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in this network. The involvement of MFG-E8 in this network indicates that it may play an im-portant role in the pathogenesis of DN partly through ERK1/2 signaling pathway.
3.4. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the protein expression of MFG-E8 in db/db mice
Western blot was carried out to confirm the protein expression of MFG-E8 detected by iTRAQ and evaluate the transfection efficiency of MFG-E8 RNAi in db/db mice. As shown inFig. 4A and D, the protein ex-pression of MFG-E8 was significantly increased in db/db mice compared to db/m mice, and normalized by GSPB2, corresponding to that of iTRAQ.Fig. 4B and E indicates MFG-E8 shRNA successfully knocked down MFG-E8 protein level by ~60% in the kidneys of db/db mice. Con-versely, the injection of exogenous recombinant MFG-E8 remarkably Fig. 6. Morphological and quantitive analysis of mesangial expansion in db/db mice by periodic acid-Schiff staining (PAS) staining at ×400 magnification. A, control db/m mice; B, untreated db/db mice; C, db/db mice treated with GSPB2 (30 mg/kg per day for 10 weeks); D, db/db mice transfected with LV–GFP; E, db/db mice transinfected with MFG-E8 shRNA; F, db/db mice injected with reMFG-E8. a, mesangial area; b, glomerular area; c, relative mesangial area. Values are expressed as mean ± SD. Asterisk (*) indicating a pb 0.05 versus group CC; Pound sign (#) indicating a pb 0.05 versus group DM.
elevated MFG-E8 protein expression (pb 0.05) (Fig. 4C and F.). LV–GFP had no significant influence on the protein level of MFG-E8 (p > 0.05). Transfection of LV–GFP to db/db mice was also confirmed by fluorescent microscopic imaging, shown in Supplemental Fig. 1.
3.5. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the renal histology of db/db mice
The histological renal alterations of db/db mice subjected to GSPB2, MFG-E8 RNAi and reMFG-E8 treatment under light microscope are depicted inFig. 5.Fig. 5A shows the renal section of db/m mice demon-strating normal architecture with normal glomeruli.Fig. 5B portrays the renal section of the diabetic group exhibiting glomerular hypertrophy, mesangial expansion related to mesangial cell proliferation and matrix accumulation, along with visceral epithelial cell proliferation. These alterations in diabetic kidneys were ameliorated to near normalcy after treatments with GSPB2, shown inFig. 5C. LV–GFP transfected db/db mice showed the similar histological changes as db/db mice (Fig. 5D). Silencing MFG-E8 significantly suppressed glomerular hypertrophy and mesangial expansion in the kidneys of db/db mice (Fig. 5E). However, ex-ogenously added reMFG-E8 to db/db mice remarkably exacerbated renal lesions, characterized by notable glomerular hypertrophy, mesangial expansion and inflammatory cell accumulation (Fig. 5F).
3.6. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the renal mesangial expansion of db/db mice
Mesangial expansion characterized by an increase in PAS-positive mesangial matrix area was accelerated in the glomeruli of db/db mice (Fig. 6B), and significantly inhibited by treatment with GSPB2 (Fig. 6C). LV–GFP transfected db/db mice showed the similar mesangial expansion as db/db mice (Fig. 6D). MFG-E8 silencing decreased whereas
exogenously added reMFG-E8 remarkably increased the PAS-positive mesangial matrix area in the kidneys of db/db mice (Fig. 6E and F).
Quantitive analysis of mesangial expansion is presented inFig. 6A–C. The PAS positive area and total glomerular area in the glomeruli of db/db mice increased by 116% and 25%, respectively, compared with that in db/m mice (Fig. 6a and b). Treatment with GSPB2 significantly reduced the extent of mesangial expansion and total glomerular area in db/db mice, by 40% and 10%, respectively (Fig. 6a and b). MFG-E8 si-lencing significantly decreased whereas exogenously added reMFG-E8 remarkably increased the extent of mesangial expansion and total glomerular area in db/db mice, shown inFig. 6a and b. The relative mesangial area calculated by mesangial area/total glomerular area ratio increased by 200% in db/db mice as compared with db/m mice. Treat-ment with GSPB2 and MFG-E8 RNAi significantly ameliorated the increase in the relative mesangial area in db/db mice compared with that in untreated db/db mice (Fig. 6c).
