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. 2024 Sep 9;25(1):297.
doi: 10.1186/s12882-024-03733-5.

"VSports手机版" P4HB regulates the TGFβ/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells

Haifeng Wang  1 , Yang Li  2 , Na Wu  2 , Chunmei Lv  2 , Yishu Wang  3
Affiliations

P4HB regulates the TGFβ/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells

Haifeng Wang et al. BMC Nephrol. .

Abstract

Background: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it's role in HG-induced TECs remains to be elucidated. VSports手机版.

Methods: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB. V体育安卓版.

Results: HG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells V体育ios版. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. .

Conclusion: P4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells VSports最新版本. .

Keywords: Epithelial-mesenchymal transition; Fibrosis; High glucose; P4HB; Renal tubular epithelial cells V体育平台登录. .

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Conflict of interest statement (VSports)

The authors declare no competing interests.

V体育官网 - Figures

Fig. 1
Fig. 1
Effect of HG induction on ROS/TGFβ/SMAD3 signaling and P4HB expression in HK-2 cells. The levels of ROS in HK-2 cells (A, 200 ×); The expression of TGFβ/SMAD3 pathway-related proteins and P4HB in HK-2 cells (B). n = 3, *** P < 0.001. HK-2, human kidney 2; ROS, Reactive oxygen species; MG, mannitol group; HG, high glucose; TGFβ, transforming growth factor beta 1; SMAD3, SMAD family member 3; p-SMAD3, phosphorylated-SMAD family member 3; P4HB, prolyl 4-hydroxylase subunit beta
Fig. 2
Fig. 2
Interference with P4HB inhibits HG-induced HK-2 cell EMT, fibrosis and TGFβ/SMAD3 signaling. The interference potency of siRNA-P4HB (A); The fluorescence intensity of N-cadherin (B, 400 ×); The fluorescence intensity of α-SMA (C, 400 ×); The expression of EMT-related proteins (D). The expression of fibrosis-related proteins (E). The expression of TGFβ/SMAD3 signaling-related proteins (F). The levels of TGFβ in the supernatant of HK-2 cells (G). n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001. HK-2, human kidney 2; α-SMA, α-smooth muscle actin; EMT, endothelial mesenchymal transition; HG, high glucose; TGFβ, transforming growth factor beta 1; SMAD3, SMAD family member 3; p-SMAD3, phosphorylated-SMAD family member 3
Fig. 3
Fig. 3
P4HB and PRMT1 bind to each other in HG-induced HK-2 cells. The expression of PRMT1 in HK-2 cells was detected by WB (A). HDOCK predicted P4HB binding to PRMT1 (B); Immunoprecipitation (IP) assay to verify P4HB and PRMT1 binding (CD). n = 3, *** P < 0.001. HK-2, human kidney 2; HG, high glucose; P4HB, prolyl 4-hydroxylase subunit beta; PRMT1, protein arginine methyltransferase 1
Fig. 4
Fig. 4
Interference with PRMT1 attenuates HG-induced HK-2 cell EMT and fibrosis. The interference potency of two siRNA-PRMT1 (A); The fluorescence intensity of N-cadherin (B, 400 ×); The fluorescence intensity of α-SMA (C, 400 ×); The expression of EMT-related proteins (D). The expression of fibrosis-related proteins (E). n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001. HK-2, human kidney 2; α-SMA, α-smooth muscle actin; EMT, endothelial mesenchymal transition; HG, high glucose; PRMT1, protein arginine methyltransferase 1
Fig. 5
Fig. 5
Interference with P4HB attenuates HG-induced HK-2 cell EMT and fibrosis by inhibiting TGFβ/SMAD3 signaling via PRMT1. The expression of P4HB and PRMT1 (A); The fluorescence intensity of N-cadherin (B, 400 ×); The fluorescence intensity of α-SMA (C, 400 ×); The expression of EMT-related proteins (D); The expression of fibrosis-related proteins (E). The expression of TGFβ/SMAD3 signaling-related proteins (F) The levels of TGFβ in the supernatant of HK-2 cells (G). n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001. HK-2, human kidney 2; α-SMA, α-smooth muscle actin; EMT, endothelial mesenchymal transition; HG, high glucose; TGFβ, transforming growth factor beta 1; SMAD3, SMAD family member 3; p-SMAD3, phosphorylated-SMAD family member 3; SNAIL, snail family transcriptional repressor 1; SLUG, snail family transcriptional repressor 2
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