ERK signaling in apolipoprotein E-deficient mice

Peptides 79 (2016) 49–57

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Peptides journal homepage: www.elsevier.com/locate/peptides

Angiotensin-converting enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in apolipoprotein E-deficient mice Lai-Jiang Chen a,b,1 , Ying-Le Xu a,1 , Bei Song a , Hui-Min Yu c , Gavin Y. Oudit d , Ran Xu a , Zhen-Zhou Zhang a,b , Hai-Yan Jin a,e , Qing Chang a , Ding-Liang Zhu a,b , Jiu-Chang Zhong a,b,∗ a State Key Laboratory of Medical Genomics, Pôle Sino-Franc¸ais de Recherches en Science du Vivant et Génomique, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension, Shanghai 200025, China b Institute of Health Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200025, China c Department of Cardiology, Guangdong General Hospital, Guangdong Academy of Medical Sciences and Guangdong Cardiovascular Institute, Guangzhou 510080, China d Division of Cardiology, Department of Medicine, University of Alberta, Mazankowski Alberta Heart Institute, Edmonton T6G 2S2, Canada e Department of Mental Health, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China

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Article history: Received 21 January 2016 Received in revised form 6 March 2016 Accepted 23 March 2016 Available online 24 March 2016 Keywords: Angiotensin-converting enzyme 2 mTOR Renal fibrosis Apolipoprotein E Atherosclerosis

a b s t r a c t Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF␤1 and collagen I and elevated renal Ang-(1–7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100 nmol L−1 ) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-␤1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1 mg mL−1 ) or mTOR inhibitor rapamycin (10 ␮mol L−1 ). These protective effects of rhACE2 were eradicated by the Ang-(1–7)/Mas receptor antagonist A779 (1 ␮mol L−1 ). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1–7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders. © 2016 Elsevier Inc. All rights reserved.

1. Introduction More than a century since the discovery of renin, the reninangiotensin system (RAS) remains a fascinating subject for

∗ Corresponding author at: 197 Ruijin 2nd Road, State Key Laboratory of Medical Genomics & Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. E-mail addresses: [email protected], [email protected] (J.-C. Zhong). 1 Equal contributors. http://dx.doi.org/10.1016/j.peptides.2016.03.008 0196-9781/© 2016 Elsevier Inc. All rights reserved.

research. Through the actions of its principal effector peptide, angiotensin (Ang) II, the activation of RAS plays an important role in the enhanced susceptibility to atherosclerotic lesions, renal inflammation and chronic kidney diseases (CKD) [1–3]. Based on its ability to degrade the vasoconstrictor Ang II and produce the vasodilator Ang-(1–7), angiotensin-converting enzyme 2 (ACE2) has been suggested to limit pathophysiologic activation of the RAS [4,5]. Activation of the ACE2/Ang-(1–7)/Mas axis is a possible alternative target for new drugs, since some beneficial effects on renal and cardiovascular function have been reported [5–8]. Studies in the kidney disease of patients with diabetes have shown