3.7. Effects of GSPB2, MFG-E8 RNAi and recombinant MFG-E8 on the glomerular ultrastructure of db/db mice
The ultrastructural changes in kidneys of db/db mice subjected to GSPB2, MFG-E8 RNAi and reMFG-E8 treatment are shown inFig. 7A–F.
Fig. 7A represents the renal ultrastructure of db/m mice showing
the normal glomerular basement membranes (GBMs), podocytes and foot processes. Ultrastructural examination of kidneys of db/db mice re-vealed irregular thickened GBMs with segmental nail-like prominencies, deformed podocytes with less Golgi complexes and mitochondria, extensively fused foot processes and slit pores, presented inFig. 7B. Treatment with GSPB2 markedly reversed the ultrastructural changes of glomeruli in db/db mice (Fig. 7C). The db/db mice transfected by LV–GFP showed similar ultrastructural changes to those in diabetic mice (Fig. 7D).The diabetic alterations in kidneys of db/db mice were re-markably attenuated by MFG-E8 silencing (Fig. 7E), but exacerbated by
Fig. 7. Electron microscopicfindings of renal ultrastructural changes in db/db mice, ×15,000. A, control db/m mice; B, untreated db/db mice; C, db/db mice treated with GSPB2; D, db/db mice transfected with LV–GFP; E, db/db mice transinfected with MFG-E8 shRNA; F, db/db mice injected with reMFG-E8. BM, basement membrane; Cap, capillary; FE, fenes-trated endothelium; FP, foot processes; Go, Golgi complex; MC, mesangial cell; Mi, mitochondria; PC, podocytes.
injection of reMFG-E8 (Fig. 7F), consistent with the observations under light microscope.
3.8. Effects of GSPB2 and MFG-E8 on the phosphorylation of ERK1/2 in db/db mice
The phosphorylation level of ERK1/2 was remarkably higher in the kidneys of db/db mice than in db/m mice, and was significantly suppressed by treatment with GSPB2, (Fig. 8A). MFG-E8 silencing signif-icantly decreased the phosphorylation level of ERK1/2 by 60% compared with db/db mice (Fig. 8B). Conversely, exogenous MFG-E8 resulted in a significantly increased level of phospho-ERK1/2 in kidneys of db/db mice (Fig. 8C), while total ERK1/2 protein level had no significant differences among these groups. These results suggested that MFG-E8 activated phosphorylation of ERK1/2, and GSPB2 down-regulated the activation of ERK1/2 in diabetic kidney.
3.9. Effects of GSPB2 and MFG-E8 on the phosphorylation of Akt in db/db mice
The phosphorylation level of Akt was remarkably increased in the kidneys of db/db mice than in db/m mice. GSPB2 treatment significantly suppressed the phosphorylation of Akt (Fig. 9A). MFG-E8 silencing sig-nificantly decreased whereas exogenous MFG-E8 remarkably increased
the phosphorylation level of Akt, without significant differences on the level of total Akt (Fig. 9B and C). These results indicated that MFG-E8 activated phosphorylation of Akt, and GSPB2 suppressed phosphoryla-tion of Akt in diabetic kidney.
