Abstract
ObjectiveTo investigate the inhibitory effect of Hirsutella sinensis (HS) on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells induced by aristolochic acid (AA) and its possible mechanism.Methods18 male Sprague-Dawley rats were randomly and equally divided into the following 3 groups: AA group, AA+HS group and control group. Urinary protein excretion and creatinine clearance (CCr) were measured. All rats were sacrificed at the end of 12th week. The pathological examination of renal tissue was performed and the mRNA and protein expression of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), cytokeratin-18 and Snail in renal cortex were determined by real time quantitative PCR and immunohistochemical staining respectively. In addition, human renal proximal tubule epithelial cells line (HKC) was divided into the following 4 groups: AA group, AA+HS group, HS control group and control group. The above mRNA and protein expression in HKC was determined by real time quantitative PCR and Western blot respectively.Results(1) CCr was significantly decreased, and the urinary protein excretion and relative area of renal interstitial fibrosis were significantly increased in the rats of AA and AA+HS group compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly lightened in the rats of AA+HS group compared to those in AA group (P<0.05). (2) The mRNA and protein expression of TGF-β1, α-SMA and Snail was significantly up-regulated and the expression of cytokeratin-18 was significantly down-regulated in the rat renal cortex as well as in the cultured HKC cells in AA and AA+HS groups compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly alleviated in AA+HS group compared to those in AA group (P<0.05 or P<0.01). (3) Knockdown endogenous Snail expression by siRNA could ameliorate AA-induced EMT of HKC cells, while overexpression of Snail by plasmid transfection diminished the antagonistic effect of HS on AA-induced EMT. These results suggest Snail might be a potential target of HS effect.ConclusionHS is able to antagonize, to some extent, tubular EMT and renal interstitial fibrosis caused by AA, which might be related to its inhibitory effects on the TGF-β1 and Snail expression.
Highlights
Renal interstitial fibrosis is the common pathway in progressive kidney disease, which leads to deterioration and eventual failure of renal function, irrespective of the diverse causes of disease [1,2]
Hirsutella sinensis (HS) is able to antagonize, to some extent, tubular epithelial-mesenchymal transition (EMT) and renal interstitial fibrosis caused by aristolochic acid (AA), which might be related to its inhibitory effects on the transforming growth factor-β1 (TGF-β1) and Snail expression
The interstitial fibrosis process is often accompanied by epithelial-mesenchymal transition (EMT) of tubular epithelial cells, by which tubular epithelial cells are converted into the phenotype of myofibroblasts and produce interstitial matrix components [1,2,3]
Summary
Renal interstitial fibrosis is the common pathway in progressive kidney disease, which leads to deterioration and eventual failure of renal function, irrespective of the diverse causes of disease [1,2]. The interstitial fibrosis process is often accompanied by epithelial-mesenchymal transition (EMT) of tubular epithelial cells, by which tubular epithelial cells are converted into the phenotype of myofibroblasts and produce interstitial matrix components [1,2,3]. Our previous in vitro studies using cell culture techniques, including cell co-culture, have revealed that tubular epithelial cells can be activated by aristolochic acid (AA) and secrete fibrogenic factors including TGF-β1, which can in turn act on renal interstitial fibroblasts through cell-cell cross talking to enhance collagen type I synthesis [6,7]. We have successfully created a rat model of chronic aristolochic acid nephropathy (CAAN) with typical interstitial fibrosis [8, 9] and demonstrated that EMT of tubular epithelial cells is involved in the development of interstitial fibrosis in the CAAN rat model in vivo [10]. In the present study, we choose the cultured tubular epithelial cells stimulated by AA and the rat model of CAAN to investigate the antagonistic effects of Hirsutella sinensis (HS) on tubular EMT
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