| BackgroundRenal interstitial fibrosis (RIF) is one of the common histopathological features of progressive renal disease of diverse etiology and eventually leads to end stage renal diseases (ESRD). Epithelial to mesenchymal transition (EMT) is the early and reversible stage of RIF. It has also been shown that RIF is more consistent predictor of functional impairment than glomerular damage. EMT and extracellular matrix (ECM) accumulation are the key histological changes of RIF. It is established that transforming growth factor-?1 (TGF-?1) plays a key role in these processes, TGF-?1/Smad2-3 is the classical pathway which is involved in TGF-?1 mediated EMT and ECM accumulation.Heat shock protein 47 (HSP47) is a collagen-binding, stress-inducible protein localized in the endoplasmic reticulum that participates in the intracellular processing, folding, assembly and secretion of procollagens. Previous studies have showed the close relationship between the increased expression of HSP47 and the progression of RIF. HSP47 is mainly expressed in the phenotypically-altered renal tubular epithelial cells and regulated by TGF-?1. Inhibition of HSP47 expression reduced the collagens deposition and ameliorated renal interstitial fibrosis. Various types of HSPs are involved in the process of EMT, but the mechanism is not fully understood. Therefore, HSP47 may play a role in the process of EMT and ECM accumulation in RIF. To this end, experimental research is carried out as follow. Chapter I The Effect of HSP47 on the Renal Tubular Cells Epithelial Mesenchymal Transition in the Rat Kidneys With Unilateral Ureteral ObstructionObjective To detect the expression of HSP47 in the kidneys with unilateral ureteral obstruction and investigate the role of HSP47 in the process of EMT and ECM accumulation.Methods Male Sprague Dawley (SD) rats weighing 280-320g were randomizely divided into sham group(n=18), unilateral ureteral obstructive (UUO) model group(n=18), UUO model with HSP47 antisense oligonucleotides (ODNs)-treat group(antisenseODNs group, n=18) and the sense ODNs-treat group(senseODNs group, n=18). The HSP47 sense or antisense ODNs were transfered into the kidney using ultrasound exposure with liposome microbubble. The obstructed kidneys were harvested 3,7 and 14 days after UUO. Changes in renal morphology were examined by HE and Masson staining. The expressions of HSP47, vimentin, zona occludens-1 (ZO-1), type I collagen were examined by immunohistochemical (IHC) in the UUO model. The expressions of HSP47, vimentin, ZO-1 and type I collagen in the 14 day groups were examined by IHC, Western blot and Real-time PCR.Results Compared with the sham group rats, the expressions of HSP47, vimentin, type I collagen were markedly upregulated in UUO model rats (p<0.05), but the expression of ZO-1 was downregulated (p <0.05), more severe tubular injury, tubulointerstitial fibrosis and inflammatory cells infiltration were also noted in UUO model rats (p< 0.05). Treated with antisense ODNs, but not sense ODNs (p>0.05), abrogated the changes in the expressions of HSP47, vimentin, ZO-1 and type?collagen (p<0.05)Conclusion HSP47 is upregulated in the progress of EMT and ECM accumulation in the UUO model. Suppression of HSP47 can reverse the EMT of renal tubular epithelial cells and inhibit the ECM accumulation Chapter?The Effect of HSP47 on the Renal Tubular EMT Induced by TGF-?1 in the Renal Tubular Epithelial CellsObjective To detect the expression of HSP47 in renal proximal epithelial cell lines (HK-2) and investigate the role of HSP47 in the progress of TGF-?1 induced EMT and ECM in HK-2 cells.Methods HK-2 cells were exposed to lOng/ml TGF-?1. The expressions of HSP47, vimentin, ZO-1, Col I and Col?were examined by Western blot and Real-time PCR, mean while, immunofluorescence for vimentin and ZO-1. Furthermore, HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-?1. Then the expressions of vimentin, ZO-1, Col I and ColIV were detected by Western blot and Real-time PCR, mean while, Western blot and immunofluorescence for HSP47.Results Stimulation of HK-2 with TGF-?1 resulted in a significant increase expressions of vimentin, Col?, Col?and decrease expression of ZO-1 (p<0.05), all in time-dependent manner. Meanwhile, TGF-(31 upregulated the protein and mRNA expression of HSP47 (p<0.05) Compared to the TGF-(31 group, inhibition of HSP47 expression in HK-2 upregulated the protein and mRNA expression of ZO-1 and downregulated the protein and mRNA expressions of vimentin, Col I, Col?(p<0.05). However, HSP47 siRNA negative control had no significant effect on the expressions of ZO-1, vimentin, Col I and ColIV (p>0.05)Conclusion TGF-?1 leads to EMT and ECM accumulation and increases the expression of HSP47 in HK-2 cells. Inhibition of HSP47 expression can reverse the EMT of HK-2 and inhibit the ECM accumulation. Chapter?HSP47 Regulates TGF-?1 Induced EMT via Modulation of the Activation of Smad3 in Renal Tubular Epithelial CellsObjective To investigate the mechanism by which HSP47 modulates the process of TGF-?1 induced EMT and ECM accumulation in renal tubular epithelial cells.Methods HK-2 cells were exposed to 10ng/ml TGF-?1. The expressions of Smad3, p-Smad3 were detected by Western blot. Furthermore, HK-2 cells were transfected with HSP47 siRNA and HSP47 siRNA negetive control before exposing to TGF-?1. Then the expressions of Smad3, p-Smad3, HSP47 were examined by Western blot.Results Stimulation of HK-2 with TGF-?1 resulted in phosphorylation of Smad3, which was peaked at 30 minutes, slightly decreased by 1 hour, and then increased again between 24 and 48 hours (p<0.05). Compared with the TGF-?1 group, inhibition of HSP47 expression down regulated the expression of p-Smad3/Smad3 in HK-2 cells (p<0.05). However, HSP47 negative control had no significant effect on the p-Smad3/Smad3 (p>0.05)Conclusion TGF-?1 leads to the upregulation of p-Smad3 in the HK-2 cells. HSP47 regulates the process of EMT and ECM accumulation via modulation of the activation of Smad3. |
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