EGFR mediates hyperlipidemia-induced renal injury via regulating inflammation and oxidative stress: the detrimental role and mechanism of EGFR activation

Abstract

Previous studies have implicated inflammation, oxidative stress, and fibrosis as key factors in the development of obesity-induced kidney diseases. Epidermal growth factor receptor (EGFR) plays an important role in cancer development. Recently, the EGFR pathway has been increasingly implicated in chronic cardiovascular diseases via regulating inflammation and oxidative stress. However, it is unclear if EGFR is involved in obesity-related kidney injury. Using ApoE-/- and C57BL/6 mice models and two specific EGFR inhibitors, we investigated the potential effects of EGFR inhibition in the treatment of obesity-related nephropathy and found that EGFR inhibition alleviates renal inflammation, oxidative stress and fibrosis. In NRK-52E cells, we also elucidated the mechanism behind hyperlipidemia-induced EGFR activation. We observed that c-Src and EGFR forms a complex, and following PA stimulation, it is the successive phosphorylation, not formation, of the c-Src/EGFR complex that results in the subsequent cascade activation. Second, we found that TLR4 regulates the activation EGFR pathway mainly through the phosphorylation of the c-Src/EGFR complex. These results demonstrate the detrimental role of EGFR in the pathogenesis of obesity-related nephropathy, provide a new understanding of the mechanism behind hyperlipidemia/FFA-induced EGFR activation, and support the use of EGFR inhibitors in the treatment of obesity-induced kidney diseases.

Keywords: c-Src; epidermal growth factor receptor; obesity; obesity-induced kidney injury; palmitate.

Figure 1. Oral administration of EGFR inhibitors suppressed HFD-induced EGFR signaling and attenuated kidney injury in ApoE^−/− mice

A. Chemical structure of 542. B. Orally administered 542 significantly inhibited EGFR signaling, including phosphorylation of EGFR, AKT and ERK, in high fat diet (HFD)-fed ApoE^−/− mice.(Shown are representative western blots, n=2 in control group; n=3 in other three groups). C-G. 542 significantly improved structural changes and renal function in kidneys of obese mice. C. H&E staining was used for the analysis of histological abnormalities, PAS staining was used for the detection of glycogen (purple) in kidney section. D-G. BUN, creatinine, and urinary protein levels, as well as kidney/body weight ratio, were measured for the renal function test. Body weight and kidney weight of mice were recorded at the time of death. Data are means ± SEM (n=8 in four groups; ns, no significance; * p<0.05, ** p<0.01; vs. HFD group; LFD, low-fat diet; HFD, high-fat diet).

Figure 2. Oral administration of EGFR inhibitors improved HFD-induced fibrosis in the kidneys of ApoE^−/− mice

A. Representative images of histological abnormalities in obese renal tissue. (400X). Masson staining was used for the detection of fibrosis (blue, respectively) in the kidney sections. B-D. Administration of 542 reduced the gene expression of the indicated genes related to fibrosis in the kidney tissue of obese mice. Renal tissues from each group were individually processed for mRNA extraction and RT-qPCR analysis. The mRNA levels of Collagen1 (B), TGF-β (C) and CTGF (D) were normalized by β-actin (n=5-7; ns, no significance; * p<0.05, ** p<0.01; vs. HFD group). E. Administration of 542 reduced the production of fibrotic cytokines and pro-apoptotic protein Bax, while increasing the expression of anti-apoptotic protein Bcl-2. Shown are representative western blots, n=2 in control group; n=3 in other three groups. (LFD, low-fat diet; HFD, high-fat diet)

Figure 3. Oral administration of EGFR inhibitors reduced HFD-induced inflammation and ROS production in the kidneys of ApoE^−/− mice

A. The administration of 542 for 2 months significantly reduced HFD-induced increases of kidney inflammation, including expression of TNF-α, and macrophage infiltration as characterized by CD68 staining. B-D. 542 also reduced the mRNA expression of TNF-α (B) and other inflammatory cytokines, such as IL-6 (C) and IL-1β (D). E. Administration of 542 also inhibited the degradation of IκB and expression of adhesion factors VCAM-1. F-I. 542 administration reduced the HFD-induced production of reactive oxygen species (ROS). Immunohistochemistry revealed that 542 decreased HFD-induced 3-NT (F) and RT-qPCR showed 542 increased mRNA levels of antioxidants, such as Nrf2 (G), NQO-1 (H) and Gclc (I), as well as the protein levels of antioxidant proteins NQO-1 and Gclc (E). (n=7/8; *p<0.05, **p<0.01, ***p<0.001; vs. HFD group; LFD, low-fat diet; HFD, high-fat diet)