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decreased ACE2 expression and increases in the ACE/ACE2 ratio in both tubulointerstitium and glomerulus, contributing to the progression of renal injury [9]. The apolipoprotein E (ApoE) knockout (KO) mouse is a well-accepted clinically relevant animal model for human atherosclerosis [10,11]. ApoE-deficient mice developed progressive kidney lesions, pronounced endothelial dysfunction, and severe hyperlipidemia resembling human type III hyperlipidemia [10–13]. Wen et al. [11] identified a striking pattern of renal injury in ApoEKO mice, characterized by glomerular macrophage infiltration with accumulation of foam cells, foci of mesangiolysis, focal intracapillary lipid deposits that resemble the human lesion of lipoprotein glomerulopathy. These findings provide a basis for the use of ApoEKO mouse as a model to define mechanisms of atherosclerotic renal injury. We and others have previously shown that genetic ACE2 deficiency exacerbates renal inflammation and atherosclerotic kidney injury in the ApoEKO mouse with larger vascular lesions in aortic atherosclerotic plaques [1,12,13] while kidney inflammation and oxidant injury is dramatically lessened by human recombinant ACE2 (rhACE2) treatment in the Ang IImediated hypertensive mice or ApoEKO mice [1,5], suggesting that ACE2 is a key modulator of atherosclerosis and kidney injury. However, little is known about the potential effects of ACE2 on renal fibrosis in arteriosclerotic cardiovascular disease. The current studies were designed to assess if ACE2 can influence tubulointerstitial fibrosis and fibrosis-associated signaling in the atherosclerosisprone ApoEKO mice. 2. Methods 2.1. Experimental animals and protocols This study was approved and conducted in the accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996) and the Guidelines for Animal Research Ethics Committee at Shanghai Jiao Tong University School of Medicine. The ApoEKO mice were backcrossed into the C57BL/6 background as previously described [1,7]. The 12-week-old male mice were used for all the experiments. The following 5 groups were created: (1) wild-type (WT) control group (n = 8); (2) ApoEKO control group (n = 8); (3) ApoEKO + rhACE2 group (n = 6); (4) ApoEKO + Ang II group (n = 8), and (5) ApoEKO + Ang II + rhACE2 group (n = 6). The ApoEKO mice were used to undergo in vivo either Ang II infusion at a dose of 1.5 mg/kg body weight daily or saline (Vehicle) for 2 weeks. The Ang II-infused ApoEKO mice were then daily treated with placebo or rhACE2 (2 mg/kg, intraperitoneal) as previously described [1,14]. Systolic blood pressure (SBP) levels of mice were measured non-invasively using the tail-cuff method. All plasma determinations were performed using a Beckman CX7 chemistry analyzer for total cholesterol (CHO) and triglycerides (TG). Ang II and Ang-(1–7) levels in renal cortex were measured by radioimmunoassay as previously described [1]. Mice were housed in the animal quarters with 12:12 h light-dark cycle and had access to standard chow and water ad libitum. Mice were anesthetized with ketamine (80 mg/kg) and xylaxine (10 mg/kg) in a prone position on an animal tray. 2.2. Quantitative real-time PCR and Western blotting analysis The renal mRNA expression of fibrosis-related genes was evaluated by quantitative real-time reverse transcription PCR as previously described [1]. Total RNA was extracted from flashfrozen renal cortex tissues using TRIzol reagent (Invitrogen, CA). The cDNA was synthesized by using the PrimeScript RT reagent kit (TAKARA). A SYBR Premix ExTaq II (TAKARA) was used

to perform the quantitative real-time PCR in ABI 7900T Real Time System (Applied. Biosystems, CA). The sequences of the primers were as follows: mouse transforming growth factor-␤1 (TGF␤1): forward 5 -GCATCCCACCTTTGCCGAG-3 , reverse 5 CACGGGAGTGGGAGCAGAA-3 ; collagen I: forward 5 -ACCCCGCCGATGTCGCTAT-3 , reverse 5 -GGAGGTCTTGGTGGTTTTGT-3 ; collagen III: forward 5 -CCCACAGCCTTCTACACCT-3 , reverse 5 CAGGGTCACCATTTCTCC-3 ; fibronectin 1 forward 5 -CACCCGTGAAGAATGAAGA-3 , reverse 5 -GGCAGGAGATTTGTTAGGA-3 and GAPDH: forward 5 -TGCGACTTCAACAGCAACTC-3 , reverse 5 ATGTAGGCCA-TGAGGTCCAC-3 . GAPDH was used as an endogenous control. All samples were run in triplicates. Standard blotting protocols were followed to examine phosphorylation signaling in renal tissues as previously described [4,15] The primary antibody against ACE2, p-Akt (Ser473 ), Akt, p-mTOR, mTOR, p-ERK1/2, ERK1/2, and GAPDH were obtained from R&D Systems (Minneapolis, MN), Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. ␤-actin was used as an endogenous control. Image Quant TL software was used for quantification of bands.

2.3. Masson’s trichrome staining and transmission electron microscope analysis To evaluate tubulointerstitial fibrosis, Masson’s Trichrome staining was performed by using five-␮m thick formalin-fixed and paraffin-embedded sections from the mouse renal cortex as before [16]. For transmission electron microscope (TEM) analysis, mouse renal cortex tissues were immediately cut into small pieces and prefixed in 2.5% glutaraldehyde as previously described [1]. The renal tubular ultrastructure was observed on a PHILIPS CM-120 Transmission Electron-Microscope (Holland) with magnifications 4200× and 13500×.

