Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis and accelerates the progression of chronic kidney disease; however, the underlying molecular mechanism is poorly understood. Angiotensin II (Ang II), the central component of RAS, is a key regulator of renal fibrogenic destruction. Here we show that epidermal growth factor receptor (EGFR) plays an important role in Ang II–induced renal fibrosis. Inhibition of EGFR activation by novel small molecules or by short hairpin RNA knockdown in Ang II–treated SV40 mesangial cells in vitro suppresses protein kinase B and extracellular signal-related kinase signaling pathways and transforming growth factor-β/Sma- and Mad-related protein activation, and abolishes the accumulation of fibrotic markers such as connective tissue growth factor, collagen IV. The transactivation of EGFR by Ang II in SV40 cells depends on the phosphorylation of proto-oncogene tyrosine-protein kinase Src (c-Src) kinase. Further validation in vivo demonstrates that EGFR small molecule inhibitor successfully attenuates renal fibrosis and kidney dysfunction in a mouse model induced by Ang II infusion. These findings indicate a crucial role of EGFR in Ang II–dependent renal deterioration, and reveal EGFR inhibition as a new therapeutic strategy for preventing progression of chronic renal diseases.
Chronic kidney disease (CKD) is now recognized to be a worldwide problem associated with significant morbidity and mortality. The progression of CKD is an irreversible process that eventually leads to end-stage renal failure, a devastating condition in which patients depend on lifelong treatment with dialysis or renal transplantation (Klahr and Morrissey, 2003). Although most patients with CKD receive the diagnosis long before they reach end-stage renal failure, no effective treatment can completely halt the progressive decline in renal functions.
The pathogenesis of CKD is characterized by progressive loss of kidney function, relentless accumulation and deposition of extracellular matrix (ECM), leading to widespread tissue fibrosis (Klahr and Morrissey, 2003). Interstitial fibrosis, a hallmark of chronic renal failure, strongly correlates with deterioration of renal function regardless of the underlying disease (Eddy, 2014). The current therapy strategy of reducing the activities of the renin-angiotensin system (RAS) at best slows but does not completely halt the progression of chronic renal fibrosis in experimental and clinical conditions (Locatelli et al., 2009).
Angiotensin II (Ang II), the central component of RAS, appears to be critical in initiating and sustaining the fibrogenic destruction of the kidney (Mezzano et al., 2001). The contribution of Ang II to the progression of renal pathology has been elegantly illustrated by genetic studies using angiotensin-converting enzyme 2 (ACE-2) knockout mice (Zhong et al., 2011; Liu et al., 2012) and by pharmacologic studies using ACE inhibitors and Ang II–receptor blockers (Ishidoya et al., 1995; Ruiz-Ortega et al., 1995; Taal and Brenner, 2000). The fibrogenic effects of Ang II are attributed to its activation of transforming growth factor-β (TGF-β) signaling (Coresh et al., 2007), the essential signaling pathway involved in extracellular matrix deposition and tissue homeostasis and repair. Suppression of Ang II–induced TGF-β stimulation may represent a promising therapeutic approach for inhibition of renal fibrosis.
Epidermal growth factor receptor (EGFR), also known as ErbB1, is a receptor tyrosine kinase from the ErbB family. EGFR protein possesses an N-terminal extracellular ligand-binding region, a conserved alpha helical transmembrane region, and a C-terminal cytoplasmic region with tyrosine kinase activity and phosphorylation sites (Herbst, 2004; Grimminger et al., 2010). Upon activation by phosphorylation, EGFR has been shown to stimulate cell growth, desensitize cells from apoptotic stimuli, and regulate angiogenesis, which are achieved by the recruitment of subsequent signaling cascades such as protein kinase B (Akt) and extracellular signal-related kinase (ERK) (Schlessinger, 2000; Grimminger et al., 2010). Currently, EGFR inhibitors such as gefitinib and erlotinib have been used clinically in the treatment of human carcinomas (Roskoski, 2014).