3.10. Effects of GSPB2 and MFG-E8 on the phosphorylation of GSK-3β in db/db mice
The phosphorylation of GSK-3β was notably increased in db/db mice compared to db/m mice, and was significantly suppressed by treatment with GSPB2, as shown inFig. 10A. Silencing the MFG-E8 gene remarkably decreased the level of phospho-GSK-3β, approximately by 50% (Fig. 10B). Oppositely, exogenous MFG-E8 remarkably increased the level of phospho-GSK-3β in db/db mice (Fig. 10C), while total GSK-3β protein level had no significant differences among these groups. These results suggested that MFG-E8 triggered the phosphorylation of GSK-3β, while GSPB2 down-regulated the phosphorylation of GSK-3β in diabetic kidney. 4. Discussion
DN is one of the major microvascular complications and accounts for a large proportion of patients under renal dialysis treatment. However, the current therapies for DN are limited to intensive insulin treatment, strict lipid control, reduction of blood hypertension and
Fig. 8. Effect of GSPB2 and MFG-E8 on the phosphorylation level of ERK1/2. A–C, representative Western blot picture showing expressions of p-ERK1/2 and total ERK1/2; D–F, densitometric quantification of p-ERK1/2 and total ERK1/2 by the NIH software ImageJ, values are expressed as mean ± SD. Asterisk (*) indicating a p b 0.05 versus group CC; pound sign (#) indicating a pb 0.05 versus group DM.
smoking cessation, which are important for preserving renal function but not sufficient to prevent renal deteriorative progression to ERSD
[2,8]. As numerous evidences have pointed to the role of oxidative stress in the development of DN[9–11], we investigated the effects of GSPB2, a natural antioxidant, on kidneys of db/db mice.
Our present results showed long-term oral treatment with GSPB2 significantly blocked the gain of body weight in db/db mice (Fig. 1), suggesting an anti-obesity effect of GSPB2 on type 2 diabetes. GSPB2 also prevented the increase in AGE level in db/db mice, suggesting an anti-non-enzymatic glycation effect of GSPB2. GSPB2 treatment in db/db mice normalized the deteriorated renal function indicated by increased serum levels of BUN, Cr, and 24 h urinary albumin (Fig. 2C, D, E). Both mesangial expansion characterized by PAS-positive area and GBM thick-ening were significantly suppressed by GSPB2 treatment in glomeruli of db/db mice (Figs. 5, 6, 7). GSPB2 reduced the glucose level of db/db mice, but not significantly, suggesting its renoprotective effects indepen-dent of anti-hyperglycemia.
Diabetic nephropathy is hallmarked by the onset of albuminuria and abnormal glomerularfiltration barrier, i.e., thickened GBMs, glomerular hypertrophy, mesangial expansion, and loss of podocytes[26]. Mesangial
expansion is regarded as one of the most striking characteristics of DN and strongly associated with ESRD in human diabetics[27–29]. The db/db mice, a rodent model for type 2 diabetes, could develop the path-ophysiological alterations and deteriorated renal functions closely simi-lar to that of human diabetic nephropathy[30,31]. In the current study, the elevated levels of BUN and Cr, increased albuminuria, and exacerbated glomerular histological alterations in db/db mice indicated the develop-ment of glomerular injury and renal dysfunction. Treatdevelop-ment with GSPB2 abrogated the renal dysfunction and mesangial expansion in db/db mice, suggesting that GSPB2 effectively reversed diabetic renal injury and prevented renal deteriorative progression to ERSD.
In diabetic kidney, accumulation of AGEs results in structural and functional alterations of some critical enzymes and proteins in mesangium and glomerular basement membranes[32]. Notably, AGEs binding to specific receptors (RAGEs) on podocytes, mesangial and tubular epithelial cells can activate intracellular signaling pathways leading to diverse consequences, including the generation of reactive oxygen species (ROS), activation of transcription factors and release of inflammatory cytokines[32–34]. In our present study, treatment with GSPB2 for 10 weeks significantly decreased the serum AGEs in db/db Fig. 9. Effect of GSPB2 and MFG-E8 on the phosphorylation level of Akt. A–C, representative Western blot picture showing expressions of p-Akt and total Akt; D–F, densitometric quantification of p-Akt and total Akt by the NIH software ImageJ, values are expressed as mean ± SD. Asterisk (*) indicating a p b 0.05 versus group CC; pound sign (#) indicating a pb 0.05 versus group DM.
mice (Fig. 2B). Previously, GSPE has been reported to ameliorate DN by decreasing serum level of AGEs and renal protein expression of RAGE
[21]. In this context, we deduced GSPB2 elicited its renoprotective effects partly due to the decrease of serum AGE level.