2.4. Culture of mouse kidney fibroblasts and measurement of superoxide production Primary mouse kidney fibroblasts (MKFs) were obtained from the 6-week-old C57BL/6 mice using the regular method reported by Lonnemann et al. [17]. The protocol of the study was approved by Animal Research Ethics Committee at Shanghai Jiao Tong University School of Medicine. Briefly, kidneys were dissected longitudinally, and then carefully cut off the cortex with a surgical blade. MKFs were dissociated from the cortex, plated onto Petri dishes in DMEM containing 15% fetal bovine serum (FBS), 100 U/ml penicillin, 100 ␮g/ml streptomycin and 2 ␮g/ml ␣-FGF and cultured in a 5% CO2 humidified incubator at 37 ◦ C. All experiments were performed on MKFs at passages 3–6. Before each experiment, MKFs were incubated in 1% FBS-DMEM for 24 h to minimize seruminduced effects. The rhACE2 (1 mg mL−1 ), Ang-(1–7)/Mas receptor antagonist A779 (1 ␮mol L−1 ; Sigma, MO), ACE2 inhibitor DX600 (0.5 ␮mol L−1 ), ERK1/2 inhibitor PD98059 (10 ␮mol L−1 ), mTOR inhibitor rapamycin (10 ␮mol L−1 ) were added to MKFs for 30 min or 2 h prior to 30-min exposure of Ang II (100 nmol L−1 ; Sigma, MO), respectively. The treated cells were further collected for TaqMan Real-time PCR, Western blotting analysis and dihydroethidium (DHE) staining. Oxidative stress is generally identified by indirect markers of the oxidant injury, such as superoxide. To evaluate superoxide production in MKFs, the oxidative fluorescent dye DHE was used as described previously [1,15]. Confocal laser scanning microscopy was used to test the identification for vimentin (green) and ␣-SMA (red) in MKFs by using multi-fluorescent method. Upon nuclear stain and mount in antifade medium containing 4 ,6 -diamidino-2-phenylindole (DAPI, blue), immunofluorescence

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Table 1 The general data in mice.

n SBP (mm Hg) KW(mg) BW (g) KW/BW ratio (mg/g) plasma TG (mmol/L) plasma CHO (mmol/L)

WTC

ApoEKO

ApoEKO+rhACE2

ApoEKO+Ang II

ApoEKO + Ang II +rhACE2

8 101 ± 3.5 138 ± 3.8 25.1 ± 1.2 5.56 ± 0.18 0.59 ± 0.05 1.48 ± 0.11

8 104 ± 2.1 135 ± 2.8 25.5 ± 0.9 5.34 ± 0.12 1.18 ± 0.07* 6.83 ± 0.47**

6 103 ± 4.3 132 ± 4.4 24.9 ± 1.7 5.38 ± 0.24 1.15 ± 0.08 6.72 ± 0.48

8 161 ± 5.6** , ## 179 ± 6.0** , ## 25.9 ± 1.3 6.96 ± 0.21** , ## 1.25 ± 0.10** 8.16 ± 0.35**

6 130 ± 3.5† 146 ± 4.6† 25.2 ± 1.0 5.83 ± 0.21† 1.19 ± 0.11 7.91 ± 0.53

SBP, systolic blood pressure; BW, body weight; KW, kidney weight; TG, triacylglycerol; CHO, cholesterol; Ang II, angiotensin II; KO, knockout; WTC, wild-type control mice; ApoE, apolipoprotein E; ACE2, angiotensin-converting enzyme 2; rhACE2, recombinant human ACE2. Results are presented as mean ± SEM. * P < 0.05 compared with WTC group. ** P < 0.01 compared with WTC group. ## P < 0.01 compared with ApoEKO control group. † P < 0.05 compared with ApoEKO + Ang II group.

Fig. 1. The levels of ACE2, Ang II and Ang-(1–7) in mice kidneys. (A and B) Representative Western blot analysis exhibiting ACE2 protein levels in the mice kidneys. ␤-actin was used as an endogenous control. (C–E) The renal levels of Ang II (C) and Ang-(1–7) (D) and the ratio of Ang-(1–7)/Ang II (E) in the kidney cortex of mice. A.U., arbitrary units; WTC, wildtype control; ApoE, Apolipoprotein E; KO, knockout; rhACE2, human recombinant ACE2; Ang II, angiotensin II;. n = 5–6. * , P < 0.05; ** , P < 0.01 compared with WT group; # , P < 0.05, compared with ApoEKO group; ␾, P < 0.05, compared with ApoEKO + Ang II group.

images were acquired using a confocal laser scanning microscope (TCS SP2; Leica Microsystems AG, Germany).