In addition to tumorigenesis, a growing number of studies have indicated that EGFR also contributes to the development and progression of renal disease in animal models of obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, and glomerulonephritis through mechanisms involved in the activation of renal interstitial fibroblast, induction of tubular atrophy, overproduction of inflammation factors, and/or promotion of glomerular and vascular injury (Advani et al., 2011; Panchapakesan et al., 2011; Zhang et al., 2014). Recent studies have brought to light that EGFR transactivation by angiotensin II and angiotensin II receptor type 1 interaction also plays a vital role in tissue fibrosis (Lautrette et al., 2005). EGFR contributes to Ang II–induced hypertrophy and fibrosis in the heart; mice with an overexpression of dominant-negative EGFR construct exhibit significantly less cardiac fibrosis compared with wild-type littermates when subjected to chronic Ang II infusion (Moriguchi et al., 1999). However, whether EGFR plays a role in Ang II–induced renal fibrosis is not clearly understood.
The classic EGFR inhibitor AG1478 [N-(3-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine], which competitively binds to the ATP pocket of EGFR, causing a conformational change to prevent EGF–EGFR interaction, is widely used in EGFR-related biologic studies (Gan et al., 2007). Using AG1478 as a leading compound, we have designed and synthesized a series of analogs as EGFR inhibitors. Among these analogs, compounds 451 [N-(4-((1H-indol-5-yl)amino)quinazolin-6-yl)acrylamide] and 557 [N-(4-((1-(4-fluorobenzyl)-1H-indol-5-yl)amino)quinazolin-6-yl)acrylamide] exhibited strong and selective EGFR-inhibitory activity at both the molecular and cellular levels, with an IC50 of 0.2 nM and 2.1 nM against recombinant EGFR kinase activity, respectively (Fig. 1A). We used AG1478, 451, and 557 to determine the effects of EGFR inhibition on Ang II–induced renal fibrosis and the underlying mechanism.
Materials and Methods
Ang II, AG1478, and PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] were purchased from Sigma-Aldrich (St. Louis, MO). The recombinant mouse EGF protein with a high-pressure liquid chromatography purity >98.0% was also bought from Sigma-Aldrich. Compounds 451 and 557 were synthesized and characterized using organic chemical methods (as described in Supplemental Figure 1), and were purified using high-pressure liquid chromatography with a purity of 99.3% and 98.5%, respectively. The structures of 451 and 557 are shown in Fig. 1. The compounds AG1478, 451, and 557 were dissolved in dimethylsulfoxide (DMSO) for in vitro experiments and were dissolved in 1% sodium carboxyl methyl cellulose (CMC-Na) for in vivo experiments. The antibodies against Akt, ERK2, Sma- and Mad-related protein (Smad), proto-oncogene tyrosine-protein kinase Src (c-Src), and phosphorylated Akt1/2/3, ERK1/2, c-Src, Smad2/3, as well as collagen IV and GAPDH were purchased from Santa Cruz Biotechnology (Dallas, TX). The antibodies against EGFR and phosphorylated EGFRtyr845 were purchased from Cell Signaling Technology (Beverly, MA), and collagen IV was purchased from Abcam (Cambridge, MA).
The immortalized mice mesangial cell line SV40 and rat tubular cell line NRK-52E were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, People’s Republic of China). Cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium and 25 mM (high-glucose) d-glucose supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin at 37°C in a humidified 5% CO2. For treatment, cells were treated as follows: PP2, 10 µM; AG1478, 10 µM; 451, 0.1 µM, 1 µM, and 10 µM; and 557, 0.1 µM, 1 µM, and 10 µM. All inhibitors were added 1 hour before Ang II or EGF incubation in all experiments. All inhibitors were dissolved in DMSO, and the control cells received an equal amount of DMSO. The cells were then incubated with Ang II (1 µM) or EGF (100 ng/ml) for 5 minutes to detect phosphorylated EGFR, with c-Src for 15 minutes for phosphorylated Akt1/2/3 and ERK1/2, 6 hours for TGF-β and phosphorylated Smad2/3, and 36 hours for collagen IV.
Short Hairpin RNA Transfection.