ERK1/2 is a member of the mitogen-activated protein kinases (MAPKs) family, which responds to various extracellular stimuli such as ROS, modulating cell growth, proliferation, apoptosis and essential biosynthetic functions[35]. Activation of ERK1/2 was involved in in-flammation and oxidative stress, leading to mesangial expansion in diabetic nephropathy[36–40]. In our present study, GSPB2 treatment inhibited the phosphorylation of ERK1/2 in kidneys of db/db mice (Fig. 8A). The phosphoinositide 3-kinase (PI3K)/Akt pathway is a cen-tral signaling cascade whose activation leads to cellular proliferation, anti-apoptotic responses, enhanced metabolism, and inflammatory response[41]. Activation of Akt has been reported to play a pivotal role in the mesangial cell survival, oxidant stress, tubular hypertrophy and mesangial expansion[42–44]. In our study, activation of Akt in kidneys of db/db mice was inhibited by GSPB2 treatment (Fig. 9A). We also found that phosphorylation of GSK-3β in kidneys of db/db mice was suppressed by GSPB2 treatment (Fig. 10A). GSK-3β, as a downstream molecule of ERK1/2 and Akt, is a constitutively active serine/threonine kinase inactivated via phosphorylation on the resi-due serine-9, accounting for a variety of biological events, including
embryonic development, cell differentiation, apoptosis, and insulin response [45]. In cultured renal mesangial cell, phosphorylation of GSK-3β mediates IGF-1-induced mesangial cell-cycle progression, hyper-trophy and extracellular matrix protein synthesis[46]. In our current study, GSPB2 treatment inhibited the phosphorylation of GSK-3β, along with EKR1/2 and Akt (Fig. 8A, 9A, 10A) in kidneys of db/db mice. Hence, we deduced that GSPB2 attenuated diabetic renal injury by regu-lation of EKR1/2, Akt and GSK-3β signaling pathway.
iTRAQ analysis identified fifty-three down-regulated and sixty up-regulated proteins after GSPB2 treatment in kidneys of db/db mice. The targeting proteins with differential abundance (Supplemental Table 1 and Table 2) played pivotal roles in a number of cellular pathways, in-cluding material metabolism related to glucose/amino acid/lipid/nucleic acid metabolism, apoptosis, oxidative stress, cell cycle and proliferation, signal transduction, and cell adhesion. In our previous study, 2-D dif-ference gel electrophoresis (2-D DIGE) has revealed that six oxidative stress proteins were back-regulated by GSPE treatment in kidneys of STZ-induced diabetic rats[20]. Although the exact mechanism by which GSPB2 reaches its intracellular targets is unclear, the targeting proteins of GSPB2 are supposed to participate in the deterioration and restoration of DN, and provide novel targets for future therapy.
MFG-E8 stood out in our proteomic analysis because it had not pre-viously been linked directly with diabetic nephropathy, although it has Fig. 10. Effect of GSPB2 and MFG-E8 on the phosphorylation level of GSK-3β. A–C, representative Western blot picture showing expressions of p-GSK-3β and total GSK-3β; D–F, densitometric quantification of p-GSK-3β and total GSK-3β by the NIH software ImageJ, values are expressed as mean ± SD. Asterisk (*) indicating a p b 0.05 versus group CC; pound sign (#) indicating a pb 0.05 versus group DM.
