| Background Tubulointerstitial fibrosis is a common final pathway of most kinds of chronic progressive renal diseases which lead to end-stage renal failure. Epithelial-mesenchymal transition is not only a critical mechanism initiating and maintaining tubulointerstitial fibrosis, but also an important regulation of tumor invasion and metastasis. Transforming Growth Factor-β(TGF-β) signals through Smads play a crucial role in regulating the process of tubular EMT and tubulointerstitial fibrosis. Snail has been reported to be a key early immediate transcription factor involved in EMT and the TGF-(3/Smads signal pathway. Resent study showed that overexpression of Snail in both tubular epithelial cells and tumor could down-regulate the expression of E-cadherin and induce EMT.Norcantharidin (NCTD), a demethylated analog of cantharidin, which is an important serine/threonine protein phosphatase inhibitor, possesses potential anticancer activity. Several studies found that NCTD inhibited the expression of Smad3, and down-regulated the activity of nuclear factor kappa B p65 in highly invasive HO-8910PM cell line. On the other hand, NCTD not only decreased the targeted gene expression involved in tumor metastasis such as VEGF, FAK and TGF-β, but also kept the balance between matrix metalloproteinase 2 and tissue inhibitor metalloproteinase. Our studies previously found that NCTD ameliorated proteinuria, inhibited the tubulointerstitial lesions which attenuated the development of renal interstitial fibrosis by inhibiting the expressions of NF-κB and CTGF in protein overload nephropathy. The study showed NCTD inhibited the expression of fibronectin in tubular epithelial cells induced by albumin. However, the detailed molecular mechanism of NCTD is still unclear.As mentioned above, we suppose that the effects of NCTD on inhibition of tubulointerstitial fibrosis maybe involved in the regulation of tubular EMT. To this end, the experimental research is carried out as follows.Chapter one Effect of NCTD on Inhibition of Epithelial-Mesenchymal Transition in Unilateral Ureteral Obstruction RatsObjective To understand the mechanism by which NCTD attenuates renal fibrosis, the effect of NCTD on inhibition of tubular EMT and renal fibrosis in UUO rats was investigated.Methods Sprague-Dawley rats were randomly divided into sham-operation, UUO and UUO treated with NCTD groups (each group: n=6). A unilateral ureteral obstruction (UUO) model was induced in male rats by ligation of the left ureter as described (Ping Xie, Lin Sun, et al. JASN,2009,Apr;20(4):807-19.). Rats were sacrificed at day 14 after surgery and the obstructed kidneys were harvested and subjected to the studies. Renal lesions and the expression of TGF-β1, E-cadherin and a-smooth muscle actin were detected by HE, Masson, immunohistochemistry, RT-PCR and Western blot, respectively.Results The progressive tubular dilatation or atrophy, interstitial mononuclear cell infiltration and tubulointerstitial fibrosis in UUO rats were observed, while the pathological changes were reduced in that of NCTD-treated group. The expressions of TGF-β1 and a-SMA increased and the expression of E-cadherin decreased in the kidneys of UUO compared with the sham-operated kidneys. However, after treatment of NCTD, these changes were partly reversed.Conclusion NCTD could attenuate the tubulointerstitial fibrosis and inhibit tubular EMT in UUO rats. The effects of NCTD on inhibition of tubulointerstitial fibrosis and EMT may associate with the inhibition of TGF-β1 overproduction in obstructive kidney. Chapter two Norcantharidin Inhibits Tubular Epithelial-Mesenchymal Transition in HK-2 by down-regulating Smad2/3 and Snail1Objective:To understand the effect of NCTD on EMT, and to investigate the expressions of signal protein Smad2/3 and transcription factor Snail1 in HK-2 after treatment with NCTD.Method:Human proximal tubular epithelial cell (HK-2) was divided into three groups:control (only FBS-free DMEM), TGF-β1 (treated with TGF-β1 5ng/ml) and NCTD (treated with TGF-β1 5ng/ml and NCTD at different dose). The expressions ofα-SMA and E-cadherin in HK-2 cells were determined by immunofluorescence, Western blot and RT-PCR, respectively. The expressions of Smad2, Smad3, Snail1 mRNA and Smad2/3, p-Smad2/3, Snail1 protein in HK-2 cells were determined by RT-PCR and Western blot, respectively.Results The expression ofα-SMA increased in HK-2 induced by TGF-β1 (5ng/ml), while that of NCTD group treated by NCTD (0.5,1.0, 2.5μg/ml) was dramatically inhibited in a dose-dependent manner. The expression of E-cadherin in HK-2 treated by TGF-β1 partly reversed by NCTD (0.5,1.0,2.5μg/ml) in a dose-dependent manner. The expressions of Smad2, Smad3 mRNA and Smad2/3 protein in HK-2 induced by TGF-β1(5ng/ml) were blocked by NCTD (0.5,1.0,2.5μg/ml) in a dose-dependent manner. The expression of p-Smad2/3 protein in HK-2 induced by TGF-β1(5ng/ml) was blocked by NCTD in the different time point (15,30,60,120min). The expressions of Snail1 mRNA and protein in HK-2 induced by TGF-β1 (5ng/ml) were suppressed by NCTD (0.5,1.0, 2.5μg/ml) in a dose-dependent manner.Conclusion NCTD may inhibit EMT of tubular cells induced by TGF-β1,which may associate with both the down-regulation of Smad2/3 expression and the inhibition of Snail1 expression. |
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