Short hairpin RNA (shRNA) (1 µg) was mixed with 3 μl Lipofectamine RNAi-MAX in 200 µl of serum-free Opti-MEM and incubated for 20 minutes at room temperature. Then the mix was added into each well containing SV40 (1 × 105) in 1 ml of Dulbecco’s modified Eagle’s medium (fetal bovine serum without antibiotics) and incubated for 48 hours.
The 6-week-old male C57BL/6 mice (n = 48) weighing 18–22 g were obtained from the Animal Centre of Wenzhou Medical University (Wenzhou, People’s Republic of China). The animals were housed with a 12-hour light/dark cycle at a constant room temperature, fed with a standard rodent diet, and provided with free access to water. The animals were acclimatized to the laboratory for at least 2 weeks before the experiments. All animal care and experimental procedures complied with the “Detailed Rules and Regulations of Medical Animal Experiments Administration and Implementation” (Order No. 1998-55, Ministry of Public Health, People’s Republic of China.), and the “Ordinance in Experimental Animal Management” (Order No. 1998-02, Ministry of Science and Technology, People’s Republic of China), and were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval Document No. 2013/APWC/0084). Protocols involving the use of animals were approved by the Wenzhou Medical University Animal Policy and Welfare Committee.
Renal fibrosis was induced in C57BL/6 mice by subcutaneous injection of Ang II (1.4 mg·kg−1 every day for 4 weeks) in phosphate buffer (pH 7.2). C57BL/6 mice were randomly divided into six groups with eight mice in each group: 1) control group: nonrenal fibrosis control mice; 2) Ang II group: Ang II–induced renal fibrosis mice that were subcutaneously injected with Ang II and orally received the vehicle (1% CMC-Na solution) daily for 4 weeks; 3) Ang II + PP2 at 20 mg·kg−1 group; 4) Ang II + AG1478 at 20 mg·kg−1 group; 5) Ang II + 557 at 5.0 mg·kg−1 group; and 6) Ang II + 557 at 20.0 mg·kg−1 group.
In the Ang II + inhibitor groups, Ang II–induced renal fibrosis mice were orally treated with the respective inhibitor (in 1% CMC-Na solution) at the indicated dosage for 4 weeks, starting at 1 day before the first Ang II injection. The Ang II group received the vehicle (1% CMC-Na solution) alone in the same schedule as compound treatment groups. After 4 weeks of treatment, animals were sacrificed under ether anesthesia, and the blood and kidney samples were collected. Kidney tissues were embedded in 4% paraformaldehyde for pathologic analysis and/or snap-frozen in liquid nitrogen for gene and protein expression analysis.
Quantitative Real-Time Polymerase Chain Reaction.
Cells or kidney tissues (20–50 mg) were homogenized in TRIzol (Invitrogen, Carlsbad, CA) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) with an Eppendorf Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany) for qRT-PCR analysis. The following primers were synthesized from Invitrogen: collagen IV, sense: TGGCCTTGGAGGAAACTTTG, and antisense: CTTGGAAACCTTGTGGACCAG; connective tissue growth factor (CTGF), sense: TGGCCTTGGAGGAAACTTTG, and antisense: CTTGGAAACCTTGTGGACCAG; TGF-β, sense: TGACGTCACTGGAGTTGTACGG, and antisense: GGTTCATGTCATGGATGGTGC; and β-actin, sense: CCGTGAAAAGATGACCCAGA, and antisense: TACGACCAGAGGCATACAG. The amount of each gene was determined and normalized to the amount of β-actin.
Cells or renal tissues (30–50 mg) were lysed, and the protein concentrations were determined by using the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA). Aliquots (about 100 μg of cellular protein) were subjected to electrophoresis and transfer to polyvinylidene fluoride membranes, which were then blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat milk. The polyvinylidene fluoride membrane was then incubated overnight with specific antibodies. After incubation with the appropriate secondary antibodies, immunoreactive proteins were visualized with electrochemiluminescence (Bio-Rad Laboratories) reagent and quantitated by densitometry. The amounts of the proteins were analyzed using Image J analysis software version 1.38e (http://imagej.nih.gov/ij/) and were normalized to their respective control.
Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin sections (5 µm) were stained with H&E. Sections were also stained with Masson trichrome stain (Nanjing KerGEN Bioengineering Institute, Jiangsu, People’s Republic of China) and Sirius Red. To estimate the extent of damage, the specimen was observed under a light microscope (400× amplification; Nikon, Tokyo, Japan).
The renal sections (5 µm) were deparaffinized and rehydrated, and then subjected to antigen retrieval in 0.01 M citrate buffer (pH 6.0) by microwaving. After blocking with 5% bovine serum albumin, the sections were incubated with anti-collagen IV antibody (1:500) overnight at 4°C, followed by the secondary antibody (1:100). The nucleus was stained with 4ʹ,6-diamidino-2-phenylindole, and sections were then viewed under the Nikon fluorescence microscope (400× amplification; Nikon).
Measurements of the Level of Serum Biomarkers.
The components of serum including the albumin, creatinine, and urine protein, were detected using commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, People’s Republic of China).
All data represent at least three independent experiments and are expressed as mean ± S.E.M. All statistical analyses were performed using GraphPad Pro Prism 5.0 (GraphPad, San Diego, CA). Student’s t test and two-way analysis of variance was employed to analyze the differences between sets of data. P < 0.05 was considered statistically significant.
AG1478, 451, and 557 Inhibited the EGFR Signaling Pathway Induced by EGF in Mesangial Cell SV40.
We first validated whether EGFR inhibitors suppressed EGFR signaling pathway in vitro. As shown in Supplemental Fig. 2, EGF stimulation for 5 minutes increased EGFR phosphorylation in SV40 cells in a dose-dependent manner. The dosage 100 ng/ml of EGF was used for the following experiments.
Renal mesangial SV40 cells were pretreated with AG1478, 451, or 557 for 1 hour, followed by incubation with EGF for 5 minutes. Western blot assay showed that renal mesangial cells had a high expression of EGFR protein (Fig. 1C). Acute EGF treatment significantly induced phosphorylation in tyrosine 845 residue of EGFR and activated its classic downstream signaling pathways, including Akt1/2/3 and ERK1/2 (Fig. 1, C–E). EGFR inhibitors, such as AG1478, 451, and 557, remarkably decreased EGFRTyr 845 phosphorylation (Fig. 1C) and its downstream Akt1/2/3 (Fig. 1D) and ERK1/2 phosphorylation (Fig. 1E) in the presence of EGF, suggesting that these inhibitors antagonized the EGF signaling pathway in renal mesangial cells.
AG1478, 451, and 557 Suppressed the Ang II–Induced EGFR Signaling Pathway in SV40 Cells.
In addition to endogenous EGFR ligands, the EGFR signaling pathway can also be transactivated by Ang II (Lautrette et al., 2005). To examine whether novel EGFR inhibitors can overcome Ang II–induced EGFR transactivation, SV40 cells were pretreated with the AG1478, 451, or 557 at various concentration for 1 hour and then stimulated with Ang II (1 μM) for 5 minutes. Ang II activated the EGFR signaling pathway, as evidenced by an increase of EGFR, Akt, and ERK phosphorylation; 451 and 557 were able to reduce EGFR, Akt, and ERK phosphorylation in a dose-dependent fashion (Fig. 2, A–C). These data indicate that EGFR inhibitors suppress Ang II–induced transactivation of the EGFR signaling pathway in SV40 cells. We observed similar results in rat renal tubular NRK-52E cells (Supplemental Fig. 3).
EGFR Inhibitors 451 and 557 Attenuate Ang II–Induced Increase in Profibrosis Proteins in Renal Cells.
Ang II is one of the major contributors to renal fibrosis, and ACE inhibitor or angiotensin receptor blockers can block the effect of Ang II on kidney fibrosis (Mezzano et al., 2001). Because EGFR inhibitors efficiently abolish the EGFR transactivation by Ang II, we further studied the effect of EGFR inhibitors on profibrosis signaling pathway in renal cells. Figure 3 shows that Ang II significantly increased TGF-β mRNA and protein expression in SV40 cells (Fig. 3, A and D). As a consequence, downstream signaling such as Samd phosphorylation as well as the expression of extracellular matrix proteins including CTGF and collagen IV were all up-regulated, as measured by both qRT-PCR and immunoblotting (Fig. 3, B and C, E and F). The EGFR inhibitors 451 and 557 were able to attenuate the TGF-β signaling and fibrotic protein expression in a dose-dependent fashion in SV40 cells (Fig. 3, A–F). Furthermore, similar results were observed in rat renal tubular NRK-52E cells (Supplemental Fig. 3). These results indicate that these novel compounds exert an antifibrosis effect in renal mesangial cells.