been shown to over-express in aorta of chronic kidney disease (CKD) rats and STZ-induced diabetic rats[22,47]. MFG-E8 is a membrane-associated glycoprotein inducing cell-specific apoptosis and inflamma-tion response[48,49], and plays a diverse biorole in various pathophys-iologic conditions, including angiogenesis, aging, tissue remodel, and tumor genesis[50–52]. In patients with CKD, elevated serum MFG-E8 level is positively associated with deteriorated renal function and increased aortic stiffness[47]. Actually, in our previous study, MFG-E8 has been shown to abundantly express in aorta of STZ-induced diabetic rats and decrease to normal after GSPE treatment[22], indicating it may be involved in the pathogenesis of diabetic vascular complications. Our present iTRAQ analysis (Supplemental Table 1) and Western blot analysis (Fig. 4A) identified that MFG-E8 was significantly elevated in diabetic kidneys, and reduced after GSPB2 treatment. Knockdown of MFG-E8 gene significantly suppressed whereas exogenous MFG-E8 exacerbated glomerular hypertrophy, mesangial expansion and thick-ening basement in db/db mice (Figs. 5, 6, 7). These results demonstrated the detrimental role of MFG-E8 in the development of diabetic nephrop-athy. Our previous vitro study has reported that protein expression of MFG-E8 was dose-dependently inhibited by GSPB2 treatment in the apoptotic process of the cultured human umbilical endothelial cells
[24]. We presumed that GSPB2 dose-dependently down-regulated the protein expression of MFG-E8 by regulating the transcription and translation of MFG-E8, after binding to specific receptors located on cell membranes.
We established the underlying molecular mechanism by which MFG-E8 accelerates diabetic kidney injury, and uncovered that MFG-E8 acted by activation of ERK1/2, Akt and GSK-3β signaling pathway. Knocking down the MFG-E8 gene remarkably suppressed whereas over-expression elevated the phosphorylation of ERK1/2, Akt and GSK-3β (Figs. 8, 9, 10). ERK1/2 acting as a downstream molecular of MFG-E8 has been reported to participate in the MFG-E8-induced inva-sion and proliferation of VSMC in aging aorta [52,53]. Actually, the links between MFG-E8, ERK1/2 and Akt signaling have been implied in the protein pathway of IPA (Fig. 3C). Both ERK1/2 and Akt pathway were activated in diabetic glomeruli mediating collagen I and MCP-1 upregulation in mesangial cells and type 2 diabetic mice[54,55]. GSK-3β is a pivotal convergence point of PI3K/Akt and ERK1/2[56,57]. Increased phospho-GSK-3β, phosphop-ERK1/2 and phospho-Akt were found in kidneys of STZ-induced diabetic rats, related with the progres-sion of DN[58]. In our previous study, GSK-3β was also found to medi-ate the apoptosis of endothelial cells induced by MFG-E8[24]. In the present study, MFG-E8 silencing inhibited whereas over-expression of MFG-E8 elevated the phosphorylation of ERK1/2, Akt, and GSK-3β (Figs. 8, 9, 10). Hence, we deduced that MFG-E8 may aggravate diabetic renal injury partially by phosphorylation of ERK1/2, Akt and GSK-3β.
Taken together, our research identified MFG-E8 along with activation of ERK1/2, Akt, and GSK-3β signaling pathway contributed to the patho-genesis of diabetic nephropathy for thefirst time. GSPB2 performed potent protective effects on DN by inhibition of MFG-E8, phosphoryla-tion of ERK1/2, Akt, and GSK-3β signaling pathway, although the molec-ular mechanism by which GSPB2 down-regulates these molecules in diabetic kidneys is unclear. 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. Acknowledgements
This work was supported by funds from the National Natural Science Foundation of China (30873145, 81000340, 81100595), Outstanding Young Scientist Research Award Fund of Shandong Province (BS2009YY046), China Postdoctoral Science Foundation (20100471520, 2011M500748), Science and Technology Development Plan of Shandong Provincial Medicine (2011HZ048) and Natural Science Foundation of Shandong Province (Y2008C100, ZR2010HQ067). The authors acknowl-edge the personnel of the Medical Science Academy of Shandong and
the personnel of the Research Center for the proteome analysis, and the Shanghai Institute for Biological Sciences, and the Chinese Academy of Sciences for their technical support. We also thank Professor Jun-Hui Zhen for helping with the histological examination.
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