3. Results 3.1. Treatment with rhACE2 prevents Ang II-induced hypertension and kidney remodeling in the ApoEKO mice with enhanced renal Ang-(1–7) levels

2.5. Statistical analysis Normally distributed data were expressed as mean ± SEM. Statistical analysis was performed using SPSS software (Version 16.0) either by Student’s t-test for comparison between groups or by ANOVA followed by the Student–Newman–Keuls test for multiple comparison testing as appropriate. Statistical significance was accepted at P < 0.05.

Compared with WT control mice, there were significant increases in plasma total CHO and TG concentrations (Table 1) and decreases in ACE2 protein (Fig. 1) in the kidneys obtained from the ApoEKO mice. There were no changes in SBP levels, kidney weight (KW), and renal Ang II and Ang-(1–7) levels between the WT and ApoEKO mice (Table 1 and Fig. 1). Gravimetric analysis showed enhanced KW and KW/body weight (BW) ratio in Ang IItreated ApoEKO mice compared with the ApoEKO mice (Table 1). Intriguingly, Ang II infusion resulted in marked elevations in SBP

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Fig. 2. Renal phosphorylated levels of Akt, mTOR and ERK1/2 in mice. The Western blot analysis showed the phosphorylated levels of Akt (Ser473 ; A, D), mTOR (B, E) and ERK1/2 (C, F) in mice kidneys. n = 5 for each group. A.U., arbitrary units; WTC, wild-type control; ApoE, Apolipoprotein E; KO, knockout; rhACE2, human recombinant ACE2; Ang II, angiotensin II; ERK1/2, extracellular signal-regulated kinase 1/2. * , P < 0.05; ** , P < 0.01 compared with WT control group or ApoEKO control group; # , P < 0.05 compared with ApoEKO + Ang II group.

and renal Ang II levels in the ApoE-mutant mice with accompanying decreases in renal ACE2 and Ang-(1–7)/Ang II ratio (Table 1 and Fig. 1). Treatment with rhACE2 had a minimal effect on renal ACE2 protein, Ang-(1–7) levels, Ang-(1–7)/Ang II ratio and circulating lipid levels in the ApoEKO mice (Table 1 and Fig. 1). Finally, rhACE2 administration remarkably prevented Ang II-induced increase in systolic blood pressure levels and facilitated the renal Ang-(1–7) levels and Ang-(1–7)/Ang II ratio in the Ang II-treated ApoEKO mice with decreased KW and KW/BW ratio (Table 1 and Fig. 1). Clearly, these observations revealed renoprotective effects of ACE2 in the Ang II-mediated pathological actions in ApoE-null mice via the modulation of renal Ang-(1–7) levels.

3.2. Treatment with rhACE2 attenuates renal fibrosis in the Ang II-infused ApoEKO mice with the suppression of the TGF-ˇ1 and mTOR/ERK1/2 signaling Ang II is a well-known activator of increased tissue fibrosis via its profibrotic effects [4]. We speculated that rhACE2 administration could rescue Ang II-mediated pathological changes in the ApoEKO kidneys. Expression analysis showed higher renal phosphorylated levels of p-Akt (Fig. 2A), p-mTOR (Fig. 2B) and p-ERK1/2 (Fig. 2C) in the ApoEKO mice when compared with WT control mice. Continuous infusion of Ang II resulted in obvious increases in phosphorylated levels of p-Akt (Ser473 ), p-mTOR and p-ERK1/2 (Fig. 2) as well as mRNA levels of TGF-␤1, collagen I and fibronectin 1 (Fig. 3) in the ApoE-mutant mice, associated with exacerbation of renal fibrosis and enhanced Collagen volume fraction (Fig. 4). More importantly, treatment with rhACE2 rescued Ang II-induced increased renal tubulointerstitial fibrosis in the Ang II-infused ApoEKO mice with downregulation of the collagen volume fraction (Fig. 4). These changes were accompanied by reduced mRNA expression of the fibrosis-associated genes TGF-␤1, collagen I and fibronectin 1 and phosphorylated levels of p-Akt (Ser473 ), p-mTOR