EGFR Is a Key Regulator of Profibrotic Signaling Activation.
To investigate whether EGFR modulates renal fibrosis, we examined the effects of shRNA knockdown of EGFR on Ang II–stimulated renal fibrotic signaling activation. Compared with scrambled vector, transfection of cells with specific shRNA against EGFR reduced EGFR protein abundance by more than 60% (Fig. 4A) in SV40 cells and attenuated fibrotic markers in the presence of Ang II (Fig. 4, B–D), indicating that EGFR mediates Ang II–induced renal profibrosis pathway.
Based on these data, we further hypothesized that EGFR activation independent from Ang II could also induce renal fibrosis. To test this hypothesis, we studied TGF-β signaling pathway as well as fibrotic markers in EGF-stimulated SV40 cells. EGF alone was able to activate TGF-β production and induce downstream signaling SMAD phosphorylation (Fig. 4, E and F, H). As a result, the extracellular matrix proteins (including CTGF and collagen IV), were up-regulated (Fig. 4, G, I, and J). Supplemental Fig. 4 also shows that EGF treatment increased the levels of p-Smad2/3, TGF-β, and collagen IV in SV40 cells in a dose-dependent manner. Pretreatment of EGFR inhibitors could down-regulate the expression the fibrotic markers stimulated by EGF (Fig. 4, E–J). Taken together, these data indicate that EGFR signaling pathway plays a key role in renal profibrosis signaling activation.
Ang II–Inducing EGFR Phosphorylation Depends on Src.
Previous studies suggested that c-Src plays an important role in Ang II–induced EGFR transactivation in type 1 diabetic mice (Taniguchi et al., 2013). To investigate whether c-Src mediates Ang II–induced EGFR transactivation in a renal fibrosis model, SV40 cells were pretreated with the c-Src inhibitor PP2 (10 µM) for 1 hour before Ang II (1 μM) stimulation. PP2 was shown to inhibit the phosphorylation of c-Src and EGFR stimulated by Ang II (Fig. 5, A–B). However, knockdown of EGFR by shRNA had no effect on Ang II–induced Src phosphorylation (Fig. 5C). These data suggest that c-Src functions as an upstream of EGFR signaling pathway and mediates Ang II–induced EGFR transactivation (Fig. 5D).
EGFR Inhibitor 557 Suppressed Ang II–Induced EGFR Signaling Pathways and Fibrosis In Vivo.
To validate the in vivo function of EGFR inhibitors, kidney fibrosis was induced in 6-week-old male C57BL/6 mice by subcutaneous injection of Ang II (1.5 mg.kg−1.d−1) for 1 month. The animals were administered PP2 (20.0 mg·kg−1), AG1478 (20.0 mg·kg−1), or compound 557 (5.0 mg·kg−1 or 20.0 mg·kg−1) by gavage at day 1 before the Ang II injection. During the animal experiments, no toxicity and abnormality were observed in all experimental mice (data not shown).
Ang II did not alter mouse body weight over the treatment period (Fig. 6A), but it did increase the kidney weight/body weight ratio (Fig. 6B), serum creatinine (Fig. 6C), urine protein (Fig. 6D), and serum albumin (Fig. 6E), indicating an impaired kidney function. Moreover, the kidney histologic staining analysis revealed elevated kidney fibrosis with Ang II treatment alone compared with the control animals (Fig. 6F). Administration of PP2, AG1478, or 557 reversed the pathologic effect of Ang II on kidney weight and function (Fig. 6, B–E). EGFR inhibitor 557 was also able to reduce kidney histologic abnormalities (H&E staining), renal fibrosis (Sirius Red and Masson Trichrome Blue staining), and collagen accumulation (anti-collagen IV staining) in Ang II–treated mice in a dose-dependent manner (Fig. 6F). The quantified data for the fibrosis degree in Masson, Sirius Red, and anti-collagen IV staining are shown in Supplemental Fig. 5.
