The International Journal of Biochemistry & Cell Biology 55 (2014) 65–71

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The Notch ␥-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-␤/Smad2/3 signaling pathway activation Zhicheng Xiao a , Jing Zhang a , Xiaogang Peng a , Yanjun Dong a , Lixin Jia a , Huihua Li b , Jie Du a,∗ a Beijing AnZhen Hospital, Capital Medical University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China b Department of Physiology and Pathophysiology, Capital Medical University, Beijing, China

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Article history: Received 24 February 2014 Received in revised form 30 July 2014 Accepted 13 August 2014 Available online 20 August 2014 Keywords: Kidney fibrosis Epithelial-to-mesenchymal transition Fibroblast Notch TGF-␤

a b s t r a c t Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 ␮g/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson’s trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1␣1/3␣1, fibronectin, and ␣-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-␤, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-␤/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Renal interstitial fibrosis is the hallmark of chronic progressive kidney disease (CKD), which leads to renal failure (Nath, 1992). Renal fibrosis is characterized by epithelial cell dysfunction, leukocyte migration, increased extracellular matrix (ECM) deposition, myofibroblast proliferation, and activation (Liu, 2011). In response to kidney damage, mature myofibroblasts are derived from various sources, including interstitial fibroblasts, pericytes, tubular

Abbreviations: DBZ, dibenzazepine; UUO, unilateral ureteral obstruction; NICD, Notch intracellular domain; ICN, intracellular domain of Notch; TECs, tubular epithelial cells; ECM, extracellular matrix; BrdU, bromodeoxyuridine; EMT, epithelial mesenchymal transition; CM, conditional media; Smad, small mother against decapentaplegic; Hes-1, hairy and enhancer of split-1. ∗ Corresponding author. Tel.: +86 10 64456030; fax: +86 10 64456094. E-mail addresses: [email protected], [email protected] (J. Du). http://dx.doi.org/10.1016/j.biocel.2014.08.009 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

epithelial cells (TECs), endothelial cells, and circulating fibrocytes (Liu, 2011). Emerging data indicate that multiple signaling pathways, such as the transforming growth factor beta (TGF␤)/Smad2/3 and Notch pathways, are involved in epithelial cell dysfunction and fibroblast activation, leading to progression of renal fibrosis (Liu, 2011). However, the molecular mechanism regulating these events remains unexplored. The Notch signaling pathway is highly conserved among all animal species. It is composed of at least 4 Notch receptors (Notch 1–4) and 5 Notch ligands (Delta-like l, 3, and 4, and Jagged 1 and 2) in vertebrates. Following ligand binding, Notch receptors undergo a series of cleavages catalyzed by the ␥-secretase complex, resulting in the release of the Notch intracellular domain (NICD); this process can be inhibited by the ␥-secretase inhibitor, dibenzazepine (DBZ) (Milano et al., 2004). The NICD then translocates into the nucleus and induces the transcription of its target genes, such as Hes1 and HeyL. Accumulating evidence indicates that Notch signaling plays a critical role in regulating cell growth, differentiation,

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apoptosis, and pattern formation in mammals (Lai, 2004). Recent studies demonstrate that Notch signaling also participates in tissue fibrosis in various diseases, including scleroderma, idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, and cardiac fibrosis (Kavian et al., 2012). Genetic deletion of the Notch pathway in TECs ameliorates renal fibrosis in the murine unilateral ureteral obstruction (UUO) model and folic acid-induced renal fibrosis. Furthermore, TEC-specific expression of active Notch1 causes renal fibrosis without extra stimulation (Bielesz et al., 2010). These data suggest that Notch signaling plays a key role in fibrosis pathogenesis. However, the precise underlying cellular mechanisms are not fully understood. In the present study, we explored the role of Notch signaling in kidney fibrosis development and whether inhibition of Notch activation by DBZ could ameliorate renal fibrosis in the murine UUO model. For the first time, we demonstrated that the Notch pathway is involved in kidney fibrosis through activation of TGF-␤/Smad2/3 signaling in TECs and myofibroblast activation. Administration of the ␥-secretase inhibitor DBZ markedly attenuated Notch activation-mediated kidney fibrosis.

