Genetic or pharmacologic inhibition of EGFR ameliorates sepsis-induced AKI

Abstract

Despite recent studies have demonstrated that the EGF receptor (EGFR) activation provided a renoprotective role during ischemic and folic acid-induced AKI, the role and regulation mechanism of EGFR in septic AKI remains unclear. Here, gefitinib, a highly selective EGFR inhibitor, abrogated LPS-induced phosphorylation of EGFR, ERK1/2, and STAT3 as well as expression of COX, eNOS, and proinflammatory cytokines in HK-2 cells. In addition, c-Src is an upstream of EGFR signaling pathway and mediates LPS-induced EGFR transactivation. In vivo, either gefitinib or genetic approaches (Wave-2 mutant mice, which have reduced EGFR tyrosine kinase activity) protected against LPS or cecal ligation and puncture (CLP) induced AKI respectively. Interestingly, the beneficial effects of gefitinib or genetic approaches were accompanied by the dephosphorylation of EGFR, ERK1/2, and STAT3, the down regulation of expression of COX, eNOS, macrophage infiltration, proinflammatory cytokines production and the renal cell apoptosis. Furthermore, mRNA array results indicated that gene families involved in cell death, inflammation, proliferation and signal transduction were down regulated in Wave-2 (Wa-2) mice. Take together, these data suggest that EGFR may mediate renal injury by promoting production of inflammatory factors and cell apoptosis. Inhibition of EGFR may have therapeutic potential for AKI during endotoxemia.

Keywords: CLP; EGFR; inflammation.

Figure 1. LPS induced activation of EGFR, ERK1/2, and STAT3 pathways in HK-2 cells

(A) HK-2 cells were treated with 10μg/ml LPS for 0, 15, 30, or 60 min to collect lysate for immunoblot analysis of expression and activation of EGFR, STAT3, and ERK1/2. (B) Densitometric analysis of the p-EGFR/EGFR, p-ERK1/2/ERK1/2, and p-STAT3/STAT3 ratio. * P<0.05 versus the other group. Data are representative of at least four separate experiments.

Figure 2. Gefitinib suppressed LPS induced ERK1/2 and STAT3 phosphorylation by blocking EGFR activation in HK-2 cells

(A) HK-2 cells were treated with 10μg/ml LPS in presence or absence of 5 nM gefitinib for 30 min to collect lysate for immunoblot analysis of expression and activation of EGFR, STAT3, and ERK1/2. (B) Densitometric analysis of the p-EGFR/EGFR, p-ERK1/2/ERK1/2, and p-STAT3/STAT3 ratio. * P<0.05 versus the other group. Data are representative of at least four separate experiments.

Figure 3. c-Src mediates the LPS induced EGFR activation in HK-2 cells

(A) HK-2 cells were treated with 10μg/ml LPS in presence or absence of 20 nmol dasatinib for 30 min to collect lysate for immunoblot analysis of expression and activation of Src and EGFR. (B) Densitometric analysis of the p-EGFR/EGFR and p-Src/Src ratio. (C) HK-2 cells were treated with 10μg/ml LPS after the transfection of EGFR siRNA or siRNA-NC for 30 min to collect lysate for immunoblot analysis of expression and activation of Src and EGFR, densitometry (D) of proteins signals on immunoblots. * P<0.05 versus mock group; # P<0.05 versus LPS group; & P>0.05 versus LPS group. Data are representative of at least four separate experiments.

Figure 4. Both ERK1/2 and STAT3 play different role in LPS induced gene expression

(A) HK-2 cells were treated with 10μg/ml LPS in presence or absence of 400 nmol U0126 for 24 h to collect lysate for immunoblot analysis of expression of COX-2 and eNOS. (B) Densitometric analysis of the COX-2/β-actin and eNOS/β-actin. (C) HK-2 cells were also treated with 10μg/ml LPS in presence or absence of 50μM S3I-201 for 24 h to collect lysate for RT-PCR analysis of expression of ICAM-1, TNF-α, and TGF-β1. (D) Densitometric analysis of the ICAM-1/GAPDH, TNF-α/GAPDH, and TGF-β1/GAPDH ratio. * P<0.05 versus mock group; #P<0.05 versus LPS group. Data are representative of at least four separate experiments.