For the mechanistic study, we examined the key signaling molecules such as c-Src, EGFR, Akt, and ERK in the Ang II–induced activation of the EGFR pathway. Treatment with 20 mg/kg of PP2 was able to attenuate the phosphorylation of Src, EGFR, Akt, and ERK caused by infusion of Ang II. High-dose 557 (20 mg/kg) suppressed the activation of the EGFR, Akt, and ERK signaling pathways but not on c-Src phosphorylation induced by Ang II; low-dose 557 (5 mg/kg) had minimal effect on the EGFR signaling pathway (Fig. 7A). These data suggest that 557 at 20 mg/kg was able to block the Ang II–induced EGFR signaling pathways in vivo. We also found that the c-Src inhibitor PP2 attenuated the production of fibrotic markers in Ang II–infused animal kidneys. Consistent with our observations about signaling molecules, high-dose 557 (20 mg/kg) was able to suppress the expression of fibrotic markers, including TGF-β, SMAD2/3, CTGF, and collagen IV (Fig. 7, B–E); low-dose 557 (5 mg/kg) only attenuated CTGF production but had no obvious effect on TGF-β or collagen IV (Fig. 7, C–E). Taken together, these pathologic and mechanistic analyses reveal that the EGFR inhibitor 557 prevents kidney abnormalities and fibrosis induced by Ang II.
CKD with end-stage renal fibrosis is a debilitating problem that has no known cure. There is growing evidence of the role of the EGFR pathway in various types of renal lesions. Increased EGFR activation and expression have been correlated with interstitial fibrosis and tubular atrophy in human renal allograft biopsies (Sis et al., 2004). EGFR activation is involved in endothelin-induced renal vascular and glomerular fibrosis (Francois et al., 2004). However, the underlying mechanism by which EGFR activation contributes to renal fibrosis remains unknown.
In our study using pharmacologic approaches, we illustrate the importance of EGFR in the development of renal fibrosis. EGFR inhibition by small molecules suppressed TGF-β up-regulation, activation of Smad2/3, and accumulation of collagen IV in a model of renal fibrosis induced by Ang II infusion; c-Src activity is required for transactivation of EGFR signaling by Ang II. In addition, we observed similarly that EGFR inhibitors attenuated Ang II–induced EGFR phosphorylation and profibrosis protein up-regulation in rat renal proximal tubular NRK-52E cells (Supplemental Fig. 3), indicating that EGFR is involved in Ang II–induced renal interstitial fibrosis.
EGFR is widely expressed in mammalian kidneys, including glomeruli, proximal tubules, and cortical and medullary collecting ducts, and expression increases in both glomeruli and tubules in response to diabetes (Gesualdo et al., 1996). Our data also show that both SV40 cells and kidney tissue have a high EGFR expression level (Figs. 1, 2, and 7), as do rat renal proximal tubular NRK-52E cells (Supplemental Fig. 3). Given recent studies indicating that angiotensin II regulates glomerular hemodynamics, filtration rate, and macromolecular permeability and contributes to fibrosis and glomerular injury (Navar, 2014), it is likely that EGFR plays a pathogenic role in multiple cell types of the nephron. Studies from Zhang et al. (2014) and other groups support EGFR as important mediator of renal repair after acute injury and also describe a detrimental role of persistent EGFR activation in progressive renal fibrosis induced by diabetes, subtotal nephrectomy, unilateral ureteral obstruction, and renovascular hypertension. Our current study indicates an important role for EGFR activation in mediating kidney injury induced by Ang II as well. Our finding of a protective role of 451 and 557 concurs with a previous study on erlotinib, a structurally different EGFR inhibitor (Chen et al., 2012b) that was also found to inhibit kidney injury induced by Ang II (Figs. 3, 6, and 7).
