- Email: [email protected]
WITHDRAWN: Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis
Accepted Manuscript Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis Mei Zhao, Lei Zhang, Lin Liu, Ya Li, Di Wang, Yi-Hui Li, Zhi-Hao Wang, Wei Zhang, Yun Zhang, Ming Zhong, Meng-Xiong Tang PII:
S0006-291X(16)31431-0
DOI:
10.1016/j.bbrc.2016.08.167
Reference:
YBBRC 36368
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 15 August 2016 Accepted Date: 29 August 2016
Please cite this article as: M. Zhao, L. Zhang, L. Liu, Y. Li, D. Wang, Y.-H. Li, Z.-H. Wang, W. Zhang, Y. Zhang, M. Zhong, M.-X. Tang, Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/ j.bbrc.2016.08.167. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis
Zhang1, Yun Zhang1, Ming Zhong1, Meng-Xiong Tang*1,2 1
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Mei Zhao1,4, Lei Zhang1, Lin Liu1,3 , Ya Li1, Di Wang1, Yi-Hui Li, Zhi-Hao Wang1,3, Wei
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese
Key
Laboratory
of
Translational
Cardiovascular
Medicine, Department
of
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Joint
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Ministry of Education and Chinese Ministry of Health; The State and Shandong Province
Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China 2
Department of Emergency, Qilu Hospital of Shandong University, Jinan, Shandong, China
3
Department of Geriatric Medicine, Qilu Hospital of Shandong University, Jinan, Shandong,
Department of Cardiology Huangdao District People's Hospital, Qingdao Shandong, China
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4
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China
* Correspondence
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Meng-Xiong Tang, Department of Emergency, Qilu Hospital of Shandong University, No.107 Wenhua West Road, Ji’nan 250012, P.R.China.
Email: [email protected]
ACCEPTED MANUSCRIPT Abstract Background: renal fibrosis is a process of excess accumulation and deposition of extracellular matrix (ECM) proteins in kidney. Transforming growth factor-beta (TGF-β) plays a vital role
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in accumulation of ECM through initiating phosphorylated Smad2/3. Whether activin receptor-like kinase 7 (ALK7), a member of TGF-β superfamily, was involved in renal interstitial fibrosis remains unclear. The aim of this study was to investigate whether ALK7
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participates in renal fibrosis via regulating phosphorylated Smad2/3 in high-fat diet (HFD)
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rats.
Methods: Forty male Sprague-Dawley (SD) rats were randomly assigned to 4 groups: control, HFD, HFD Vehicle and HFD ALK7-siRNA. The metabolic index, ALK7 expression and collagen deposition in renal tissue were measured, and renal fibrosis was evaluated by stained
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with hematoxylin and eosin, Masson's trichrome and periodic acid-Schiff. Results: HFD induced insulin resistant state accompanied by increased blood glucose, cholesterol and triglyceride levels in rats. HFD enhanced the expression of ALK7 which
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involved the renal fibrosis, which was accompanied by increased phosphorylated Smad2/3.
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Additionally, ALK7 gene silencing reduced renal fibrosis along with decreased Smad2/3 phosphorylation.
Conclusion: ALK7 gene silencing plays a protective role in renal fibrosis in rats with HFD and might be an effective treatment target of HFD-induced renal fibrosis.
Key words activin receptor-like kinase 7; renal fibrosis; high fat diet; insulin resistance; Smad2/3
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Introduction Renal fibrosis including tubulointerstitial fibrosis and glomerulosclerosis[1,2] is the common
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manifestation of Chronic kidney disease (CKD). Renal fibrosis is characterized by the excessive accumulation and deposition of extracellular matrix (ECM) components such as collagen IV, the major constituent in renal tissue. A long-term high-fat diet (HFD) plays an
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important role in the pathogenesis of renal fibrosis[3], but the regulatory mechanisms
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underlying these events remain elusive.
It has been reported that transforming growth factor-beta (TGF-β) had an important effect in the development and progression of tissue fibrosis[4,5], which was mainly through activating Smad family (Smad 2, 3 and 4)[6,7]. Activin receptor-like kinase 7 (ALK7), a member of
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TGF-β superfamily, can induce the activation of Smads or other molecules and further participated in cell proliferation, differentiation and apoptosis[8,9,10]. ALK7 has been confirmed to be associated with body fat change, carbohydrate metabolism and lipid in
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adipose tissue[11], which exacerbated the progression of renal fibrosis. Additionally, ALK7,
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together with ALK5 and ALK6, has been identified as the potent mediator to regulate renal interstitial fibrosis[12]. There was study showing that fibrosis process induced by Angiotensin II and other profibrogenic factors was correlated with Smad2/3 signaling pathway[13]. Besides that, our former study suggested that ALK7 mediates high glucose-induced cardiomyoblast apoptosis through activation of Smad2/3[14]. However, whether ALK7 is involved in HFD-induced renal fibrosis via regulating Smad2/3 is unclear. A long-term HFD can cause the insulin resistance[15], and insulin resistance plays a vital role
ACCEPTED MANUSCRIPT in promoting the process of renal fibrosis. In HFD-induced insulin resistance state, whether ALK7 downregulation could delay the pathophysiologic proceeding of renal fibrosis need to be confirmed. Additionally, we wonder whether ALK7 is complicated in the process of renal
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fibrosis by regulating phosphorylation levels of Smad2/3 in a rat model with long-term HFD.
