Abstract
IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.
Highlights
Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs)
Inhibitory effect of transforming growth factor-β1 (TGF-β1) on miR-133b The real-time PCR results of miR-133b expression analysis showed that the stimulation of Human renal proximal tubular epithelial (HK2) cells with miR-133b inhibits epithelial-mesenchymal transition (EMT) of HK2 cells induced by transforming growth factor (TGF)-β1 Normal HK2 cells or HK2 cells transfected with miR-133b mimic/NC-miR for 24 h were stimulated with 8 ng/mL of TGF-β1 for 48 h
Immunofluorescence results showed that the fluorescence intensity of α-smooth muscle actin (SMA), collagen 3A1 (Col3A1), fibronectin, and connective tissue growth factor (CTGF) increased and that of E-cadherin decreased in cells treated with TGF-β1 as compared to that in control cells
Summary
An important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). MiR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). Renal interstitial fibrosis is an important pathological feature of kidney aging [1], and bone marrow mesenchymal stem cells (MSCs) play an important role in the regulation of renal interstitial fibrosis. We found that miR-133b-3p was underexpressed in MSC-EVs of aged rats; miR-133b could inhibit the epithelial-mesenchymal transition (EMT) of renal tubular cells induced by transforming growth factor (TGF)-β1 [6]. MiR-133b of MSC-EVs derived from aged rats has been suggested to exhibit important intervening effects on renal fibrosis [7] The inhibitory effects of MSC-EVs against renal fibrosis decreased with age. miR-133b of MSC-EVs derived from aged rats has been suggested to exhibit important intervening effects on renal fibrosis [7]
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