Figure 4. EGFR silencing inhibited PA-induced activation of EGFR signaling, inflammation, oxidation and fibrosis in NRK-52E cells

A. PA-stimulated time-dependent activation of EGFR signaling. NRK-52E cells were stimulated with PA (100uM) for different various time periods (5, 15, 30, 60 and 120 min). Western blot analysis was then used to examine EGFR activation, including EGFR, AKT and ERK phosphorylation. B. Following PA stimulation for 15 min, total proteins were extracted from the cell lysate, and the phosphorylation levels of EGFR, AKT and ERK were examined by Western blot analysis using GAPDH as a loading control. C. Following PA stimulation for 30 min, the total proteins were extracted from the cell lysate, and the level of IκBα were examined by Western blot analysis with GAPDH as a loading control. After PA stimulation for 2 h, the levels of adhesion factors VCAM-1 and ICAM-1 were examined also by Western blot with GAPDH as a loading control. F. NRK-52E cells were pre-treated with Si-EGFR for 24h to block the EGFR gene and then stimulated with PA for 6h. Flow Cytometry was used for detecting the ROS. G. After PA stimulation for 12 h, antioxidant proteins NQO-1 and Gclc were examined by Western blot with GAPDH as a loading control. J. After PA stimulation for 24 h, fibrotic factors TGF-β and Collagen4, pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were examined by Western blot with GAPDH as a loading control. D-E, H-I, K-L. NRK-52E cells were pre-treated with Si-EGFR for 24h to block the EGFR gene and then stimulated with PA for 24h. Total RNAs were extracted, and the mRNA levels of inflammatory, oxidant and fibrotic cytokines, such as TNF-α (D), IL-6 (E), Gclc (G), HO-1 and NQO-1 (H), TGF-β (J), CTGF and Collagen1 (K), were detected by real-time qPCR. (*p<0.05, **p<0.01, ***p<0.001; vs. the PA Group).

Figure 5. TLR4 regulated PA-induced phosphorylation of EGFR and c-Src in the EGFR/c-Src complex in NRK-52E cells (TLR4 silencing inhibits PA-induced phosphorylation of c-Src in the EGFR/c-Src complex and EGFR signaling in NRK-52E cells)

A. c-Src is upstream of EGFR. NRK-52E cells were pre-treated with AG (inhibitor of EGFR) and PP2 (inhibitor of c-Src) for 1 h and then stimulated with PA for 15 min. Using Western blot analysis, we detected the c-Src and EGFR phosphorylation. Both AG and PP2 suppressed EGFR phosphorylation, but while PP2 inhibited PA-induced c-Src phosphorylation, AG had no effect. B. Immunofluorescence revealed that EGFR binds with c-Src without any stimulation. C-D. In the c-Src/EGFR complex, PA stimulated the phosphorylation of c-Src and then EGFR. Co-immunoprecipitation showed that in the complex, PA induced the phosphorylation of EGFR first 5 min after PA stimulation and s-Src 15 min after stimulation. E. Through co-immunoprecipitation, we further revealed that PP2 inhibited the phosphorylation of c-Src, whereas AG had no effect on c-Src phosphorylation in the c-Src/EGFR complex. F. Western blot analysis showing the efficiency of using SiRNA to silence TLR4 in NRK-52E cells. G. Silencing of TLR4 blocked PA-induced activation of EGFR signaling, inhibiting EGFR, c-Src, AKT and ERK phosphorylation. H. Silencing of TLR4 also significantly blocked PA-induced phosphorylation of c-Src in the EGFR/c-Src complex.

Figure 6. Inhibition of AKT and ERK attenuates PA-induced inflammation, ROS production, fibrosis and apoptosis in NRK-52E cells

A. The efficiency of AKT inhibitor MK and ERK inhibitor PD when used the same concentration. B-C. MK and PD attenuated PA-induced inflammation and oxidation. MK and PD inhibited the degradation of IκB and production of ROS. MK and PD also increased the expression of antioxidant proteins NQO-1 and Gclc. D. MK and PD also inhibited PA-induced expression of fibrotic factors TGF-β, Collagen4, and MMP9 pro-apoptotic proteins Bax and anti-apoptotic protein Bcl-2. E. The proposed mechanism of TLR4/c-Src/EGFR signaling in obesity-related glomerulopathy (ORG).