Role of the transcription factor erythroblastosis virus E26 oncogen homolog-1 (ETS-1) as mediator of the renal proinflammatory and profibrotic effects of angiotensin II

Abstract

Angiotensin II (Ang II) plays a major role in the pathogenesis of end-organ injury in hypertension via its diverse hemodynamic and nonhemodynamic effects. Erythroblastosis virus E26 oncogen homolog-1 (ETS-1) is an important transcription factor recently recognized as an important mediator of cell proliferation, inflammation, and fibrosis. In the present studies, we tested the hypothesis that ETS-1 is a common mediator of the renal proinflammatory and profibrotic effects of Ang II. C57BL6 mice (n=6 per group) were infused with vehicle (control), Ang II (1.4 mg/kg per day), Ang II and an ETS-1 dominant-negative peptide (10 mg/kg per day), or Ang II and an ETS-1 mutant peptide (10 mg/kg per day) via osmotic minipump for 2 or 4 weeks. The infusion of Ang II resulted in significant increases in blood pressure and left ventricular hypertrophy, which were not modified by ETS-1 blockade. The administration of ETS-1 dominant-negative peptide significantly attenuated Ang II-induced renal injury as assessed by urinary protein excretion, mesangial matrix expansion, and cell proliferation. Furthermore, ETS-1 dominant-negative peptide but not ETS-1 mutant peptide significantly reduced Ang II-mediated upregulation of transforming growth factor-β, connective tissue growth factor, and α-smooth muscle actin. In addition, ETS-1 blockade reduced several proinflammatory effects of Ang II, including macrophage infiltration, nitrotyrosine expression, and NOX4 mRNA expression. Our studies suggest that ETS-1 is a common mediator of the proinflammatory and profibrotic effects of Ang II-induced hypertensive renal damage and may result in the development of novel strategies in the treatment and prevention of end-organ injury in hypertension.

Figure 1

Angiotensin II (Ang I)I increases cortical ETS-1 expression. A, Representative confocal photomicrographs showing low basal expression of ETS-1 (green) in control kidney cortex, which is predominantly glomerular and increased by Ang II. B, The renal cortical ETS-1 protein expression increases after 2 weeks of Ang II as assessed by Western blot (n=6, *P<0.05 vs control) and returns to baseline after 4 weeks of Ang II. The expression of ETS-1 was not significantly modified by treatment with either ETS-1 dominant-negative (ETS-1 DN) or ETS-1 mutant (ETS-1 MU) peptide. C, Densitometric analysis for ETS-1 showing significant increases in ETS-1 protein expression after 2 but not after 4 weeks of Ang II. Neither ETS-1 DN nor ETS-1 MU modified ETS-1 protein expression. Data expressed as mean±SEM are normalized to GAPDH (*P<0.05 vs control; #P<0.05 vs Ang II group; n=6). D, The infusion of Ang II for 2 weeks resulted in increases in cortical ETS-1 mRNA expression as assessed by real-time reverse transcriptase polymerase chain reaction (n=6; *P<0.05 vs control) and returns to baseline levels after 4 weeks of Ang II.

Figure 2

ETS-1 blockade reduces matrix expansion in angiotensin II (Ang II)–infused mice. A, Representative photomicrographs of sections stained with periodic acid Schiff to assess extracellular matrix (pink stain). B, Quantitative analysis of glomerular matrix expansion from mice infused with Ang II for 4 weeks with and without concomitant infusion of ETS-1 dominant-negative (ETS-1 DN) or ETS-1 mutant (ETS-1-MU) peptide. ETS-1 DN inhibited the Ang II–induced mesangial expansion (n=6 per group; *P<0.05 vs control; #P<0.05 vs Ang II).

Figure 3

ETS-1 blockade reduces cortical α-smooth muscle actin (α-SMA) expression in angiotensin II (Ang II)–infused mice. A, Representative photomicrographs of immunohistochemistry sections for α-SMA; Brown staining is indicative of positive staining that was evident in glomerular and interstitial areas. B, Quantitative analysis of glomerular α-SMA immunohistochemistry from mice infused with Ang II for 4 weeks with and without concomitant infusion of ETS-1 dominant-negative (ETS-1 DN) or ETS-1 mutant (ETS-1-MU) peptide. ETS-1 DN inhibited the expression of α-SMA induced by Ang II (n=6 per group; *P<0.05 vs control; #P<0.05 vs Ang II).

