Early Growth Response 1 (Egr1) Is a Transcriptional Activator of NOX4 in Oxidative Stress of Diabetic Kidney Disease

Abstract

Background NADPH oxidase 4 (NOX4) plays a major role in renal oxidative stress of diabetic kidney disease (DKD). NOX4 was significantly increased in Egr1-expressing fibroblasts, but the relationship between Egr1 and NOX4 in DKD is unclear. Methods For the evaluation of the potential relationship between Egr1 and NOX4, both were detected in HFD/STZ-induced mice and HK-2 cells treated with TGF-β1. Then, changes in NOX4 expression were detected in HK-2 cells and mice with overexpression and knockdown of Egr1. The direct relationship between Egr1 and NOX4 was explored via chromatin immunoprecipitation (ChIP). Results We found increased levels of Egr1, NOX4, and α-SMA in the kidney cortices of diabetic mice and in TGF-β1-treated HK-2 cells. Overexpression or silencing of Egr1 in HK-2 cells could upregulate or downregulate NOX4 and α-SMA. ChIP assays revealed that TGF-β1 induced Egr1 to bind to the NOX4 promoter. Finally, Egr1 overexpression or knockdown in diabetic mice could upregulate or downregulate the expression of NOX4 and ROS, and α-SMA was also changed. Conclusion Our study provides strong evidence that Egr1 is a transcriptional activator of NOX4 in oxidative stress of DKD. Egr1 contributes to DKD by enhancing EMT, in part by targeting NOX4.

<p>Egr1 Knockdown Combined with an ACE Inhibitor Ameliorates Diabetic Kidney Disease in Mice: Blockade of Compensatory Renin Increase</p>

“…C57BL6/J male mice (Animal Center of Guangdong province, 3-4 w, 15-16 g) were used to construct the DKD model as described previously. 15 In total, 24 DKD mice were randomly assigned to four groups at 12 w. Our previous study showed that DKD mice models could be conducted successfully, and the gene expression of Egr1 was significantly higher in DKD mice than in controls at 12 w. 15 E-DM were treated with oral enalapril (5 mg/ 150 mL water). EG-DM were treated with enalapril and pGPU6-shEgr1 plasmid, GV-DM were treated with pGPU6 vector plasmid, and the remainder were controls.…”

“…The pGPU6-shEgr1 plasmid (1 μg/g, Genepgarma, China, 5′-GCTGCTTCATCGTCTTCCTCT-3′) was rapidly injected into the tail vein through by hydrodynamic gene delivery every week for 4 weeks. 15,19,20 All interventions lasted for 4 weeks (12-16w). The mice were housed in separate cages in a temperature-controlled room with a 12-h light-dark cycle in a specific pathogen free environment.…”

“…Real-time quantitative PCR was performed in a Biosystems 4800 Real-Time PCR System as described previously. 15 The relative expression of each gene was estimated using the comparative 2 −ΔΔCt method, with β-actin as the reference. The PCR primers used in this study are listed in Table 1.…”

“…13 Our previous studies confirmed that Egr1 gene and protein expression in DKD rats and mice were increased compared to the non-DKD group. 14,15 Furthermore, it has been demonstrated that Egr1 can promote the development of DKD by binding to the TGF-β and NOX4 promoters. 14,15 Furthermore, transient expression of Egr1 has been shown to be increased in HK-2 cells after TGF-β1 stimulation, and high glucose is known to promote Egr1 expression by downregulating microRNA-181a-5p, thereby promoting DKD renal fibrosis.…”

“…14,15 Furthermore, it has been demonstrated that Egr1 can promote the development of DKD by binding to the TGF-β and NOX4 promoters. 14,15 Furthermore, transient expression of Egr1 has been shown to be increased in HK-2 cells after TGF-β1 stimulation, and high glucose is known to promote Egr1 expression by downregulating microRNA-181a-5p, thereby promoting DKD renal fibrosis. 16 Klotho inhibits DKD renal fibrosis by inhibiting Egr1.…”