Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1

Abstract

DNA methylation plays a pivotal role in the progression of renal fibrosis. Methyl-CpG-binding domain protein 2 (MBD2), a protein reader of methylation, is involved in the development of acute kidney injury (AKI) caused by vancomycin. However, the role and mechanism of action of MBD2 in renal remain unclear. In this study, MBD2 mediated extracellular matrix (ECM) production induced by TGF-β1 in Boston University mouse proximal tubule (BUMPT) cells，and upregulated the expression EGR1 to promote ECM production in murine embryonic NIH 3T3 fibroblasts. ChIP analysis demonstrated that MBD2 physically interacted with the promoter region of the CpG islands of EGR1 genes and then activated their expression by inducing hypomethylation of the promoter region. In vivo, PT-MBD2-KO attenuated unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis via downregulation of EGR1, which was demonstrated by the downregulation of fibronectin (FN), collagen I and IV, α-SMA, and EGR1. Injection of MBD2-siRNA attenuated the UUO- and I/R-induced renal fibrosis. Those molecular changes were verified by biopsies from patients with obstructive nephropathy (OB). These data collectively demonstrated that inhibition of MBD2 reduces renal fibrosis via downregulating EGR1, which could be a target for treatment of fibrotic kidney disease.

Keywords: EGR1; I/R; MBD2; MT: Bioinformatics; UUO; renal fibrosis.

Graphical abstract

Figure 1

MBD-2 was induced by TGF-β1 or UUO in BUMPT cells or mice kidneys BUMPT cells were treated with 5 ng/mL TGF-β1 for 0–48 h. Male C57BL/6J mice were subjected to UUO for examination on days 0–7. Whole-cell lysates were collected at the time points and analyzed by western blotting with antibodies to MBD2. Left kidneys were removed for immunoblot and immunohistochemistry analysis of MBD2 at the indicated time points. (A and C) Immunoblot analysis of MBD2 at indicated time points. (B and D) Analysis of the gray-scale image between them. (E and F) Immunohistochemistry of MBD2. These data are representative of at least four separate experiments shown as means ± SD (n = 6). #p < 0.05 versus hour or day 0 or sham group. Original magnification, ×400. Scale bar: 100μm.

Figure 2

MBD2 was involved in ECM accumulation induced by TGF-β1 in BUMPT cells BUMPT cells were transfected with MBD2 siRNA or MBD2 plasmid and then treated with 5 ng/mL TGF-β1 for 24 h to collect total cell lysates for immunoblot analysis of MBD2, FN, and Col I and IV. (A) transfection of control plasmid. (B and C) Representative immunoblots. (D and E) Densitometry analysis of proteins signals, and normalized to internal control of GAPDH. These data are representative of at least four separate experiments shown as means ± SD (n = 6). #p < 0.05 versus scramble or control group. ∗p < 0.05 versus TGF-β1 group. Original magnification, ×200. Scale bar: 50μm.

Figure 3

EGR1 mediated the promoting fibrosis role of MBD2 during BUMPT cells treated with TGF-β1 or co-culture of murine embryonic NIH 3T3 fibroblasts, and BUMPT cells BUMPT cells were treated with 5 ng/mL TGF-β1 for 0–48 h or transfected with EGR1 siRNA and then treated with 5 ng/mL TGF-β1 for 24 h. (A) Immunoblot analysis of EGR1 at indicated time points. (B) Analysis of the gray-scale image between them. (C) Immunoblot analysis of EGR1, FN, and Col I and IV. (D) Analysis of the gray-scale image between them. (E) Immunoblot analysis of SMAD3, p-SMAD3, AP-1, TGF-β, ERK1/2, and p-ERK1/2. (F) Analysis of the gray-scale image between them. (G) The murine embryonic NIH 3T3 fibroblasts were treated with the supernatant from BUMPT cells transfected with plasmid of MBD2 plus with or without EGR1 neutralizing antibody for 24 h, which indicated by the co-culture model diagram of BUMPT cells and murine embryonic NIH 3T3 fibroblasts. (H) Immunoblot analysis of FN, Col I and IV, and α-SMA. (I) Analysis of the gray-scale image between them. (J) Concentration of EGR1 by ELISA. Data are expressed as means ± SD (n = 6). #p < 0.05 versus scramble or control group. ∗p < 0.05 versus TGF-β1 or MBD2 group.

Figure 4

MBD2 directly binds to CpG islands of promoter of EGR1 and positively activates transcription of them by hypomethylation of promoter (A) The patterns of CpG islands of EGR1 promoter and five pairs of primer were predicted by the software of MethPrimer 2.0. (B) ChIP assays represents the binding sites of MBD2 interaction with CpG islands of promoter of EGR1. (C) qRT-PCR confirmed the expression of CpG islands of promoter of EGR1. (D) Relative luciferase activity of MBD2 or MBD2 mutation plasmids co-transfected with methylated EGR1 pCpGI plasmid in BUMPT cells. (E) CpG-DNA methylation of the EGR1 promoter region. (F and H) Representative immunoblots of MBD2 and EGR1. (G and I) Densitometry analysis of proteins levels, and normalized to internal control of GAPDH. Data are expressed as means ± SD (n = 6). #p < 0.05 versus scramble or control group. ∗p < 0.05 versus TGF-β1 group.

Figure 5

Generation and features of the PT-MBD2-KO mouse model (A) Breeding procedure for the creation of PT-MBD2-KO mice. (B) PCR-based genotyping of wild-type and floxed alleles of MBD2 and PEPCK-Cre allele. (C) Representative immunoblots for the expression of MBD2 and GAPDH. (D) Densitometry analysis of proteins signals, and normalized to internal control of GAPDH. Data are expressed as means ± SD (n = 6). #p < 0.05 versus sham group. ∗p < 0.05 versus PT-MBD2-WT with UUO group.

Figure 6

PT-MBD2-KO mice attenuated UUO-induced renal fibrosis The left ureter of PT-MBD2-WT and PT-MBD2-KO littermate mice was ligated to establish UUO model for 7 days. (A) Representative H&E staining. (B) Masson trichrome staining shows interstitial collagen deposition (blue). (C–F) Immunohistochemistry of FN, Col I and IV, and α-SMA. (G) Quantify tubulointerstitial fibrosis in the kidney cortex. (H) Quantification of immunohistochemistry staining. (I) Representative immunoblots for the expression of FN, Col I and IV, α-SMA, EGR1, and GAPDH. (J) Densitometry analysis of proteins signals, and normalized to internal control of GAPDH. Data are expressed as means ± SD (n = 6). #p < 0.05 versus sham group. ∗p < 0.05 versus PT-MBD2-WT UUO group. Original magnification, ×400. Scale bar: 100μm.