2. Materials and methods 2.1. Antibodies and reagents Antibodies to Notch4, alpha-smooth muscle actin (␣-SMA) and fibronectin were purchased from Abcam Inc.(Cambridge, MA); antibodies to pan-cadherin and Notch3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to Hes1 were from Millipore Biosciences (Billerica, MA); antibodies to cleaved Notch1, Notch2, E-cadherin, TGF-␤, Smad2/3, phospho-Smad2/3, GAPDH, and horseradish peroxidase-linked anti-mouse, goat or rabbit IgG antibody were from Cell Signaling Technology (Beverly, MA); anti-HeyL antibodies and anti-TGF-␤1 neutralizing antibodies were from R&D Systems (Minneapolis, MN). ␥-secretase inhibitor dibenzazepine (DBZ) was purchased from Santa Cruz Biotechnology. Penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies (Carlsbad, CA). Other reagents were purchased from Sigma–Aldrich (St. Louis, MO).

2.3. Primary culture of renal tubular epithelial cells Tubular epithelial cells were isolated as described previously (Cheng et al., 2010). Minced kidneys were washed in three changes of cold phosphate buffered saline (PBS) containing 1 mM EDTA and were digested in 0.25% trypsin solution in a shaking incubator at 37 ◦ C for 2 h. Trypsin was neutralized with Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum. The suspension was triturated by pipetting and passed through a 100-mm cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA). The filtrate, consisting mostly of dispersed renal tubules, was plated onto culture dishes (Nalge Nunc International, Naperville, IL, USA). The cells were cultured at 37 ◦ C in a CO2 incubator with the media changed every 2 days. 2.4. Primary culture of renal fibroblasts The cortical tissue of murine kidneys was minced into small pieces (1 mm3 per plate) and plated onto culture dishes. They were flooded with Dulbecco’s modified Eagle’s medium and 20% fetal calf serum supplemented with penicillin-streptomycin and L-valine (Sigma–Aldrich) and incubated at 37 ◦ C in a CO2 incubator with the media changed every 2 days (Kelynack et al., 2000). 2.5. Histopathology and immunohistochemistry Kidneys from WT mice treated with or without DBZ fixed in 10% formalin were routinely processed and paraffin embedded. Kidney sections (4 ␮m) were then stained with Masson’s trichrome reagent (Cheng and Du, 2007). For immunofluorescence, frozen kidney sections were labeled with primary antibodies against ␣-SMA (1:500 dilution), pan-cadherin (1:100 dilution) or Hes1 (1:200 dilution) and then incubated with fluorescein isothiocyanate (FITC)and tetramethylrhodamine isothiocyanate-conjugated secondary antibody (1:500). For immunohistochemistry, kidney sections were stained with primary antibodies against ␣-SMA (1:500 dilution) as described previously (Li et al., 2012; Yang et al., 2012). Images were viewed and captured with a confocal laser scanning microscope (TCS 4D, Leica; Heidelberg, Germany) and a Nikon Labophot 2 microscope (Nikon, Tokyo, Japan). 2.6. Quantitative real-time PCR analysis

2.2. Mouse models of kidney fibrosis Male wild-type (WT) mice (C57BL/6 background) were bred and maintained in the Laboratory of Animal Experiments at Anzhen Hospital affiliated to Capital Medical University. The mice were given a standard diet. Unilateral ureteral obstruction (UUO) was performed in adult (8–12 weeks) mice as described previously, and sham-operated mice were used as controls (Cheng et al., 2010). Briefly, under anesthesia by ketamine/xylazine (100/10 mg/kg i.p), the left ureter was ligated twice using 4–0 nylon surgical sutures at the level of the lower pole of kidney. DBZ (dissolved in DMSO) was administered intraperitoneally (250 ␮g/100 g/d) one day prior to the operation and once per day. Different doses of DBZ (between 100 and 500 ␮g/100 g/d) were adopted in various studies (Bielesz et al., 2010; Droy-Dupre et al., 2012; Zheng et al., 2013). We tested doses responses, and found that administration of DBZ at the dose of 250 ␮g/100 g/d effectively inhibited Notch signaling without obvious side effects. After 7 days, all animals were euthanized by overdose pentobarbital (100 mg/kg) at the end of each treatment period. The study protocol was approved by the Ethical Committee of Capital Medical University and conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85–23, 1996).

