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. 2020 Oct 9;9(5):683-692.
doi: 10.1093/toxres/tfaa067. eCollection 2020 Sep.

Manganese exposure induces permeability in renal glomerular endothelial cells via the Smad2/3-Snail-VE-cadherin axis

Peng Gao  1 , Yutian Tian  2 , Qi Xie  1 , Liang Zhang  3 , Yongjian Yan  2 , Dongmei Xu  4
Affiliations

Manganese exposure induces permeability in renal glomerular endothelial cells via the Smad2/3-Snail-VE-cadherin axis

Peng Gao et al. Toxicol Res (Camb). .

Abstract

Manganese (Mn) is an essential micronutrient. However, it is well established that Mn overexposure causes nervous system diseases. In contrast, there are few reports on the effects of Mn exposure on glomerular endothelium. In the present study, the potential effects of Mn exposure on glomerular endothelium were evaluated. Sprague Dawley rats were used as a model of Mn overexposure by intraperitoneal injection of MnCl2·H2O at 25 mg/kg body weight. Mn exposure decreased expression of vascular endothelial-cadherin, a key component of adherens junctions, and increased exudate from glomeruli in Sprague Dawley rats. Human renal glomerular endothelial cells were cultured with different concentration of Mn. Exposure to 0.2 mM Mn increased permeability of human renal glomerular endothelial cell monolayers and decreased vascular endothelial-cadherin expression without inducing cytotoxicity. In addition, Mn exposure increased phosphorylation of mothers against decapentaplegic homolog 2/3 and upregulated expression of zinc finger protein SNAI1, a negative transcriptional regulator of vascular endothelial-cadherin. Our data suggest Mn exposure may contribute to development of glomerular diseases by inducing permeability of glomerular endothelium.

Keywords: VE-cadherin; adherens junction; glomerular endothelial cells; glomerular filtration barrier; manganese.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
Effects of Mn exposure on VE-cadherin expression in a rat model. (A) Representative H&E stain of glomerular tissue from rats treated with or without Mn. Protein exudates in the Mn exposure group are indicated with black arrows. (B) Relative VE-cadherin mRNA expression in rat glomeruli measured by qRT-PCR (**P < 0.01 vs. control). (C) VE-cadherin expression in rat glomeruli was analysed by western blot. (D) Relative expression of VE-cadherin (**P < 0.01 vs. control). (E) Detection of VE-cadherin expression in rat glomeruli by immunohistochemistry. (F) Positive areas of VE-cadherin staining in glomeruli (*P < 0.05 vs. control).
Figure 2
Figure 2
Effects of Mn on permeability of HRGEC monolayers. HRGEC monolayers were treated with 0, 0.04, 0.2, 1.0, and 5.0 mM Mn for 24 h, and then analysed by FITC-dextran transwell assay (n = 6, *P < 0.05, **P < 0.01 vs. control).
Figure 3
Figure 3
Effects of Mn on growth and apoptosis of HRGECs. (A) Representative image of flow cytometric analysis of HRGECs. Cells were stained with annexin V/PI following exposure with or without Mn for 24 h. (B) Rate of apoptosis of HRGECs (n = 3, **P < 0.01 vs. control). (C) HRGECs treated with 0, 0.04, 0.2, 1.0, and 5.0 mM Mn for 24 h, and then analysed by MTT assay (n = 6, **P < 0.01 vs. control).
Figure 4
Figure 4
Mn treatment inhibits expression of VE-cadherin in HRGECs. (A) HRGECs were treated with 0, 0.04, 0.2, 1.0, or 5.0 mM Mn for 24 h. VE-cadherin expression was analysed by western blot (n = 3, **P < 0.01 vs. control). (B) Relative VE-cadherin mRNA expression measured by qRT-PCR in HRGEC monolayers exposure to different concentrations of Mn for 24 h (n = 3, *P < 0.05, **P < 0.01). (C) Immunofluorescence staining of VE-cadherin on HRGEC monolayers treated with or without 0.2 mM Mn for 24 h.
Figure 5
Figure 5
Mn treatment increases phosphorylation of Smad2/3 and inhibits VE-cadherin expression. (A) HRGECs were treated with 0.2 mM Mn for 24 h. Phosphorylation of Smad2/3 and total Smad2/3 were analysed by western blot. (B) Phospho-Smad3/total Smad3 (**P < 0.01 vs. control). (C) Phospho-Smad2/total Smad2 (n = 3, **P < 0.01 vs. control). (D) Expression of Snail and VE-cadherin were analysed by western blot. (E) Relative expression of Snail (**P < 0.01 vs. control). (F) Relative expression of VE-cadherin (n = 3, **P < 0.01 vs. control).
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