Materials and Methods
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1. Animals
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The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (Document No. 55, 2001) and was approved by the Animal Care and Use Committee of Shandong University. Forty male Sprague-Dawley (SD) rats (120-140 g) were purchased from the experimental animal center of Shandong University of
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Traditional Chinese Medicine (Ji'nan, China). The animals were kept at the temperature of 22 °C with 12-h light-dark cycles. After 1 week of acclimatization, the rats were randomly assigned to 2 groups: control group (normal chow; 20% protein, 3% fat, 3% fiber, 74%
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carbohydrates; Beijing HFK Bio-Technology) and HFD group (high-fat diet; 34.5% fat,
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17.5% protein, 48% carbohydrates; Beijing HFK Bio-Technology). The HFD group was further randomized into 3 subgroups (n=10 each): HFD group, Vehicle group for injection at week 17 with empty virus and ALK7-siRNA group for injection at week 17 with ALK7-siRNA adenovirus. Animals were injected at a dosage of 2.5*1010 plaque-forming units of adenovirus or vehicle via the jugular vein. Adenovirus transfer was repeated in 2 weeks. The rats were killed at the 22nd week. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US
ACCEPTED MANUSCRIPT National Institutes of Health and approved by the Ethics Committee for Animal Research of Shandong Province in China. 2. Blood analyses
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We collected blood samples after rats overnight fasting. We used the Bayer 1650 blood chemistry analyzer (Bayer, Tarrytown, NY) to test total cholesterol (TC), triglyceride (TG), and fasting blood glucose (FBG) and used enzyme-linked immunosorbent assay to analyze fasting insulin (FINS) levels. The insulin sensitivity index (ISI) = [ln(FBG*fasting insulin) 1]
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−
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was calculated. 3. Histology and morphometric analysis
At the end of the experiment, we killed the rats and excised the kidney tissue. Tissues were fixed in 4% paraformaldehyde and made into 4-µm sections for hematoxylin and eosin (H&E)
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and Masson's trichrome staining or 3-µm sections for periodic acid-Schiff (PAS) staining. Interstitialfibrosis was calculated and evaluated by dividing the area of trichrome-stained interstitium by total cortical area. Glomerulosclerosis was expressed as the ratio of the total
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area of PAS-positive staining in the glomeruli/total area of the glomeruli. ImagePro Plus v5.0
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was used to evaluate the interstitialfibrosis and glomerulosclerosis. 4. Immunohistochemical staining Sections were immunoblotted with antibody for collagen IV (all 1:1000; Abcam) for 4°C overnight. Then, they were incubated with secondary antibody for 30 min at room temperature. After incubation with DAB solution (Beijing Zhong Shan Golden Bridge Biological Technology, China), the sections were counterstained with hematoxylin. We used a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan) and Image-Pro Plus 5.0
ACCEPTED MANUSCRIPT software to view and analyze the results, respectively. 5. Western blot analysis Protein samples of kidney tissue was separated by SDS-PAGE and transferred to
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polyvinylidene difluoride membrane. Membranes were immunoblotted with antibodies for ALK7 (monoclonal antibody; R&D, Minneapolis, MN, USA), phospho-Smad2 or phospho-Smad3 (Cell Signaling Technology, Beverly, MA, USA) for overnight. After
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incubated with horseradish peroxidase-conjugated secondary antibody (1:4000), the
6. Statistical analysis
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immunoreactive bands were visualized by using enhanced chemiluminescence (ECL).
Data are reported as mean ± SD and analyzed by software SPSS 18.0. Differences of groups were compared using the One-way ANOVA Analysis or Student's t-test.
Results
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considered statistically significant.
p<0.05 was
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1. Metabolism of rats with HFD
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At the end of the experiment, TC, TG, FBG and FINS levels of HF group were remarkably higher than those of control group (TC: 4.51 ± 0.95 vs. 1.24 ± 0.12; TG: 4.58 ± 0.49 vs. 0.44 ± 0.07; FBG: 19.19 ± 1.21 vs. 5.74 ± 0.92; FINS: 16.41 ± 0.48 vs. 13.64 ± 0.78; respectively, Fig. 1A-D). While the ISI decreased and the IRI increased in HF group compared with the control ( (ISI: −5.75 ± 0.09 vs. −4.35 ± 0.11; IRI: 14.01 ± 1.26 vs. 3.46 ± 0.38; respectively, Fig. 1E-F). So HFD could induce insulin resistance in rat. 2. Pathologic features of rats with HFD
ACCEPTED MANUSCRIPT Renal fibrosis was embodied in tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis and collagen deposition in tubular compartments and glomeruli was more obvious in HF group than control group (Fig. 2A1-2; Fig. 3A1-2 and C1-2). Tubulointerstitial fibrosis of
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HF group was significantly greater than that of control group (CVF%: 3.38 ± 0.20 vs. 0.59 ± 0.21; P<0.05, Fig. 3A1-2), and HFD-induced mesangial expansion in glomeruli revealed stronger compared to that of control group (Glomerulus' sclerosis index%: 12.67 ±1.17 vs.
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5.57 ± 0.71; P<0.05, Fig. 3C1-2). In summary, the data above suggested that HFD is closely
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related to renal fibrosis. 3. Activated ALK7/Smads signaling in rats with HFD
In Figure 4A-B, there was a significant increase in the level of renal ALK7 protein expression in HF group. Along with the up-regulation of ALK7, the phosphorylated Smad2 and Smad3
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were also increased (Fig. 4C, D and E). Meanwhile, HFD elevated the protein expression of collagen IV compared with control group (Fig. 4F-G). 4. Effect of ALK7 silencing on metabolism and insulin sensitivity
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ALK7 siRNA transfection had no notable adverse effects and no deaths in rats. The levels of
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TC, TG, FBG and FINS were significantly lower in ALK7 siRNA group than vehicle treatment group (TC: 3.30 ± 0.46 vs. 5.13 ± 0.59; TG: 3.33 ± 0.38 vs. 4.94 ± 1.36; FBG: 13.58 ± 1.04 vs. 19.93 ± 1.60; FINS: 13.23 ± 1.03 vs. 16.07 ± 0.73; respectively, Fig. 1A-D). In addition, ISI was significantly elevated and IRI was significantly decreased in ALK7 siRNA in comparison to vehicle treatment (ISI: −5.19 ± 0.11 vs. −5.77 ± 0.10; IRI: 7.98 ± 0.82 vs. 14.24 ± 1.47; respectively, Fig. 1E-F). In conclusion, ALK7 silencing might play a vital role in improving insulin resistance.