and p-ERK1/2 in the kidneys (Figs. 2 and 3). However, there were no changes in collagen III mRNA levels among groups (Fig. 3). 3.3. ACE2 is a negative regulator in tubulointersitial fibrosis and adverse renal injury in the ApoEKO mice in response to Ang II Consistent with exacerbation in renal fibrosis, increased tubulointersitial collagen contents and aggravated tubule ultrastructure injury (Fig. 5) were observed in the Ang II-infused ApoEKO mice when compared with the ApoEKO control mice by the TEM analyses. These ultrastructure changes were characterized with renal cell necrosis and vacuolar degenerational mitochondrias (white star), as well as renal tubular epithelial cell pyknosis with disruption, tubulointersitial fibrosis (black arrow) and inflammatory lesions (black triangle), contributing to progression of kidney damage (Fig. 5). Notably, in response to rhACE2 treatment, renal tubular ultrastructure injury was remarkably alleviated in the Ang IIinfused ApoEKO mice with reduction in tubulointersitial collagen contents (Fig. 5E). 3.4. Treatment with rhACE2 and mTOR inhibitor rapamycin suppresses Ang II-mediated activation of the mTOR/ERK1/2 signaling and superoxide generation in MKFs As shown in Fig. 6, we firstly identified the fibroblastic marker vimentin protein in cultured mouse kidney fibroblasts observed with multi-fluorescent confocal laser scanning microscopy. In cultured kidney fibroblasts, exposure to Ang II (100 nmol L−1 ) resulted in significant increases in superoxide production (Fig. 6K and L) and phosphorylated levels of mTOR (Fig. 6I) and ERK1/2 (Fig. 6J), which were largely aggravated by pharmacological inhibition of ACE2 with DX600 (0.5 ␮mol L−1 ). Intriguingly, pre-treatment with rhACE2 (1 mg mL−1 ) or mTOR inhibitor rapamycin (10 ␮mol L−1 ) dramatically prevented Ang II-induced increases in superoxide generation (Fig. 6K and L) and mRNA levels of TGF-␤1 (Fig. 6E),

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Fig. 3. Effects of rhACE2 treatment on renal levels of fibrosis-related genes in mice. The real-time PCR analysis revealed the mRNA levels of fibrosis-related genes TGF-␤1 (A), collagen I (B), collagen III (C) and fibronectin 1 (D) in the kidneys of WT and ApoEKO mice. n = 6-8. GAPDH was used as an endogenous control. R.E.=relative expression; Ang II, angiotensin II; TGF-␤1, transforming growth factor-␤1. * , P < 0.05; ** , P < 0.01 compared with WT group; # , P < 0.01, compared with ApoEKO group;  , P < 0.01 compared with ApoEKO + Ang II group.

Fig. 4. Effects of rhACE2 treatment on kidney fibrosis in the ApoEKO mice. Representative Massons trichrome staining images revealed renal fibrosis (A–E) and collagen volume fraction (F) in the WT and ApoEKO mice (×100 magnification). n = 4 for each group. Ang II, angiotensin II; KO, knockout. * , P < 0.05; ** , P < 0.01 compared with WT group; # , P < 0.05, compared with ApoEKO group;  , P<0.05, compared with ApoEKO + Ang II group.

collagen I (Fig. 6F) and fibronectin (Fig. 6H) in cultured MKFs with marked reduction of phosphorylated levels of p-mTOR (Fig. 6I) and p-ERK1/2 (Fig. 6J). The actions of rhACE2 on superoxide generation and the mTOR/ERK signaling were completely suppressed by the administration of the Mas receptor antagonist A779 (1 ␮mol L−1 ), suggesting the beneficial effect of rhACE2 on the Ang II-mediated pathological actions in the kidney fibroblasts via the Ang-(1–7)/Mas receptor signaling (Fig. 6). However, rhACE2 or rapamycin had no effect on collagen III (Fig. 6G) mRNA expression. ACE2 serves as a negative regulator of Ang II-mediated renal fibrosis and oxidative stress in the kidneys via the modulation of TGF-␤, and mTOR-

ERK1/2 signaling, ultimately resulting in attenuation of adverse renal injury in the ApoEKO mice. Our data point to ACE2 as an important modulator of renal fibrosis and structural damage and a novel target for kidney-protective therapies.

