miR-200a Prevents renal fibrogenesis through repression of TGF-β2 expression

Abstract

Objective: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

Research design and methods: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

Results: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

Conclusions: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

FIG. 1.

TGF-β2 induces EMT-like changes in proximal tubular epithelial cells. A: NRK52E cells were cultured in the presence of TGF-β2 (10 ng/ml, 3 days). Light microscopy images (×20) demonstrate that TGF-β2 causes a loss of the typical epithelial morphology to larger and more irregular shaped cells typical of the myofibroblast phenotype. B: After treatment with TGF-β2 (10 ng/ml, 3 days), gene expression levels were assessed by real-time QPCR. A significant increase was observed in the expression of profibrotic factors (TGF-β1, CTGF), fibrotic genes (αSMA, fibronectin [Fib], collagen [Col] I and IV), and PAI-1, but E-cadherin (E-cad) was significantly reduced (*P < 0.05 compared with control). C: Western analysis demonstrated that αSMA and Col I were both significantly elevated at the protein level after TGF-β2 treatment, while E-cadherin was decreased. D: The results from the Western analysis in C are shown as a graph (*P < 0.05 compared with control).

FIG. 2.

TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A: NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (*P < 0.05 compared with control). B: The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (*P < 0.0005 compared with control). C: TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (*P < 0.0005 compared with control). D: NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (*P < 0.05 compared with control). E: Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (*P < 0.05 and #P < 0.001 compared with control).

FIG. 3.

TGF-β1-induced changes in miRNA expression. A: NRK52E cells were cultured in the presence of TGF-β1 (10 ng/ml, 3 days) before miRNA expression levels were assessed by real-time QPCR. A significant decrease was observed in miR-200a but not in miR-141 (*P < 0.03 compared with control). B: SB432542 also attenuated the TGF-β1-induced reduction of miR-200a (*P < 0.02 compared with control). C: Treatment of NRK52E cells with TGF-β2 (10 ng/ml, 3 days) also caused reduction in the expression of miR-200a, miR-200b, and miR-200c (*P < 0.05 compared with miR-control).

FIG. 4.

miR-200a represses the expression of ECM proteins. A: NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (*P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B: Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (*P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (*P < 0.05 compared with control). C: NRK52E cells were cotransfected with the p(CAGA)₁₂ SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (*P < 0.00005 compared with miR-C control; #P < 0.0005 compared with miR-C with TGF-β1; $P < 0.002 compared with miR-200a control). D: Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (*P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (*P < 0.05 compared with miR-C).

FIG. 5.

miR-141a shares the same seed sequence with miR-200a and represses the expression of ECM proteins. A: Alignment of the miR-141/200a sequences and the targeted area of the 3′UTR of TGF-β2 (http://www.targetscan.org). Also shown is the altered sequence of the mutant 3′UTR of TGF-β2. B: Proximal tubular cells were transfected with either miR-control or miR-141 (100 nmol/l), and the expression of certain genes was assessed by real-time QPCR. As with miR-200a, miR-141 was able to significantly reduce the expression of αSMA, fibronectin (Fibr), and collagen I (Col I) and resulted in increased expression of E-cadherin (E-cad) (*P < 0.05 compared with control). The expression of TGF-β2 was also significantly reduced.

FIG. 6.

The TGF-β2 3′UTR is regulated by miR-141/200a. A: NRK52E cells were transfected with TGF-β2 3′UTR luciferase reporter plasmid (1 μg), β-galactosidase plasmid (0.2 μg), and either miR-control (miR-C), miR-141, or miR-200a (100 nmol/l), and cells were analyzed for β-galactosidase and luciferase activity after 3 days. Both miR-141 and miR-200a were able to significantly repress luciferase activity from the TGF-β2 3′UTR (*P < 0.05 compared with control transfected cells). B: No activity of miR-141 and miR-200a against the mutant TGF-β2 3′UTR was observed. C: miR-141 and (D) miR-200a were able to prevent the increased luciferase activity induced by TGF-β1 on the TGF-β2 3′UTR. E: Total TGF-β2 was significantly decreased at the protein level as measured by ELISA and (F) at the mRNA level as measured by QPCR in NRK52E cells, 3 days after transfection with miR-200a (*P < 0.05, compared with control).

FIG. 7.

Changes in gene and miR-200a expression in diabetic mouse kidney cortex. A: Immunohistochemical analysis demonstrated increased expression for αSMA, collagen IV (Col IV), and fibronectin (Fibr) in the diabetic mouse kidney cortex compared with control. B: mRNA was extracted from the renal cortex of control and 10-week diabetic apoE mice (n = 8 per group). Gene expression was assessed by real-time QPCR for a number of genes, revealing significantly increased expression of αSMA, fibronectin, and collagen IV at the RNA level (*P < 0.01 and #P < 0.05 compared with control). C: Expressions of TGF-β1 and TGF-β2 were also elevated at the mRNA level in diabetic mouse kidney cortex (*P < 0.05 and #P < 0.01 compared with control). D: The increased expressions of TGF-β1 and TGF-β2 were associated with decreased expressions of miR-141 and miR-200a in diabetic kidney (*P < 0.05 compared with control). E: Relative expression levels of miR-141 and miR-200a in NRK52E cells and mouse kidney cortex. (*P < 0.005 compared with control). (A high-quality color representation of this figure is available in the online issue.)

FIG. 8.

Changes in miR-141/200a expression in the kidney in the adenine-induced renal fibrosis model. A: Trichrome staining of tissue sections from renal cortex from control and adenine-fed C57bl6 mice after 4 weeks of treatment. Blue staining indicates high levels of collagen in the adenine-fed mouse kidney compared with control. B: mRNA was extracted from the renal cortex of control and adenine-fed C57bl6 mice (n = 4 per group). Gene expression was assessed by real-time QPCR, revealing significantly increased expression of collagen I, collagen III, fibronectin, vimentin, TGF-β1, and TGF-β2. C: The increased expression of TGF-β1, TGF-β2, and collagen was associated with decreased expression of miR-141 and miR-200a but not the appropriate control, RNU6B, in kidney cortex (*P < 0.05 compared with control). (A high-quality color representation of this figure is available in the online issue.)