ACCEPTED MANUSCRIPT 5. Effect of ALK7 silencing on renal fibrosis Under the intervention of ALK7 siRNA, the alleviation was found in renal fibrosis (Fig. 2A3-4). Tubulointerstitial fibrosis and glomerulosclerosis were ameliorated respectively with
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ALK7 siRNA than vehicle treatment (CVF%: 1.27 ± 0.16 vs. 3.13 ± 0.16; P<0.05, Fig. 3A3-4; Glomerulus' sclerosis index%: 6.77 ±0.55 vs. 12.40 ± 0.96; P<0.05, Fig. 3C3-4). In a word, ALK7 silencing could improve renal fibrosis.
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6. ALK7 silencing down-regulated the expressions of phosphorylated Smad2/3 and collagen
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IV.
With ALK7 siRNA treatment, the protein expression of ALK7 exhibited lower level than the vehicle treatment (Fig. 4A and B). While phosphorylated Smad2/3 and collagen IV were significantly reduced respectively in HF group with TRIB3 siRNA than vehicle (Fig. 4C-G).
Discussion
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fibrosis in HFD rats.
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So, we may conclude that ALK7/Smad2 and 3 participate in the collagen synthesis of renal
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In the present study, we found that ALK7 participated in the progression of HFD-induced rat renal fibrosis. Insulin resistance-upregulated ALK7 accelerated collagen synthesis and renal fibrosis probably via mediating phosphorylated Smad2/3. However, ALK7 gene silencing ameliorated collagen deposition and renal fibrosis by downregulating Smad2/3 signaling. Renal fibrosis, characterized by tubulointerstitial fibrosis and glomerulosclerosis[1,2], revealed the accumulation and deposition of ECM protein[16]. Insulin resistance exerted an crucial effect on the pathogenesis of renal fibrosis, and could be induced by a long-term
ACCEPTED MANUSCRIPT HFD[15]. In our study, we successfully built rat insulin-resistant model by HFD with the characteristics of increased TC, TG, FBG and FINS and decreased ISI. It has been proved that TGF-β system contributed to the development of renal fibrosis and
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TGF-β/Smads was a critical signaling pathway that participated in collagen synthesis and renal fibrosis[3]. Previous researches[12] reported that ALK7 as a member of TGF-β superfamily was relevant to the rat renal interstitial fibrosis. In HFD rats, the level of renal
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ALK7 protein expression was increased significantly in comparison to control rats.
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Concomitant with the overexpression of ALK7, the phosphorylated Smad2 and Smad3 were also increased. Consistent with the previous study[17], we demonstrated that in an insulin-resistant state, there were the excessive collagen deposition and tubulointerstitial fibrosis and glomerulosclerosis in rat renal tissue. Therefore, the activation of ALK7 might be
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inclined to promoting the progression of renal fibrosis via elevating the phosphorylation levels of Smad2 and Smad3 in insulin-resistance. There were studies revealing that ALK7 promoted fat accumulation and exacerbated insulin
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resistance by GDF3 pathway[18]. The mutation of ALK7 in mice resulted in the decrease of
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fat accumulation, and improved glucose tolerance and insulin sensitivity[19]. Our previous research revealed that ALK7 gene polymorphism is related closely to metabolic syndrome in Chinese females[20]. In our study, we found that the levels of TC, TG, FBG and FINS were significantly lower and insulin resistance was significantly improved in HF rats with ALK7 siRNA than vehicle treatment. Based on the theory that ALK7 aggravated insulin resistance-induced renal fibrosis, we speculated that downregulation of ALK7 could reverse the progression of renal fibrosis. So
ACCEPTED MANUSCRIPT we used ALK7 siRNA adenovirus to keep ALK7 gene silencing in rats. Protein expression of ALK7 was decreased in ALK7 siRNA than vehicle treatment, and ALK7 silencing abolished the increased phosphorylation levels of Smad2 and Smad3 induced by HFD. Simultaneously,
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ALK7 gene silencing alleviated the renal collagen deposition and pathological changes including tubulointerstitial fibrosis and glomerulosclerosis. The data above suggest that ALK7 silencing could delay renal fibrosis in insulin-resistant state via down-regulating
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phosphorylation levels of Smad2 and Smad3. ALK7/Smad2 and 3 was recognized as the
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crucial signaling pathway in insulin resistance-induced renal fibrosis. So ALK7/Smad2 and 3 signaling pathway might be an effective treatment target which prevented renal fibrosis
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induced by insulin resistance.
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the research grants from the National Natural Science Foundation of China (81070192, 81100605, 81270352, 81270287, 81300168, 81471036,
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81470560 and 81570400), the Natural Science Foundation of Shandong Province (ZR2014HQ037), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP 20130131120065), and Key research and development program of
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Shandong Province (2015GSF118062)
ACCEPTED MANUSCRIPT Abbreviations ECM, extracellular matrix; TGF-β, Transforming growth factor-beta; ALK7, activin receptor-like kinase 7; HFD, high-fat diet; CKD, Chronic kidney disease; TC, total
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cholesterol; TG, triglyceride; FBG, fasting blood glucose; FINS, fasting insulin; ISI, insulin sensitivity index; IRI, insulin resistance index; H&E, hematoxylin and eosin; PAS, periodic
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acid-Schiff.
ACCEPTED MANUSCRIPT References [1] F. Eitner, J. Floege, Novel insights into renal fibrosis, Curr Opin Nephrol Hypertens 12 (2003) 227-232.
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[2] G. Remuzzi, T. Bertani, Pathophysiology of progressive nephropathies, N Engl J Med 339 (1998) 1448-1456.