4. Discussion Renal tubulointerstitial fibrosis is the final common pathway and inevitable outcome of a wide variety of progressive chronic kidney diseases and atherosclerosis-related kidney injury, which have been linked with abnormal activation of the RAS [1,5,18,19]. The

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Fig. 5. Effects of rhACE2 treatment on tubulointersitial fibrosis and tubular ultrastructural injury in the kidneys of ApoEKO mice. The renal tubular ultrastructural changes were observed in the WTC (A), ApoEKO (B), ApoEKO + rhACE2 (C), ApoEKO + Ang II (D), and ApoEKO + Ang II + rhACE2 mice (E) by transmission electron microscope analysis (×4200 and ×13500 magnifications). Compared with the ApoEKO mice, renal ultrastructure injury was aggravated in the Ang II-infused ApoEKO mice. These ultrastructure changes were characterized with renal cell necrosis and vacuolar degenerational mitochondrias (white star), renal tubular epithelial cell pyknosis with disruption, as well as tubulointersitial collagen and fibrosis (black arrow) and inflammatory lesions (black triangle). ApoE, Apolipoprotein E; ACE2, angiotensin-converting enzyme 2.

RAS has emerged as a key component responsible for collagen synthesis and fibrosis through activation of mitogen-activated protein kinases/ERK, Akt-mTOR and TGF-␤ signaling pathways [5,7,18,19]. The present study uncovered the insights into the beneficial effects of ACE2 overexpression on kidney fibrosis in the atherosclerosisprone ApoEKO mice. To our knowledge, this is the first report of the relationship between the ACE2 and mTOR-ERK1/2 signaling in the absence of ApoE status. ACE2, a newly discovered member of the RAS family, is an essential regulator in maintaining the balance between Ang II degradation and Ang-(1–7) generation locally within the kidney [1,14–16,20,21]. Our previous studies revealed regulatory effects of ACE2 on renal inflammation, oxidative injury and glomerular dysfunction, focusing on roles of the nephrin, NOX4 and Tumor necrosis factor-␣ (TNF␣)/TNFRSF1A signaling pathways [1,23,24]. We demonstrated that renal expression of nephrin was lower, and proinflammatory factors were higher in the ApoE/ACE2 double-KO mice, including interleukin (IL)-1␤, IL-6, IL-17A, RANTES, ICAM-1, TNF␣ and TNFRSF1A. In contrast, treatment with rhACE2 was able to rescue Ang II-mediated renal dysfunction and glomerular ultrastructure injury in the Ang IIinfused ApoEKO mice with decreased plasma levels of creatinine and blood urea nitrogen. In addition, rhACE2 treatment appeared to show counterregulation against Ang II-mediated reduction of nephrin and activation of the TNF-␣-TNFRSF1A signaling, and these mechanisms may be involved in regulatory effects of ACE2 in renal inflammation and kidney dysfunction the ApoE-deficient mice [1,23,24]. However, little was elucidated upon its anti-fibrotic roles and regulatory mechanism. Thus, we analysed the regulatory roles of ACE2 in the renal fibrosis, and phosphorylation status of Akt/mTOR/ERK1/2 cascade signaling in the atherosclerosis-prone ApoEKO mice in this work. The present study showed that downregulation of renal ACE2 protein was observed in the ApoEKO mice, and Ang II infusion resulted in a marked increase in renal tubulointerstitial fibrosis in the ApoE-null mice with lower renal ACE2 levels and Ang-(1–7)/Ang II ratio. These changes were associated with enhanced expression of fibrosis-inducible gene TGF-␤ and collagen I in the ApoEKO kidneys, with no change in collagen III levels. Treatment with rhACE2 had a minimal effect on renal Ang-(1–7) levels in the ApoEKO mice. These results suggest that the potent and high-capacity ability of neprilysin and Ang II-activated ACE system to metabolize Ang-(1–7) are likely the major determinants of steady-state renal Ang-(1–7) levels in the ApoE-mutant status without Ang II stimulation [5]. Importantly, recombinant human ACE2 treatment dramatically ameliorated Ang II-mediated hypertension, kidney remodeling and tubulointerstitial fibrosis in the Ang II-treated ApoE-mutant mice with downreguration of mTORERK1/2 signaling and the upregulation of renal Ang-(1–7) levels and Ang-(1–7)/Ang II ratio in the kidney. Our data suggest that