Figure 5. Effect of gefitinib on LPS-induced renal tissue damage, BUN, and serum creatinine in mice

Male C57 mice were injected with 10 mg/kg LPS with or without 100 mg/kg gefitinib. As a control, the mice were injected with saline. Kidney tissues and blood samples were collected at 24 h after treatment. (A) Kidney sections were stained with hematoxylin and eosin to assess tubular damage. Tubular damage scores of kidney cortex (B), tubular damage scores of OSOM (C), BUN (D), and serum creatinine (E). Data are shown as mean ± S.E.M. (n=10). Data are presented as mean ± SEM; * P<0.05 versus saline group; #P<0.05 versus LPS group. Data are represe ntative of at least four separate experiments. Original magnification, x200 in A.

Figure 6. Gefitinib inhibited infiltration of macrophages in a LPS induced AKI model

Male C57 mice were injected with 10 mg/kg LPS with or without 100 mg/kg gefitinib. As a control, the mice were injected with saline. Kidney tissues were collected at 24 h after treatment. (A) Representative macrophage immunohistochemistry from kidney cortical tissues of different groups at 24h (original magnification, ×400). (B) Quantitation of macrophage-positive cells per total cross-sectional area was performed from each group (n=10). Data are presented as mean ± SEM; * P<0.05 versus saline group; #P<0.05 versus LPS group. Data are representative of at least four separate experiments. Original magnification, x200 in A.

Figure 7. Gefitinib suppressed phosphorylation of ERK1/2 and STAT3, and expression of COX-2 and eNOS by blocking EGFR activation in a LPS induced AKI model

Male C57 mice were injected with 10 mg/kg LPS with or without 100 mg/kg gefitinib. As a control, the mice were injected with saline. Kidney tissues were collected at 24 h after treatment. (A) Immunoblot analysis of expression and activation of EGFR, STAT3, and ERK1/2, and expression of COX-2 and eNOS. (B-F) Densitometric analysis of the p-EGFR/EGFR, p-ERK1/2/ERK1/2, p-STAT3/STAT3, COX-2/β-actin, and eNOS/β-actin ratio. Data are presented as mean ± SEM; * P<0.05 versus saline group; #P<0.05 versus LPS group. Data are representative of at least four separate experiments.

Figure 8. Gefitinib suppressed expression of ICAM-1, TNF-α, and TGF-β1 by blocking EGFR activation in a LPS induced AKI model

Male C57 mice were injected with 10 mg/kg LPS with or without 100 mg/kg gefitinib. As a control, the mice were injected with saline. Kidney tissues were collected at 24 h after treatment. (A) RT-PCR analysis of expression of ICAM-1, TNF-α, TGF-β1, and IL-1β. (B-E) Densitometric analysis of the ICAM-1/GAPDH, TNF-α/GAPDH, and TGF-β1/GAPDH, IL-1β/ GAPDH ratio. Data are presented as mean ± SEM; * P<0.05 versus saline group; #P<0.05 versus LPS group. Data are representative of at least four separate experiments.

Figure 9. CLP induced AKI is attenuated in Wa-2 mice

Wild-type and Wa-2 littermate mice were subjected to CLP. Sham-operated mice were used as a control. Kidney tissues and blood samples were collected at 18 h after treatment. (A) Kidney sections were stained with hematoxylin and eosin to assess tubular damage. Tubular damage scores of kidney cortex (B), tubular damage scores of OSOM (C), BUN (D), and serum creatinine (E). Data are shown as mean ± S.E.M. (n=8); * P<0.05 WT/Sham group; #P<0.05 versus CLP group. Data are representative of at least four separate experiments. Original magnification, x200 in A.

Figure 10. CLP induced renal cell apoptosis is reduced in Wa-2 mice

Wild-type and Wa-2 littermate mice were subjected to CLP. Sham-operated mice were used as a control. Kidney tissues were collected at 18 h after treatment. (A) TUNEL assay and cleaved caspase3 to reveal apoptosis (original magnification, ×200). Quantification of TUNEL-positive cells (B) and cleaved caspase3 (C) in CLP-treated cortical tissues. Data were expressed as means ± SD; * P<0.05 versus Wa-2 with CLP group. Data are representative of at least four separate experiments.