[3] J. Lu, N. Bankovic-Calic, M. Ogborn, M.H. Saboorian, H.M. Aukema, Detrimental
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Han:SPRD-cy rats, J Nutr 133 (2003) 180-186.
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effects of a high fat diet in early renal injury are ameliorated by fish oil in
[4] E.P. Bottinger, TGF-beta in renal injury and disease, Semin Nephrol 27 (2007) 309-320. [5] S. Rosenkranz, TGF-beta1 and angiotensin networking in cardiac remodeling, Cardiovasc Res 63 (2004) 423-432.
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[6] M. Kretzschmar, J. Massague, SMADs: mediators and regulators of TGF-beta signaling, Curr Opin Genet Dev 8 (1998) 103-111. [7] C.M. Zimmerman, R.W. Padgett, Transforming growth factor beta signaling mediators
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and modulators, Gene 249 (2000) 17-30.
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[8] F. Zhao, F. Huang, M. Tang, X. Li, N. Zhang, A. Amfilochiadis, Y. Li, R. Hu, T. Jin, C. Peng, Q. Wang, Nodal induces apoptosis through activation of the ALK7 signaling
pathway in pancreatic INS-1 beta-cells, Am J Physiol Endocrinol Metab 303 (2012) E132-143.
[9] P. Bertolino, R. Holmberg, E. Reissmann, O. Andersson, P.O. Berggren, C.F. Ibanez, Activin B receptor ALK7 is a negative regulator of pancreatic beta-cell function, Proc Natl Acad Sci U S A 105 (2008) 7246-7251.
ACCEPTED MANUSCRIPT [10] O. Andersson, M. Korach-Andre, E. Reissmann, C.F. Ibanez, P. Bertolino, Growth/differentiation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity, Proc Natl Acad Sci U S A 105 (2008)
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7252-7256. [11] L.M. Carlsson, P. Jacobson, A. Walley, P. Froguel, L. Sjostrom, P.A. Svensson, K. Sjoholm, ALK7 expression is specific for adipose tissue, reduced in obesity and
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correlates to factors implicated in metabolic disease, Biochem Biophys Res Commun
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382 (2009) 309-314.
[12] N. Shen, H. Lin, T. Wu, D. Wang, W. Wang, H. Xie, J. Zhang, Z. Feng, Inhibition of TGF-beta1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis, Kidney Int 84 (2013) 64-77.
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[13] T.H. Lan, X.Q. Huang, H.M. Tan, Vascular fibrosis in atherosclerosis, Cardiovasc Pathol 22 (2013) 401-407.
[14] L. Liu, W.Y. Ding, J. Zhao, Z.H. Wang, M. Zhong, W. Zhang, Y.G. Chen, Y. Zhang, L.
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Li, M.X. Tang, Activin receptor-like kinase 7 mediates high glucose-induced H9c2
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cardiomyoblast apoptosis through activation of Smad2/3, Int J Biochem Cell Biol 45 (2013) 2027-2035.
[15] L. Kang, J.E. Ayala, R.S. Lee-Young, Z. Zhang, F.D. James, P.D. Neufer, A. Pozzi, M.M. Zutter, D.H. Wasserman, Diet-induced muscle insulin resistance is associated with
extracellular matrix remodeling and interaction with integrin alpha2beta1 in mice, Diabetes 60 (2011) 416-426. [16] Y. Liu, Renal fibrosis: new insights into the pathogenesis and therapeutics, Kidney Int 69
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96 (2014) 80-84. [18] S. Yogosawa, T. Izumi, Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor gamma, in lean and obese adipocytes,
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Adipocyte 2 (2013) 246-250.
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[19] S. Yogosawa, S. Mizutani, Y. Ogawa, T. Izumi, Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor gamma and C/EBPalpha, Diabetes 62 (2013) 115-123. [20] W. Zhang, H. Wang, W. Zhang, R. Lv, Z. Wang, Y. Shang, Y. Zhang, M. Zhong, Y. Chen,
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M. Tang, ALK7 gene polymorphism is associated with metabolic syndrome risk and
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cardiovascular remodeling, Arq Bras Cardiol 101 (2013) 134-140.
ACCEPTED MANUSCRIPT Figure legends Figure. 1. Serum markers for control, high-fat (HF) diet, vehicle group and ALK7-siRNA group. A: total cholesterol (TC), B: triglyceride (TG), C: fasting blood glucose (FBG), D:
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fasting insulin (FINS) concentrations and E: insulin sensitivity index (ISI), and F: insulin resistance (IRI) are shown. Data are mean ± SD. n = 6 per group. *p<0.05 vs. control; and #
p<0.05 vs. HFD + Vehicle.
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Figure. 2. Pathologic features of rats fed with a high-fat diet and injected with ALK7-siRNA.
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A1-4: Kidney sections were stained with hematoxylin and eosin (H&E; scale bar: 50 mm). B. The body weight at the ends of the experiments. Data are mean ± SD. n = 6 per group. *
p<0.05 vs. Control.
Fig. 3. Tubulointerstitial fibrosis and Glomerulosclerosis in rats fed with a high-fat diet and
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injected with ALK7-siRNA. A1-4: Masson's trichrome staining shows tubulointerstitial fibrosis (scale bar: 50 mm). B. Quantitative analysis of collagen volume fraction (CVF%). C1-4: periodic acid-Schiff (PAS) staining shows glomerulosclerosis (scale bar: 20 mm). D.
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Quantitative analysis of glomerulus sclerosis index (%). Data are mean ± SD. n = 6 per group. p<0.05 vs. control; and #p<0.05 vs. HFD + Vehicle.
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*
Fig. 4. ALK7/Smads signaling is involved in HFD-induced collagen synthesis in rats. Representative
immunoblots
with
anti-ALK7,
anti-phosphorylated-Smad2
and
anti-phosphorylated-Smad3 antibodies are shown (A and C). Western blot analyses of protein level of ALK7 (B), phosphorylated-Smad2 (D), and phosphorylated-Smad3 (E). F: Representative immunohistochemical staining showing collagen IV (scale bar: 50 mm). G: Immunohistochemical analysis of collagen IV. Data are mean ± SD. n = 6. *p<0.05 vs.
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control; and #p<0.05 vs. HFD + Vehicle.
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ACCEPTED MANUSCRIPT Highlights 1. Activation of ALK7 was inclined to promoting the progression of renal fibrosis in insulin-resistance;
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2. ALK7 involved progression of renal fibrosis via elevating the phosphorylation levels of Smad2 and Smad3;
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3. Downregulation of ALK7 could reverse the progression of renal fibrosis.
ACCEPTED MANUSCRIPT Conflicts of interest
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There are no conflicts of interest.
S0006-291X(16)31431-0
DOI:
10.1016/j.bbrc.2016.08.167
Reference:
YBBRC 36368
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 15 August 2016 Accepted Date: 29 August 2016
Please cite this article as: M. Zhao, L. Zhang, L. Liu, Y. Li, D. Wang, Y.-H. Li, Z.-H. Wang, W. Zhang, Y. Zhang, M. Zhong, M.-X. Tang, Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/ j.bbrc.2016.08.167. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Protective role of activin receptor-like kinase 7 gene silencing in renal fibrosis
Zhang1, Yun Zhang1, Ming Zhong1, Meng-Xiong Tang*1,2 1
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Mei Zhao1,4, Lei Zhang1, Lin Liu1,3 , Ya Li1, Di Wang1, Yi-Hui Li, Zhi-Hao Wang1,3, Wei
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese
Key
Laboratory
of
Translational
Cardiovascular
Medicine, Department
of
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Joint
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Ministry of Education and Chinese Ministry of Health; The State and Shandong Province
Cardiology, Qilu Hospital of Shandong University, Jinan, Shandong, China 2
Department of Emergency, Qilu Hospital of Shandong University, Jinan, Shandong, China
3
Department of Geriatric Medicine, Qilu Hospital of Shandong University, Jinan, Shandong,
Department of Cardiology Huangdao District People's Hospital, Qingdao Shandong, China
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4
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China
* Correspondence
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Meng-Xiong Tang, Department of Emergency, Qilu Hospital of Shandong University, No.107 Wenhua West Road, Ji’nan 250012, P.R.China.
Email: [email protected]
ACCEPTED MANUSCRIPT Abstract Background: renal fibrosis is a process of excess accumulation and deposition of extracellular matrix (ECM) proteins in kidney. Transforming growth factor-beta (TGF-β) plays a vital role
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in accumulation of ECM through initiating phosphorylated Smad2/3. Whether activin receptor-like kinase 7 (ALK7), a member of TGF-β superfamily, was involved in renal interstitial fibrosis remains unclear. The aim of this study was to investigate whether ALK7
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participates in renal fibrosis via regulating phosphorylated Smad2/3 in high-fat diet (HFD)
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rats.
Methods: Forty male Sprague-Dawley (SD) rats were randomly assigned to 4 groups: control, HFD, HFD Vehicle and HFD ALK7-siRNA. The metabolic index, ALK7 expression and collagen deposition in renal tissue were measured, and renal fibrosis was evaluated by stained
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with hematoxylin and eosin, Masson's trichrome and periodic acid-Schiff. Results: HFD induced insulin resistant state accompanied by increased blood glucose, cholesterol and triglyceride levels in rats. HFD enhanced the expression of ALK7 which
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involved the renal fibrosis, which was accompanied by increased phosphorylated Smad2/3.
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Additionally, ALK7 gene silencing reduced renal fibrosis along with decreased Smad2/3 phosphorylation.
Conclusion: ALK7 gene silencing plays a protective role in renal fibrosis in rats with HFD and might be an effective treatment target of HFD-induced renal fibrosis.
Key words activin receptor-like kinase 7; renal fibrosis; high fat diet; insulin resistance; Smad2/3
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Introduction Renal fibrosis including tubulointerstitial fibrosis and glomerulosclerosis[1,2] is the common
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manifestation of Chronic kidney disease (CKD). Renal fibrosis is characterized by the excessive accumulation and deposition of extracellular matrix (ECM) components such as collagen IV, the major constituent in renal tissue. A long-term high-fat diet (HFD) plays an
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important role in the pathogenesis of renal fibrosis[3], but the regulatory mechanisms
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underlying these events remain elusive.
It has been reported that transforming growth factor-beta (TGF-β) had an important effect in the development and progression of tissue fibrosis[4,5], which was mainly through activating Smad family (Smad 2, 3 and 4)[6,7]. Activin receptor-like kinase 7 (ALK7), a member of
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TGF-β superfamily, can induce the activation of Smads or other molecules and further participated in cell proliferation, differentiation and apoptosis[8,9,10]. ALK7 has been confirmed to be associated with body fat change, carbohydrate metabolism and lipid in
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adipose tissue[11], which exacerbated the progression of renal fibrosis. Additionally, ALK7,
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together with ALK5 and ALK6, has been identified as the potent mediator to regulate renal interstitial fibrosis[12]. There was study showing that fibrosis process induced by Angiotensin II and other profibrogenic factors was correlated with Smad2/3 signaling pathway[13]. Besides that, our former study suggested that ALK7 mediates high glucose-induced cardiomyoblast apoptosis through activation of Smad2/3[14]. However, whether ALK7 is involved in HFD-induced renal fibrosis via regulating Smad2/3 is unclear. A long-term HFD can cause the insulin resistance[15], and insulin resistance plays a vital role
ACCEPTED MANUSCRIPT in promoting the process of renal fibrosis. In HFD-induced insulin resistance state, whether ALK7 downregulation could delay the pathophysiologic proceeding of renal fibrosis need to be confirmed. Additionally, we wonder whether ALK7 is complicated in the process of renal
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fibrosis by regulating phosphorylation levels of Smad2/3 in a rat model with long-term HFD.
Materials and Methods
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1. Animals
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The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (Document No. 55, 2001) and was approved by the Animal Care and Use Committee of Shandong University. Forty male Sprague-Dawley (SD) rats (120-140 g) were purchased from the experimental animal center of Shandong University of
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Traditional Chinese Medicine (Ji'nan, China). The animals were kept at the temperature of 22 °C with 12-h light-dark cycles. After 1 week of acclimatization, the rats were randomly assigned to 2 groups: control group (normal chow; 20% protein, 3% fat, 3% fiber, 74%
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carbohydrates; Beijing HFK Bio-Technology) and HFD group (high-fat diet; 34.5% fat,
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17.5% protein, 48% carbohydrates; Beijing HFK Bio-Technology). The HFD group was further randomized into 3 subgroups (n=10 each): HFD group, Vehicle group for injection at week 17 with empty virus and ALK7-siRNA group for injection at week 17 with ALK7-siRNA adenovirus. Animals were injected at a dosage of 2.5*1010 plaque-forming units of adenovirus or vehicle via the jugular vein. Adenovirus transfer was repeated in 2 weeks. The rats were killed at the 22nd week. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US
ACCEPTED MANUSCRIPT National Institutes of Health and approved by the Ethics Committee for Animal Research of Shandong Province in China. 2. Blood analyses
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We collected blood samples after rats overnight fasting. We used the Bayer 1650 blood chemistry analyzer (Bayer, Tarrytown, NY) to test total cholesterol (TC), triglyceride (TG), and fasting blood glucose (FBG) and used enzyme-linked immunosorbent assay to analyze fasting insulin (FINS) levels. The insulin sensitivity index (ISI) = [ln(FBG*fasting insulin) 1]
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−
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was calculated. 3. Histology and morphometric analysis
At the end of the experiment, we killed the rats and excised the kidney tissue. Tissues were fixed in 4% paraformaldehyde and made into 4-µm sections for hematoxylin and eosin (H&E)
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and Masson's trichrome staining or 3-µm sections for periodic acid-Schiff (PAS) staining. Interstitialfibrosis was calculated and evaluated by dividing the area of trichrome-stained interstitium by total cortical area. Glomerulosclerosis was expressed as the ratio of the total
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area of PAS-positive staining in the glomeruli/total area of the glomeruli. ImagePro Plus v5.0
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was used to evaluate the interstitialfibrosis and glomerulosclerosis. 4. Immunohistochemical staining Sections were immunoblotted with antibody for collagen IV (all 1:1000; Abcam) for 4°C overnight. Then, they were incubated with secondary antibody for 30 min at room temperature. After incubation with DAB solution (Beijing Zhong Shan Golden Bridge Biological Technology, China), the sections were counterstained with hematoxylin. We used a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan) and Image-Pro Plus 5.0
ACCEPTED MANUSCRIPT software to view and analyze the results, respectively. 5. Western blot analysis Protein samples of kidney tissue was separated by SDS-PAGE and transferred to
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polyvinylidene difluoride membrane. Membranes were immunoblotted with antibodies for ALK7 (monoclonal antibody; R&D, Minneapolis, MN, USA), phospho-Smad2 or phospho-Smad3 (Cell Signaling Technology, Beverly, MA, USA) for overnight. After
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incubated with horseradish peroxidase-conjugated secondary antibody (1:4000), the
6. Statistical analysis
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immunoreactive bands were visualized by using enhanced chemiluminescence (ECL).
Data are reported as mean ± SD and analyzed by software SPSS 18.0. Differences of groups were compared using the One-way ANOVA Analysis or Student's t-test.
Results
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considered statistically significant.
p<0.05 was
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1. Metabolism of rats with HFD
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At the end of the experiment, TC, TG, FBG and FINS levels of HF group were remarkably higher than those of control group (TC: 4.51 ± 0.95 vs. 1.24 ± 0.12; TG: 4.58 ± 0.49 vs. 0.44 ± 0.07; FBG: 19.19 ± 1.21 vs. 5.74 ± 0.92; FINS: 16.41 ± 0.48 vs. 13.64 ± 0.78; respectively, Fig. 1A-D). While the ISI decreased and the IRI increased in HF group compared with the control ( (ISI: −5.75 ± 0.09 vs. −4.35 ± 0.11; IRI: 14.01 ± 1.26 vs. 3.46 ± 0.38; respectively, Fig. 1E-F). So HFD could induce insulin resistance in rat. 2. Pathologic features of rats with HFD
ACCEPTED MANUSCRIPT Renal fibrosis was embodied in tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis and collagen deposition in tubular compartments and glomeruli was more obvious in HF group than control group (Fig. 2A1-2; Fig. 3A1-2 and C1-2). Tubulointerstitial fibrosis of
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HF group was significantly greater than that of control group (CVF%: 3.38 ± 0.20 vs. 0.59 ± 0.21; P<0.05, Fig. 3A1-2), and HFD-induced mesangial expansion in glomeruli revealed stronger compared to that of control group (Glomerulus' sclerosis index%: 12.67 ±1.17 vs.
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5.57 ± 0.71; P<0.05, Fig. 3C1-2). In summary, the data above suggested that HFD is closely
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related to renal fibrosis. 3. Activated ALK7/Smads signaling in rats with HFD
In Figure 4A-B, there was a significant increase in the level of renal ALK7 protein expression in HF group. Along with the up-regulation of ALK7, the phosphorylated Smad2 and Smad3
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were also increased (Fig. 4C, D and E). Meanwhile, HFD elevated the protein expression of collagen IV compared with control group (Fig. 4F-G). 4. Effect of ALK7 silencing on metabolism and insulin sensitivity
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ALK7 siRNA transfection had no notable adverse effects and no deaths in rats. The levels of
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TC, TG, FBG and FINS were significantly lower in ALK7 siRNA group than vehicle treatment group (TC: 3.30 ± 0.46 vs. 5.13 ± 0.59; TG: 3.33 ± 0.38 vs. 4.94 ± 1.36; FBG: 13.58 ± 1.04 vs. 19.93 ± 1.60; FINS: 13.23 ± 1.03 vs. 16.07 ± 0.73; respectively, Fig. 1A-D). In addition, ISI was significantly elevated and IRI was significantly decreased in ALK7 siRNA in comparison to vehicle treatment (ISI: −5.19 ± 0.11 vs. −5.77 ± 0.10; IRI: 7.98 ± 0.82 vs. 14.24 ± 1.47; respectively, Fig. 1E-F). In conclusion, ALK7 silencing might play a vital role in improving insulin resistance.
ACCEPTED MANUSCRIPT 5. Effect of ALK7 silencing on renal fibrosis Under the intervention of ALK7 siRNA, the alleviation was found in renal fibrosis (Fig. 2A3-4). Tubulointerstitial fibrosis and glomerulosclerosis were ameliorated respectively with
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ALK7 siRNA than vehicle treatment (CVF%: 1.27 ± 0.16 vs. 3.13 ± 0.16; P<0.05, Fig. 3A3-4; Glomerulus' sclerosis index%: 6.77 ±0.55 vs. 12.40 ± 0.96; P<0.05, Fig. 3C3-4). In a word, ALK7 silencing could improve renal fibrosis.
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6. ALK7 silencing down-regulated the expressions of phosphorylated Smad2/3 and collagen
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IV.
With ALK7 siRNA treatment, the protein expression of ALK7 exhibited lower level than the vehicle treatment (Fig. 4A and B). While phosphorylated Smad2/3 and collagen IV were significantly reduced respectively in HF group with TRIB3 siRNA than vehicle (Fig. 4C-G).
Discussion
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fibrosis in HFD rats.
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So, we may conclude that ALK7/Smad2 and 3 participate in the collagen synthesis of renal
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In the present study, we found that ALK7 participated in the progression of HFD-induced rat renal fibrosis. Insulin resistance-upregulated ALK7 accelerated collagen synthesis and renal fibrosis probably via mediating phosphorylated Smad2/3. However, ALK7 gene silencing ameliorated collagen deposition and renal fibrosis by downregulating Smad2/3 signaling. Renal fibrosis, characterized by tubulointerstitial fibrosis and glomerulosclerosis[1,2], revealed the accumulation and deposition of ECM protein[16]. Insulin resistance exerted an crucial effect on the pathogenesis of renal fibrosis, and could be induced by a long-term
ACCEPTED MANUSCRIPT HFD[15]. In our study, we successfully built rat insulin-resistant model by HFD with the characteristics of increased TC, TG, FBG and FINS and decreased ISI. It has been proved that TGF-β system contributed to the development of renal fibrosis and
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TGF-β/Smads was a critical signaling pathway that participated in collagen synthesis and renal fibrosis[3]. Previous researches[12] reported that ALK7 as a member of TGF-β superfamily was relevant to the rat renal interstitial fibrosis. In HFD rats, the level of renal
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ALK7 protein expression was increased significantly in comparison to control rats.
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Concomitant with the overexpression of ALK7, the phosphorylated Smad2 and Smad3 were also increased. Consistent with the previous study[17], we demonstrated that in an insulin-resistant state, there were the excessive collagen deposition and tubulointerstitial fibrosis and glomerulosclerosis in rat renal tissue. Therefore, the activation of ALK7 might be
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inclined to promoting the progression of renal fibrosis via elevating the phosphorylation levels of Smad2 and Smad3 in insulin-resistance. There were studies revealing that ALK7 promoted fat accumulation and exacerbated insulin
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resistance by GDF3 pathway[18]. The mutation of ALK7 in mice resulted in the decrease of
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fat accumulation, and improved glucose tolerance and insulin sensitivity[19]. Our previous research revealed that ALK7 gene polymorphism is related closely to metabolic syndrome in Chinese females[20]. In our study, we found that the levels of TC, TG, FBG and FINS were significantly lower and insulin resistance was significantly improved in HF rats with ALK7 siRNA than vehicle treatment. Based on the theory that ALK7 aggravated insulin resistance-induced renal fibrosis, we speculated that downregulation of ALK7 could reverse the progression of renal fibrosis. So
ACCEPTED MANUSCRIPT we used ALK7 siRNA adenovirus to keep ALK7 gene silencing in rats. Protein expression of ALK7 was decreased in ALK7 siRNA than vehicle treatment, and ALK7 silencing abolished the increased phosphorylation levels of Smad2 and Smad3 induced by HFD. Simultaneously,
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ALK7 gene silencing alleviated the renal collagen deposition and pathological changes including tubulointerstitial fibrosis and glomerulosclerosis. The data above suggest that ALK7 silencing could delay renal fibrosis in insulin-resistant state via down-regulating
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phosphorylation levels of Smad2 and Smad3. ALK7/Smad2 and 3 was recognized as the
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crucial signaling pathway in insulin resistance-induced renal fibrosis. So ALK7/Smad2 and 3 signaling pathway might be an effective treatment target which prevented renal fibrosis
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induced by insulin resistance.
ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the research grants from the National Natural Science Foundation of China (81070192, 81100605, 81270352, 81270287, 81300168, 81471036,
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81470560 and 81570400), the Natural Science Foundation of Shandong Province (ZR2014HQ037), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP 20130131120065), and Key research and development program of
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Shandong Province (2015GSF118062)
ACCEPTED MANUSCRIPT Abbreviations ECM, extracellular matrix; TGF-β, Transforming growth factor-beta; ALK7, activin receptor-like kinase 7; HFD, high-fat diet; CKD, Chronic kidney disease; TC, total
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cholesterol; TG, triglyceride; FBG, fasting blood glucose; FINS, fasting insulin; ISI, insulin sensitivity index; IRI, insulin resistance index; H&E, hematoxylin and eosin; PAS, periodic
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acid-Schiff.
ACCEPTED MANUSCRIPT References [1] F. Eitner, J. Floege, Novel insights into renal fibrosis, Curr Opin Nephrol Hypertens 12 (2003) 227-232.
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[2] G. Remuzzi, T. Bertani, Pathophysiology of progressive nephropathies, N Engl J Med 339 (1998) 1448-1456.
[3] J. Lu, N. Bankovic-Calic, M. Ogborn, M.H. Saboorian, H.M. Aukema, Detrimental
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Han:SPRD-cy rats, J Nutr 133 (2003) 180-186.
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effects of a high fat diet in early renal injury are ameliorated by fish oil in
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[6] M. Kretzschmar, J. Massague, SMADs: mediators and regulators of TGF-beta signaling, Curr Opin Genet Dev 8 (1998) 103-111. [7] C.M. Zimmerman, R.W. Padgett, Transforming growth factor beta signaling mediators
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ACCEPTED MANUSCRIPT [10] O. Andersson, M. Korach-Andre, E. Reissmann, C.F. Ibanez, P. Bertolino, Growth/differentiation factor 3 signals through ALK7 and regulates accumulation of adipose tissue and diet-induced obesity, Proc Natl Acad Sci U S A 105 (2008)
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[13] T.H. Lan, X.Q. Huang, H.M. Tan, Vascular fibrosis in atherosclerosis, Cardiovasc Pathol 22 (2013) 401-407.
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Li, M.X. Tang, Activin receptor-like kinase 7 mediates high glucose-induced H9c2
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cardiomyoblast apoptosis through activation of Smad2/3, Int J Biochem Cell Biol 45 (2013) 2027-2035.
[15] L. Kang, J.E. Ayala, R.S. Lee-Young, Z. Zhang, F.D. James, P.D. Neufer, A. Pozzi, M.M. Zutter, D.H. Wasserman, Diet-induced muscle insulin resistance is associated with
extracellular matrix remodeling and interaction with integrin alpha2beta1 in mice, Diabetes 60 (2011) 416-426. [16] Y. Liu, Renal fibrosis: new insights into the pathogenesis and therapeutics, Kidney Int 69
ACCEPTED MANUSCRIPT (2006) 213-217. [17] W.Y. Ding, W.B. Li, Y. Ti, X.P. Bi, H. Sun, Z.H. Wang, Y. Zhang, W. Zhang, M. Zhong, Protection from renal fibrosis, putative role of TRIB3 gene silencing, Exp Mol Pathol
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96 (2014) 80-84. [18] S. Yogosawa, T. Izumi, Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor gamma, in lean and obese adipocytes,
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Adipocyte 2 (2013) 246-250.
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[19] S. Yogosawa, S. Mizutani, Y. Ogawa, T. Izumi, Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor gamma and C/EBPalpha, Diabetes 62 (2013) 115-123. [20] W. Zhang, H. Wang, W. Zhang, R. Lv, Z. Wang, Y. Shang, Y. Zhang, M. Zhong, Y. Chen,
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M. Tang, ALK7 gene polymorphism is associated with metabolic syndrome risk and
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cardiovascular remodeling, Arq Bras Cardiol 101 (2013) 134-140.
ACCEPTED MANUSCRIPT Figure legends Figure. 1. Serum markers for control, high-fat (HF) diet, vehicle group and ALK7-siRNA group. A: total cholesterol (TC), B: triglyceride (TG), C: fasting blood glucose (FBG), D:
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fasting insulin (FINS) concentrations and E: insulin sensitivity index (ISI), and F: insulin resistance (IRI) are shown. Data are mean ± SD. n = 6 per group. *p<0.05 vs. control; and #
p<0.05 vs. HFD + Vehicle.
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Figure. 2. Pathologic features of rats fed with a high-fat diet and injected with ALK7-siRNA.
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A1-4: Kidney sections were stained with hematoxylin and eosin (H&E; scale bar: 50 mm). B. The body weight at the ends of the experiments. Data are mean ± SD. n = 6 per group. *
p<0.05 vs. Control.
Fig. 3. Tubulointerstitial fibrosis and Glomerulosclerosis in rats fed with a high-fat diet and
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injected with ALK7-siRNA. A1-4: Masson's trichrome staining shows tubulointerstitial fibrosis (scale bar: 50 mm). B. Quantitative analysis of collagen volume fraction (CVF%). C1-4: periodic acid-Schiff (PAS) staining shows glomerulosclerosis (scale bar: 20 mm). D.
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Quantitative analysis of glomerulus sclerosis index (%). Data are mean ± SD. n = 6 per group. p<0.05 vs. control; and #p<0.05 vs. HFD + Vehicle.
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*
Fig. 4. ALK7/Smads signaling is involved in HFD-induced collagen synthesis in rats. Representative
immunoblots
with
anti-ALK7,
anti-phosphorylated-Smad2
and
anti-phosphorylated-Smad3 antibodies are shown (A and C). Western blot analyses of protein level of ALK7 (B), phosphorylated-Smad2 (D), and phosphorylated-Smad3 (E). F: Representative immunohistochemical staining showing collagen IV (scale bar: 50 mm). G: Immunohistochemical analysis of collagen IV. Data are mean ± SD. n = 6. *p<0.05 vs.
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control; and #p<0.05 vs. HFD + Vehicle.
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ACCEPTED MANUSCRIPT Highlights 1. Activation of ALK7 was inclined to promoting the progression of renal fibrosis in insulin-resistance;
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2. ALK7 involved progression of renal fibrosis via elevating the phosphorylation levels of Smad2 and Smad3;
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3. Downregulation of ALK7 could reverse the progression of renal fibrosis.
ACCEPTED MANUSCRIPT Conflicts of interest
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There are no conflicts of interest.