ACE2 is a key negative modulator of atherosclerosis-related kidney fibrosis and Ang II-driven adverse renal injury in a murine model for the development of atherosclerosis via the modulation of the Ang-(1–7)/Ang II imbalance. A large number of clinical trials and animal studies have indicated that antagonism of the RAS by ACE inhibitors or AT1 receptor blockers (ARB) is the cornerstone of treatment of patients with progressive chronic kidney diseases and atherosclerosis [1,2,4,25–27]. The elevation in renal Ang-(1–7) levels coupled with the decrease in renal Ang II levels in response to rhACE2 treatment is likely a key mechanism of its renoprotective benefits of rhACE2 in the Ang II-infused ApoEKO mice. As a critical enzyme in the metabolism of Ang II, ACE2 serves to directly balance the levels of Ang II and Ang (1–7). The ACE2/Ang-(1–7) plays a critical role in the control of renal physiology and its altered expression is linked to major pathophysiological changes of the kidney [1,4,15,21,22]. Enhancing ACE2 actions such as rhACE2 has emerged as a potential therapeutic strategy, functioning as an endogenous ACE inhibitor or natural ARB [1,6,8,28]. The present study provides the evidence for reno-protective roles of rhACE2 in the pathological fibrosis in the ApoE-mutant mice by blocking the mTOR signaling. The mTOR is a major effector of protein synthesis, cell growth, lipogenesis and energy metabolism [18,29,30]. Recently, novel regulation of mTOR signaling has been identified in various pathological conditions, including activation of macrophages and myofibroblasts [18,31], pathogenic roles in diabetic nephropathy [32], and proinflammatory and pro-atherosclerotic effects in the ApoEKO mice [29,30,33], indicating the importance of mTOR signaling in the regulation of atherosclerosis and kidney fibrosis. In this work, we demonstrated that phosphorylated levels of mTOR were higher in the atherosclerosis-prone ApoEKO mice. Notably, Ang II infusion resulted in marked increases in phosphorylated levels of Akt, mTOR and ERK1/2 signaling in the ApoEKO kidneys, which were remarkably reversed by administration of rhACE2. Intriguingly, inhibition of mTOR signaling by rapamycin has been shown to prevent or delay the pathogenesis of atherosclerosis [29,33,34] and ameliorate the interstitial inflammation, kidney fibrosis, and loss of renal function associated with CKD [18,35]. To evaluate more direct effects of ACE2 and mTOR inhibition on Ang II signaling independent of a blood pressure–lowering effect, we examined the impact of rhACE2 or rapamycin on Ang II effects in cultured mouse kidney fibroblasts. Exposure to fibroblasts with Ang II led to marked increases in phosphorylated levels of mTOR and ERK1/2, which were largely aggravated by pharmacological inhibition of ACE2 with DX600. Conversely, treatment with rhACE2 or rapamycin dramatically prevented the Ang II-mediated increased superoxide production and enhanced mTOR and ERK1/2 phosphorylation signaling, along with reduced mRNA levels of TGF-␤1, collagen

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Fig. 6. Treatment with rhACE2 and rapamycin reverses Ang II-mediated activation of the mTOR and ERK signaling and superoxide generation in cultured MKFs. (A–D) The identification of vimentin (A), ␣-SMA (B), DAPI (C), and merge (D) in cultured primary mouse kidney fibroblasts (MKFs) with confocal laser scanning microscopy. Green: vimentin; Red: ␣-SMA; Blue: DAPI, 4 ,6 -diamidino-2-phenylindole; (E–H), The real-time PCR analysis revealed the mRNA levels of TGF-␤1 (E), collagen I (F), collagen III (G) and fibronectin (H) in MKFs. n = 6. GAPDH was used as an endogenous control. (I–L) Representative Western blotting (I and J) and dihydroethidium (DHE) fluorescence images (K) and relative fluorescence values (L) exhibited phosphorylated levels of mTOR (I) and ERK1/2 (J) and superoxide generation in the MKFs treated with Ang II (100 nmol L−1 ) in the absence and presence of rhACE2 (1 mg mL−1 ), the Mas receptor antagonist A779 (1 ␮mol L−1 ), ACE2 inhibitor DX600 (0.5 ␮mol L−1 ), ERK1/2 inhibitor PD98059 (10 ␮mol L−1 ), and mTOR inhibitor rapamycin (10 ␮mol L−1 ). n = 5 for each group. R.E., relative expression; A.U., arbitrary units; rhACE2, human recombinant ACE2; Ang II, angiotensin II; TGF-␤1, transforming growth factor-␤1; ERK1/2, extracellular signal-regulated kinase 1/2. ** , P < 0.01 compared with control group; # , P < 0.05; ## , P < 0.01 compared with Ang II group; ␾, P < 0.05 compared with Ang II + rhACE2 group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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I and fibronectin. The actions of rhACE2 on superoxide production and the mTOR/ERK signaling were completely suppressed by the administration of A779, a specific Ang-(1–7)/Mas receptor antagonist, suggesting the beneficial effect of rhACE2 on the Ang II-mediated pathological actions in kidney fibroblasts via the Ang-(1–7)/Mas receptor signaling. Previous research reported that renovascular hypertension increased Mas receptors expression and nitric oxide production in the rats carotid which, consequently increased Ang-(1–7)-vasorelaxant response and A-779 reduced the vasodilator effect of Ang-(1–7) in renovascular hypertension [6]. In another in vivo study [36], the Mas receptor antagonist A779 (200 ng/kg/min) has also shown to prevent the beneficial effects of rhACE2 on the Ang II-induced hypertrophy and diastolic dysfunction, indicating that blocking Ang-(1–7)/Mas receptor action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II. Intriguingly, Ang-(1–7)-treated ApoEKO mice showed improved renal function and oxidant injury when conmpared with untreated ApoEKO mice, which were strikingly abolished with A779 [10]. These results highlight a key renoprotective role of the ACE2/Ang-(1–7), and increased ACE2/Ang-(1–7) action represents a potential therapeutic strategy for atherosclerosis and kidney diseases. Our data underscore renoprotective effects of ACE2/Ang(1–7) with the anti-fibrotic properties through normalization of the mTOR/ERK phosphorylation signaling pathways. 5. Conclusions In summary, the present study revealed the ability of ACE2 to alleviate renal fibrosis and kidney structural injury resulting from the atherosclerosis-prone ApoE-mutant mice. Our results demonstrate that downregulation of renal ACE2 protein is observed in the ApoEKO mice, and Ang II infusion results in a marked increase in renal tubulointerstitial fibrosis in the ApoE-null mice with greater increases in phosphorylated levels of mTOR and ERK1/2. In contrast, rhACE2 treatment appears to show counterregulation against Ang II-mediated activation of phosphorylated mTOR, ERK1/2, and TGF-␤ cascade in both ApoE-mutant kidneys and mouse kidney fibroblasts. Suppression of the mTOR signaling by rhACE2 is responsible for amelioration of kidney fibrosis and improvement of adverse renal injury in the ApoE-deficient mice with enhancement of renal ACE2 levels and Ang-(1–7)/Ang II ratio. Our findings also identify a molecular crosstalk between the mTOR signaling and renal fibrosis via ACE2. Targeting ACE2/Ang-(1–7) or mTOR signaling has potential therapeutic importance for preventing and treating atherosclerotic renal injury and CKD. Some novel mTOR inhibitors with less toxicity and improved specificity might offer better perspectives for safe, long-term treatment of atherosclerosis and many other fibrosis-related disorders. A better understanding of the interactions between ACE2/Ang-(1–7) and mTOR signaling in kidney might help to find out a way to halt the fibrotic progression and renal injury in the atherosclerosis-related kidney diseases. Competing interests The authors have declared that no competing interests exist. Authors’ contributions L.J.C., G.Y.O. and J.C.Z. conceived the study design, performed the data analysis and wrote the main manuscript text. L.J.C., Y.L.X., B.S., R.X., Z.Z.Z., H.Y.J. and Q.C. carried out the experiments and participated in the acquisition of data, analysis and interpretation. H.M.Y and D.L.Z. provided critical advice and performed the data analysis. All authors reviewed and approved the final manuscript.

Acknowledgements This work was supported by the National Major Research Plan Training Program (91339108), the National Basic Research Program of China (2014CB542300), the National Natural Science Foundation of China (81170246, 81370362 & 81273599), Canadian Institutes of Health Research (CIHR) Heart & Stroke Foundation to GYO, Shanghai Pujiang Talents Program of Shanghai Science and Technology Committee (11PJ1408300), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant (20152509) and Scientific Research Project of Health Bureau of Shanghai (201440368).

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