Total RNA was extracted from mouse kidney or cultured cells by use of TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. A total of 2 ␮g RNA were reversely transcribed and used to synthesize first-strand cDNA with moloney murine leukemia virus reverse transcriptase. Quantitative real-time PCR (qPCR) was performed with an iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with SYBR Green JumpStart Taq ReadyMix (Takara, Otsu, Shiga, Japan) (Pan et al., 2012). The primer sequences for mouse Notch 1–4, Hes1, HeyL, Col1␣1, Col3␣1, fibronectin, TGF-␤1, PDGF-B, CTGF, and GAPDH were described in Table 1. 2.7. Western blot analysis Whole kidneys or cortical tissues from UUO or sham mice were homogenized in lysis buffer (20 mM Tris, 1% TritonX-100, 0.05% SDS, 5 mg of sodium deoxycholate, 150 mM NaCl and 1 mM PMSF) containing protease/phosphatase inhibitor cocktail. Fiftysixty gram protein samples were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad). The membranes were incubated with primary antibody against Notch1 (1:1000), Notch2 (1:1000), Notch3 (1:500), Notch4 (1:1000), Hes1 (1:1000), HeyL (1:1000), fibronectin (1:1000), E-cadherin (1:1000), TGF-␤ (1:1000), Smad2/3, phospho-Smad2/3 (1:1000), ␣-SMA,

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Table 1

Genes Notch1 Notch2 Notch3 Notch4 Hes1 HeyL Col 1␣1 Col 3␣1 TGF-␤1 PDGFB CTGF GAPDH FN

Forward primers 5 -CGTGGTCTTCAAGCGTGATG-3 5 -CAATGCTGGCAACACCCATC-3 5 -ATGTGCAAATGGAGGTCGGT-3 5 -GCCAAGGTCAGGAACACAGA-3 5 -ACCCCAGCCAGTGTCAACA-3 5 -GGGAGAGGAGGCGATTGAA-3 5 -GACTGGAAGAGCGGAGAGTACTG-3 5 -GGGAATGGAGCAAGACAGTCTT-3 5 -ATGCTAAAGAGGTCACCCGC-3 5 -CCCACAGTGGCTTTTCATTT-3 5 -ACCGCACAGAACCACCACTCT-3 5 -TGAAGGTCGGTGTGAACGG-3 5 -CCGCCGAATGTAGGACAAGA-3

(1:2000) or GAPDH (1:1000) at 4 ◦ C overnight and then with IRDyeconjugated secondary antibodies (1:5000) for 1 h. Images were quantified by use of the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). To quantify the protein signals, we subtracted background, normalized the value to GAPDH. As for the phospho-specific protein, we normalized the signal to the amount of total target protein or GAPDH (Ma et al., 2012). 2.8. Infection of primary epithelial cells and cell proliferation assay Primary epithelial cells were infected with adenovirus expressing the pHB-Ad-MCMV-GFP control or pHB-Ad-MCMV-GFPNotch1 ICD at MOI of 50 (Hanbio corporation, China). Infection efficiency, estimated under fluorescence microscope by the presence of GFP-positive cells, ranged more than 95%. After 72 h of infection, the conditioned medium was collected and added to serum-starved fibroblasts for additional 24 h. Fibroblast proliferation was detected by immunohistochemical staining using anti-bromodeoxyuridine (BrdU) monoclonal antibodies according to the manufacturer’s protocol (Roche Applied Science). Gene expression of fibroblasts was determined by qPCR analysis. Protein levels of primary epithelial cells were tested by western blot.

Reverse primers 5 -AGCTCTTCCTCGTGGCCATA-3 5 -CTGGCACTCGTCCACTTCAT-3 5 -CACTGAACTCTGGCAAACGC-3 5 -CACCCAGTTCTGTCTCGCAT-3 5 -TGTGCTCAGAGGCCGTCTT-3 5 -GCTAGGAGGCTGCCAAACTACA-3 5 -CCTTGATGGCGTCCAGGTT-3 5 -TGCGATATCTATGATGGGTAGTCTCA-3 5 -TGCTTCCCGAATGTCTGACG-3 5 -GTGAACGTAGGGGAAGTGGA-3 5 -TGGCAGGCACAGGTCTTGATGA-3 5 -CGTGAGTGGAGTCATACTGGAA-3 5 -GCCAACAGGATGACATGAAATG-3

trichrome staining demonstrated enhanced collagen deposition (stained blue) and tubular dilation in UUO kidneys as compared with controls (Fig. 1A). qPCR analysis revealed that the mRNA levels of Notch 1, 3, 4 and its downstream effectors Hes1 and HeyL were significantly upregulated in UUO mice compared with those of the control group (Fig. 1B). Through western blot analysis and quantification, we observed that the protein levels of intracellular domain (ICN) of Notch 1–4, Hes1, and HeyL were increased after UUO (Fig. 1C). Furthermore, immunofluorescent staining confirmed that Hes1 protein was highly expressed in UUO kidneys and colocalized with the TECs (Fig. 1D), but not in ␣-smooth muscle actin (SMA)-positive myofibroblasts (Fig. 1E). These results suggest that Notch signaling is activated in TECs after UUO injury.

2.9. Blocking TGF-ˇ1 signaling with neutralizing antibodies in vitro Primary epithelial cells were infected with adenovirus expressing the pHB-Ad-MCMV-GFP control or pHB-Ad-MCMV-GFPNotch1 ICD at 50 MOI as above. Conditional media were incubated with anti-TGF-␤1 neutralizing antibody (R&D Systems; Minneapolis, MN; 1.0 ␮g/mL) or control mouse IgG1 (Sigma–Aldrich; 1.0 ␮g/mL) and then cultured fibroblasts for 24 h (Zhang et al., 2013). 2.10. Statistical analysis Data are presented as mean ± SEM. Differences between groups were analyzed by Student’s t-test or ANOVA followed by the Newman–Keuls multiple-comparison test by use of GraphPad Prism 5.0. P < 0.05 was considered statistically significant. 3. Results 3.1. Notch signaling is activated in UUO mice To determine whether the Notch signaling pathway is involved in kidney fibrosis, we first examined the expression of Notchrelated genes in UUO mice. Seven days after UUO, Masson’s

Fig. 1. Epithelial Notch signaling pathway is activated in kidney after UUO injury. (A) Representative Masson trichrome staining in the kidneys from control or UUO mice. Scale bars: 50 ␮m. (B) qPCR analysis of relative mRNA expression of Notch 1–4, Notch target genes Hes1 and HeyL in the kidneys from control or UUO mice. (C) Western blot analysis of intracellular domain of Notch 1–4 (ICN 1–4), Hes1, and HeyL (left) and quantification (right) in the kidneys from control or UUO mice. (D and E) Double immunofluorescence staining of Hes1 (red) and pan-cadherin (green) or ␣-SMA (green) in the kidneys from control or UUO mice. Scale bars: 25 ␮m. Data are expressed as mean ± SEM (n = 4 mice per group).*P < 0.05 vs. control.

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Fig. 2. Notch ␥-secretase inhibitor ameliorates renal fibrosis after UUO injury. (A) Western blot analysis of Notch intracellular domain NICD and GADPH expression (left) and quantification (right) in the kidneys from control or UUO mice treated with or without DBZ. (B) Representative Masson trichrome staining (left) and quantification (right) of the kidneys from control or UUO mice treated with or without DBZ. Scale bars: 50 ␮m. (C) Western blot analysis of fibronectin and GADPH expression (left) and quantification (right) in the kidneys from control or UUO mice treated with or without DBZ. (D) Relative mRNA levels of collagen 1␣1, collagen 3␣1, and fibronectin in the kidneys from control or UUO mice treated with or without DBZ. Data are expressed as mean ± SEM (n = 4 mice per group).*P < 0.05 vs. UUO group.

3.2. The Notch inhibitor DBZ ameliorates renal fibrosis after UUO To explore whether Notch signaling plays an important role in renal fibrosis, mice were pretreated with the ␥-secretase inhibitor DBZ (250 ␮g/100 g/d) and then subjected to UUO. After 7 days, NICD expression was significantly increased in UUO mice compared with that in controls, and this effect was markedly attenuated by DBZ treatment (Fig. 2A). To assess the effect of the Notch inhibitor DBZ on UUO-induced kidney fibrosis, Masson’s trichrome staining was performed on renal tissues. The UUO-induced increase in collagen deposition in the kidney tissues was significantly decreased by DBZ treatment relative to control mice (Fig. 2B). Moreover, western blot and qPCR demonstrated that the expression of fibrotic markers, including collagen 1␣1, collagen 3␣1, and fibronectin, was markedly decreased in DBZ-treated mice compared with controls after UUO (Fig. 2C and D). There were no significant differences in the fibrotic area or expression of fibrotic markers between the two groups after sham treatment (Fig. 2B–D). These results indicate that inhibition of Notch signaling suppresses kidney fibrosis after UUO. 3.3. The Notch inhibitor DBZ inhibits activation of myofibroblasts after UUO, independent of epithelial-mesenchymal transition Epithelial–mesenchymal transition (EMT) plays a critical role in the development of renal fibrosis (Rastaldi, 2006); therefore, we

Fig. 3. Notch ␥-secretase inhibitor attenuates activation of myofibroblasts via inhibition of TGF-␤ signaling after UUO injury. (A) Representative immunohistochemical staining of ␣-SMA expression (left) and quantification (right) in the kidneys from control or UUO mice treated with or without DBZ. Scale bars: 50 ␮m. (B) Western blot analysis of ␣-SMA, E-cadherin and GADPH expression (left) and quantification (right) in whole kidneys from control or UUO mice treated with or without DBZ. (C) Western blot analysis of ␣-SMA, E-cadherin and GADPH expression (left) and quantification (right) in cortical tissues from control or UUO mice treated with or without DBZ. (D) Western blot analysis of TGF-␤ expression and phosphorylated Smad2/3 protein levels (left) and quantification (right) in whole kidneys from control or UUO mice treated with or without DBZ. Data are expressed as mean ± SEM (n = 4 mice per group).*P < 0.05 vs. UUO group. NS: no significant difference.

next assessed the effect of the Notch inhibitor DBZ on the expression of the EMT marker E-cadherin and mesenchymal marker ␣-SMA in the kidney after UUO injury. The number of ␣-SMApositive myofibroblasts and protein level of ␣-SMA expression were significantly increased in UUO mice as compared with those in sham mice, and this action was effectively attenuated by DBZ administration (Fig. 3A and B). Protein was extracted from cortex region or whole kidneys for western blot. However, we failed to observe a significant change in the expression of E-cadherin protein between UUO and sham groups after DBZ or vehicle treatment (Fig. 3B and C). These results suggest that the effects of Notch on renal fibrosis appear to be dependent on the proliferation or differentiation of myofibroblasts, independent of EMT after UUO. 3.4. The Notch inhibitor DBZ inhibits the TGF-ˇ signaling pathway Because the TGF-␤/Smad signaling pathway is involved in the progression of renal fibrosis (Lan and Chung, 2012), we next tested whether DBZ affected this pathway in UUO mice. As shown in Fig. 3D, TGF-␤ expression and Smad2 and Smad3 phosphorylation were significantly upregulated in UUO mice as compared with the control group. These changes were significantly attenuated in

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Fig. 4. Activation of Notch in tubular epithelial cells promotes the proliferation and activation of fibroblasts in vitro. Primary tubular epithelial cells (TECs) were infected with adenovirus expressing GFP protein (Ad-GFP) or intracellular domain of Notch1 (Ad-NICD). Seventy-two hours later, conditioned media (CM) were harvested for fibroblast culture. (A) BrdU staining (green) of fibroblasts after 24 h culture with conditioned medium from Ad-GFP- or Ad-NICD-infected TECs. Bar graph showed the percentage of BrdU-positive cells. (B) qPCR analysis of collagen 1␣1, collagen 3␣1, and fibronectin mRNA expression in fibroblasts after 24 h culture with conditioned medium from Ad-GFP- or Ad-NICD-infected TECs. Data are expressed as mean ± SEM (n = 3).*P < 0.05 vs. Ad-GFP TECs.

DBZ-treated UUO mice, indicating that inhibition of Notch signaling suppresses the activation of the TGF-␤ signaling pathway in UUO mice. 3.5. Notch activation in epithelial cells promotes the proliferation and differentiation of fibroblasts TECs are postulated to contribute to pericyte–myofibroblast transition (Wu et al., 2013). To determine whether activation of Notch in TECs regulates myofibroblast activation, primary TECs were infected with an adenovirus overexpressing green fluorescent protein (Ad-GFP) or the active form of Notch1 NICD (Ad-NICD). Seventy-two hours later, we harvested the culture supernatants from infected TECs and applied conditional media (CM) to cultured fibroblasts. The BrdU incorporation assay and qPCR analysis of fibroblasts showed that the number of BrdU-positive cells and expression of collagen 1␣1, collagen 3␣1, fibronectin mRNA were markedly increased in fibroblasts cultured with supernatants from Ad-NICD-infected TECs as compared with Ad-GFP-infected cells (Fig. 4A and B). These results suggest that Notch activation in TECs promotes the proliferation and activation of fibroblasts. To elucidate how Notch activation in TECs influences fibroblast activation, we examined the expression of several growth factors, including TGF-␤1, connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF)-␤ in NICD- or GFPinfected TECs, which are known to stimulate fibroblast proliferation and activation. As shown in Fig. 5A, no significant changes in the expression of CTGF or PDGF-␤ were observed between the two groups. However, the expression of TGF-␤1 mRNA was significantly upregulated (>4-fold) in NICD-infected TECs as compared with AdGFP-infected cells. This result is further confirmed by increased TGF-␤ precursor protein in primary epithelial cells after Ad-NICD transfection, as compared with Ad-GFP transfection (Fig. 5B). To examine whether TGF-␤1 mediates Notch-induced fibroblast proliferation and activation, fibroblasts were treated with conditional media from NICD- or GFP-infected TECs in the presence of a TGF-␤1 neutralizing antibody or IgG control. As shown

Fig. 5. Notch signaling enhances activation of fibroblasts through TGF-␤1 signaling in vitro. (A) qPCR analysis of TGF-␤1, PDGF-B, and CTGF mRNA expression in primary tubular epithelial cells (TECs) infected with Ad-GFP- or Ad-NICD after 72 h culture. (B) Western blot analysis of TGF-␤ precursor and GAPDH and quantification in primary tubular epithelial cells (TECs) infected with Ad-GFP- or Ad-NICD after 72 h culture. (C) Fibroblasts were cultured with conditional media from NICD- or GFPinfected TECs for 24 h, in the presence of TGF-␤1 neutralizing antibody (TGF-␤1 Ab, 1.0 ␮g/mL) or IgG control. BrdU staining (green) of fibroblasts was performed. The percentage of BrdU-positive cells (green) was determined. (D and E) Fibroblasts were pre-treated and cultured as in C. qPCR analysis of fibronectin, collagen 1␣1 and collagen 3␣1 mRNA expression in fibroblasts. Data are expressed as mean ± SEM (n = 3).*P < 0.05 vs. Ad-NICD TECs. NS: no significant difference.

in Fig. 5C–E, treatment with an anti-TGF-␤1 neutralizing antibody markedly attenuated the expression of collagen 1␣1, collagen 3␣1, fibronectin mRNA in the supernatants of Ad-NICD-infected cells. In contrast, the anti-TGF-␤1 neutralizing antibody had no effect on fibroblast proliferation. Collectively, these results suggest that Notch activation in TECs induces the differentiation of fibroblasts via enhanced production of TGF-␤1. 4. Discussion In the present study, we demonstrated that Notch signaling was activated in the kidney after UUO, which was characterized by significant upregulation of ICN 1–4 and their target genes Hes1 and HeyL. Administration of the Notch ␥-secretase inhibitor DBZ markedly inhibited UUO-induced renal fibrosis and the expression of collagen 1␣1/3␣1, fibronectin, and ␣-SMA. These effects were involved with Notch-mediated TGF-␤ signaling activation in TECs. Accumulating evidence indicates that Notch signaling has a critical role in the pathogenesis of various inflammatory diseases (Djudjaj et al., 2012; Hans et al., 2012; Zheng et al., 2013). Notch ␥-secretase inhibitors have been used to reduce the formation of atherosclerotic lesions and abdominal aortic aneurysms (Zheng et al., 2013), reverse pulmonary hypertension (Qiao et al., 2012),

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attenuate rat hepatic fibrosis (Chen et al., 2012), and exert antifibrotic effects in different murine models of systemic sclerosis (Dees et al., 2011). Recent studies show that Notch signaling is activated in TECs in patients with kidney diseases (Bielesz et al., 2010). The expression of the active form of Notch NICD in the tubulointerstitium is correlated with the extent of tubulointerstitial fibrosis in various renal diseases (Murea et al., 2010). The expression levels of Notch1, Notch2, and Jagged1 on podocytes also correlate with the degree of albuminuria and glomerulosclerosis (Sharma et al., 2011). Moreover, Notch3-deficient animals are protected from UUO-induced tubular fibrosis through inhibition of chemokine synthesis and monocyte infiltration (Djudjaj et al., 2012). Importantly, genetic deletion of Notch signaling in TECs ameliorates renal fibrosis, while expression of cleaved Notch1 in epithelial cells is sufficient to induce fibrosis without extra stimulation (Bielesz et al., 2010). However, the molecular mechanisms by which Notch activation promotes renal fibrosis were previously unexplored. Our study demonstrated that Notch activation by UUO enhances the expression of collagen and fibronectin, resulting in renal fibrosis (Fig. 2). These effects are markedly attenuated by DBZ, suggesting a profibrotic function of Notch signaling in the obstructed kidney. Among the mechanisms responsible for the development of renal fibrosis, EMT is associated with this process (Kriz et al., 2011). This process is characterized by the loss of epithelial adhesive molecules, for example, E-cadherin (Liu, 2011). However, the role of Notch signaling in the regulation of EMT and renal fibrosis remains controversial. One study shows that activation of Notch signaling promotes EMT through the induction of Snail (Matsuno et al., 2012). Another study also reveals that activation of Notch signaling induces EMT in vitro, but does not affect EMT in vivo (Bielesz et al., 2010). Moreover, Notch3-deficient mice are protected from tubular injury and cell loss with significantly reduced fibrosis induced by UUO via an EMT-independent pathway (Djudjaj et al., 2012). Recent study indicates that only 5% of myofibroblasts are derived from EMT (LeBleu et al., 2013). And some researches failed to observe the loss of E-cadherin after UUO (Bielesz et al., 2010, Djudjaj et al., 2012). In this study, we explored whether EMT contributes to renal fibrosis. As shown by Fig. 3B and C, the expression of ␣-SMA was increased after UUO, indicating extensive activation of myofibroblasts. But no decline of E-cadherin was observed after UUO, suggesting EMT plays a small role in renal fibrosis. In addition, DBZ treatment did not influence E-cadherin expression in control or obstructed kidneys, indicating that Notch activation promotes renal fibrosis in an EMT-independent manner in vivo. It has been reported that fibroblast proliferation and differentiation play critical roles in the formation of fibrosis. TGF-␤/Smad2/3 is a major signaling pathway that stimulates renal fibrosis (Lan and Chung, 2012). Accumulating evidence indicates that TECs can directly influence the proliferation and differentiation of fibroblasts through secretion of profibrogenic growth factors, including TGF-␤1 and CTGF (Yang et al., 2010). Previous reports indicates that Notch interferes with TGF-␤ signaling pathway. Smad3 can interact with NICD directly and promote the transcription of Hes1 in vivo and in vitro (Blokzijl et al., 2003). TGF-␤ can also induce EMT through upregulating Hes1 and Jagged1, which can be ceased by Notch signaling inhibition (Zavadil et al., 2004). Depending on different cell contexts, this cross-talk may turn to antagonism. For example, in the neonatal cardiac stromal cells, TGF-␤ stimulation decreases Notch1 expression and promotes the fibroblast-myofibroblast transition. Pharmacological inhibition of endogenous Notch1 signaling by ␥-seretase inhibitor DAPT, potentiates this process (Sassoli et al., 2013). In addition, Notch activation can induce the expression of TGF-␤1 and phosphorylated Smad3 in RLE-6TN cells (Aoyagi-Ikeda et al., 2011). Overexpression of NICD results in upregulation of TGF-␤1 mRNA in renal TECs (Bielesz

et al., 2010). The current results also demonstrate that Notch activation markedly upregulates TGF-␤1 expression (>4-fold) and not CTGF or PDGF-␤ expression (Fig. 5A), indicating that TGF-␤1 may mediate the effects of Notch signaling in fibroblast proliferation and differentiation. Existing research shows that stimulating TECs with TGF-␤1 induces the production of CTGF, which increases the expression of collagen I and fibronectin in tubulointerstitial fibroblasts (Okada et al., 2005). A recent study also demonstrates that TGF-␤1 induces G2/M growth arrest in cultured renal epithelial cells, which stimulates the production of PDGF-␤ and TGF-␤1 to promote pericyte proliferation and differentiation into myofibroblasts (Wu et al., 2013). However, whether Notch activation in TECs regulates fibroblast proliferation and differentiation remains unclear. We approached this question by coculturing fibroblasts with conditioned medium from Ad-GFP- or Ad-NICD-infected TECs, respectively, and found that overexpression of NICD markedly promotes the proliferation and activation of fibroblasts (Fig. 4A and B). Moreover, treatment of cells with a TGF-␤1 neutralizing antibody markedly attenuates fibroblast activation but does not affect fibroblast proliferation induced by NICD-infected TECs (Fig. 5C–E). Previous report suggests that FGF can promote the proliferation of fibroblast (Xiao et al., 2012). Other study indicates that under visfatin stimulation, Notch1 binds to the promoter region of FGF2 and promote the expression of FGF-2 in endothelial cells (Bae et al., 2011). Thus, we speculated that Notch activation in tubular epithelial cells might promote the secretion of FGF-2, to induce the proliferation of fibroblast. Taken together, these results demonstrate that activation of Notch signaling plays an important role in the development of renal fibrosis via a TGF-␤1-dependent mechanism in TECs. 5. Conclusions In conclusion, our study provides direct evidence that activation of Notch signaling in TECs significantly stimulates expression of TGF-␤1, which promotes fibroblast differentiation and leads to renal fibrosis after UUO injury. Conversely, the Notch inhibitor DBZ markedly attenuates these effects. Urinary tract infection contributes to renal scaring and renal failure (Vachvanichsanong, 2007). Since preventive administration of DBZ effectively inhibited interstitial fibrosis, it may be a promising treatment preventing CKD in people with urinary tract infection. Funding This study was supported by grants from the National Natural Science Foundation of China (31090363, 81230006), the Chinese Ministry of Science and Technology (2012CB945104), and Beijing collaborative innovative research center for cardiovascular diseases (PXM2013 014226 07 000088). Disclosure The authors have declared that no competing interests exist. Acknowledgement We thank Dr. Shulan Qiu for image analysis of the confocal laserscanning microscope and thank Congcong Zhang for her help on experimental techniques. References Aoyagi-Ikeda K, Maeno T, Matsui H, Ueno M, Hara K, Aoki Y, et al. Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-{beta}-Smad3 pathway. Am J Respir Cell Mol Biol 2011;45:136–44.

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