Figure 4

ETS-1 blockade reduced angiotensin II (Ang II)–induced transforming growth factor-β (TGF-β) upregulation. A, Representative Western blots for TGF-β. B, Densitometry data analysis for TGF-β protein expression. ETS-1 dominant-negative (ETS-1 DN) peptide inhibited the Ang II–induced TGF-β overexpression in kidney cortex. Data expressed as mean±SEM are normalized to GAPDH (*P<0.05 vs control; #P<0.05 vs Ang II group; n=6). C, TGF-β mRNA expression as assessed by real-time polymerase chain reaction after 2 and 4 weeks of Ang II infusion. ETS-1 DN inhibited Ang II–induced TGF-β mRNA expression in kidney cortex. Data are expressed as mean±SEM and normalized to GAPDH (*P<0.05 vs control; #P<0.05 vs Ang II group; n=6). ETS-1 MU indicates ETS-1 mutant.

Figure 5

ETS-1 blockade reduced angiotensin II (Ang II)–induced connective tissue growth factor (CTGF) upregulation. A, Representative Western blots for CTGF. B, Densitometry data analysis for CTGF protein expression. ETS-1 dominant-negative (ETS-1 DN) peptide inhibited the Ang II–induced CTGF overexpression in kidney cortex. Data are expressed as mean±SEM and are normalized by GAPDH (*P<0.05 vs control; #P<0.05 vs Ang II group; n=6). C, CTGF mRNA expression as assessed by real-time polymerase chain reaction after 2 and 4 weeks of Ang II infusion. ETS-1 DN inhibited the Ang II–induced CTGF mRNA expression in mice kidney. Data are expressed as means±SEM and normalized to GAPDH (*P<0.05 vs control; #P<0.05 vs Ang II group; n=6). ETS-1 MU indicates ETS-1 mutant.

Figure 6

ETS-1 blockade reduces angiotensin II (Ang II)–induced macrophage infiltration. A, Representative photomicrographs of immunohistochemical staining for F4/80-positive cells (macrophages). Reddish brown color in the cytoplasm indicates positive stain. Positive staining for macrophages was found in tubulointerstitial and glomerular areas (arrows). B, Quantitative analysis of immunohistochemical staining for F4/80-positive cells. In mice infused with Ang II, the number of positive cells increased significantly (*P<0.01 vs control). The administration of ETS-1 dominant-negative (ETS-1 DN) peptide decreased macrophage infiltration in response to Ang II, whereas the ETS-1 mutant (ETS-1 MU) peptide had no effect (#P<0.05 vs Ang II; n=6 per group).

Figure 7

ETS-1 blockade reduces angiotensin II (Ang II)–induced cell proliferation. A, Representative photomicrographs of immunohistochemical staining for Ki-67–positive cells as a marker of cell proliferation. Nuclear reddish brown color is considered positive. Positive cells were found in tubulointerstitial area and glomerular areas (arrows). B, Quantitative analysis of immunohistochemical staining for Ki-67 cells. In mice infused with Ang II, the number of Ki-67–positive cells increased significantly (*P<0.01, control vs Ang II). The administration of ETS-1 dominant-negative (ETS-1 DN) peptide decreased cell proliferation in response to Ang II, whereas the ETS-1 mutant (ETS-1 MU) peptide had no effect (#P<0.05 vs Ang II; n=6 per group).

Figure 8

ETS-1 blockade reduces angiotensin II (Ang II)–induced nitrotyrosine formation and NOX4 expression. A, Representative photomicrographs of immunofluorescence for nitrotyrosine. B, Quantitative analysis of the intensity of cortical nitrotyrosine immunofluoroscence from mice infused with Ang II for 4 weeks with and without concomitant infusion of ETS-1 dominant-negative (ETS-1 DN) or ETS-1 mutant (ETS-1-MU) peptide showing significant increase in nitrotyrosine in response to Ang II, which is significantly reduced by ETS-1 DN but not by ETS-1 MU (n=6 per group; *P<0.05 vs control; #P<0.05 vs Ang II). C, NOX4 mRNA expression as assessed by real-time polymerase chain reaction after 2 and 4 weeks of Ang II, which is increased by Ang II and reduced by ETS-1 DN but not by ETS-MU (*P<0.01 vs control; #P<0.01 vs Ang II and Ang II+ETS-1 MU).