Pathologic kidney fibrosis is a consequence of maladaptive alterations and is an important pathologic process in diverse kidney diseases. A role for RAS has been well documented in both diabetic and nondiabetic progressive renal injury, and the ameliorative effects of RAS inhibition retard not only glomerular but also tubulointerstitial injury in progressive disease (van der Meer et al., 2010; Miyata et al., 2014; Yacoub and Campbell, 2015). Activation of angiotensin II receptor type 1 by Ang II induces TGF-β signaling and renal fibrosis via several signaling molecules, including tyrosine kinases such as Akt and mitogen-activated protein kinases (MAPKs) (Lautrette et al., 2005). The EGFR pathway activation has also been shown to contribute to the development of kidney fibrosis (Tang et al., 2013). Abrogation of EGFR kinase activity by selective pharmacologic inhibitors or gene deletion significantly protects against kidney fibrosis (Francois et al., 2004; Liu et al., 2013). For example, transactivation of EGFR by high glucose leads to collagen synthesis in mesangial cells, and EGFR inhibition by erlotinib attenuates the development of diabetic nephropathy in type 1 diabetes (Zhang et al., 2014). Our results reveal that the EGFR inhibition by either three small-molecule inhibitors or by shRNA silencing could attenuate kidney fibrosis in Ang II–treated SV40 cells (Figs. 3 and 4, A–D). Administration of AG1478 and 557 also significantly reversed Ang II–induced kidney fibrosis in a mouse model in vivo (Figs. 6 and 7). These results indicate that EGFR plays a critical role in mediating the induction of kidney fibrosis by Ang II, which may be one of mechanisms by which EGFR inhibition attenuates a variety of kidney diseases.
EGFR mediates renal fibrosis possibly through its prominent downstream Akt and MAPK signaling pathways. Growing evidence supports a potential role for active Akt in the process of renal fibrosis and kidney dysfunction in view of its targeted effects on multiple pathogenic pathways. It is reported that Akt2 contributes to the epithelial-to-mesenchymal transition and to interstitial fibrosis after unilateral ureteral obstruction (UUO) (Lan et al., 2014). Further evidence of the involvement of the phosphoinositide 3-kinase (PI3K)–Akt signaling pathway in renal damage has been provided by a recent study that reported that early pharmacologic down-regulation of the PI3K–Akt signaling pathway reduced not only the profibrotic interstitial cells but also the potential number of tubular cells that have been described as responsible for excessive interstitial matrix production at later stages of UUO (Yoon et al., 2014).
The MAPK pathways are well characterized in regulating ECM expression. A previous report showed that blockade of ERK after the emergence of established fibrosis is effective for reducing subsequent renal fibrosis in the UUO model (Pat et al., 2005). Downstream targets of ERK and PI3K–Akt include transcription factors such as SMAD2/3, which regulates the expression of fibrotic genes such as TGF-β, procollagen I, procollagen IV, and CTGF (Coresh et al., 2007). Our data demonstrate that the phosphorylation of both ERK1/2 and Akt is elevated in Ang II–treated mesangial cells and mice, accompanied with fibrosis. Three EGFR inhibitors markedly inhibited both ERK1/2 and Akt activation in vivo and in vitro in response to Ang II. These findings confirm Akt and ERK as pivotal regulators of kidney fibrosis and suggest that the profibrotic effect of EGFR in kidney may be mediated by its downstream Akt and ERK signaling pathways.
Activation of EGFR also triggers the TGF-β/Smad signaling pathway (Fig. 4), the major mechanism that regulates the accumulation of extracellular matrix proteins, collagen, and fibronectin in renal fibrosis. Previous studies have shown that blockade of TGF-β diminishes Ang II–induced ECM production (Ruiz-Ortega et al., 2007). In our study, inhibition of EGFR reduced the production of TGF-β, activation of Smad2/3, and accumulation of collagen. These findings verify that the profibrotic effect of EGFR in kidney depends on the TGF-β/Smad signaling pathways.
A limitation of our study is the missing in vivo blood pressure data. Ang II injection could increase the blood pressure of mice. However, a recent study demonstrated that treatment with EGFR inhibitor erlotinib attenuated Ang II–induced vascular remodeling and cardiac hypertrophy but not hypertension in mice, indicating that the mechanisms by which Ang II elevates blood pressure and enhances end-organ damage may be distinct (Takayanagi et al., 2015). Another independent group found that Ang II–induced hypertrophy in cerebral arterioles involves EGFR signaling, which is independent of blood pressure (Chan et al., 2015). Although these previous studies have confirmed that EGFR inhibition has no effect on Ang II–induced blood pressure, there may be differences from experiment to experiment. The beneficial effect of EGFR inhibition on renal histology may be independent of blood pressure.
The mechanism by which Ang II activates EGFR signaling is still unclear. Activation of EGFR occurs either by binding with ligands, such as EGF and heparin bound-EGF, or by transactivation. Previous studies demonstrated that Ang II induces c-Src activation (Chen et al., 2012a). In addition, c-Src-dependent transactivation of EGFR signaling pathway is essential for collagen synthesis in mesangial cells from diabetic mice (Taniguchi et al., 2013). In the present study, we showed that the c-Src inhibitor PP2 significantly blocked Ang II–induced EGFR phosphorylation, and EGFR shRNA had no effect on Ang II–induced c-Src phosphorylation. These data indicate that c-Src functions as an upstream signaler to mediate EGFR transactivation by Ang II (Fig. 5D). Another important finding is that EGFR-stimulated renal fibrosis can also be Ang II–independent, as evidenced by the fact that EGF stimulation alone induced kidney fibrosis in SV40 cells (Fig. 4, E–J). This suggests that EGFR autoactivation or EGFR overexpression resulted from genetic mutation, which occurs frequently in cancer genesis and development and may also be a risk factor for kidney diseases, although no tumorigenesis was observed in the kidneys. Future studies should explore EGFR as a biomarker or a therapeutic target for kidney diseases or even as a cross-linker for kidney diseases and cancer, especially EGFR-positive cancer.
In summary, we have demonstrated that EGFR plays a key role in kidney fibrosis, and EGFR inhibition by either small-molecule inhibitors or genetic silencing can protect against Ang II–induced kidney fibrosis. Based on observations from the literature and our findings, Fig. 5D depicts a model for the signaling mechanisms linking Ang II, EGFR, and kidney fibrosis. Ang II transactivates EGFR through c-Src, which further stimulates Akt and ERK signaling pathways to induce the TGF-β/SMAD pathway and fibrotic gene expression. Pharmacologic approaches that target EGFR may be a promising therapeutic strategy for kidney diseases. Although several EGFR specific inhibitors and antibodies have been used for anticancer treatment clinically, the extent to which inhibition of EGFR is clinically applicable to the prevention and treatment of kidney diseases in human patient remains to be determined.
Participated in research design: G. Liang, Wang, Qian, Peng.
Conducted experiments: Qian, Qiu, Xu, Zhang, D. Liang, Zou.
Contributed new reagents or analytic tools: Huang, Hu.
Performed data analysis: G. Liang, Peng.
Wrote or contributed to the writing of the manuscript: G. Liang, Peng, Skibba.
- Received July 25, 2015.
- Accepted October 27, 2015.
Y.Q. and K.P. contributed equally to this work.
This work was supported by the National Natural Science Funding of China (81503123, 81502912, and 21472142), Zhejiang Provincial Natural Science Funding (LQ15H120005, LQ14H310003, and LY13H060007), and High-Level Innovative Talent Funding of Zhejiang Department of Health (2010-017).
- angiotensin-converting enzyme
- protein kinase B
- Ang II
- angiotensin II
- chronic kidney disease
- sodium carboxyl methyl cellulose
- proto-oncogene tyrosine-protein kinase Src
- connective tissue growth factor
- extracellular matrix
- epidermal growth factor
- epidermal growth factor receptor
- extracellular signal-related kinase
- mitogen-activated protein kinase
- phosphoinositide 3-kinase
- quantitative reverse transcription polymerase chain reaction
- renin-angiotensin system
- short hairpin RNA
- Sma- and Mad-related protein
- transforming growth factor-β
- unilateral ureteral obstruction
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics