Effect And Significance Of Smurf2 On Renal Interstitial Fibrosis

The ubiquitin-proteasome system (UPS) is the major nonlysosomal pathway for intracellular protein degradation, generally requiring a covalent linkage of one or more chains of polyubiquitins to the protein intended for degradation. It has become clear that the UPS plays major roles in regulating many cellular processes, including the cell cycle, immune responses, apoptosis, cell signaling, and protein turnover under normal and pathological conditions, as well as in protein quality control by removal of damaged, oxidized, and/or misfolded proteins.Degradation of a protein via the ubiquitin-proteasome pathway involves two discrete and successive steps: 1) tagging of the substrate by covalent attachment of multiple ubiquitin molecules and 2) degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin. This last process is mediated by ubiquitin recycling enzymes , including Ubiquitin activating enzymeE1,Ubiquitin conjugating enzymeE2,Ubiquitin-protein ligaseE3. Thus E3s play a key role in the ubiquitin-mediated proteolytic cascade since they serve as the specific recognition factors of the downstream system 26S proteasome complex.Smad ubiquitination regulatory factor 2 (Smurf2), a member of E3 ubiquitin-protein ligase family for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7.Overexpression of transforming growth factorβ(TGF-β) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. We investigated the effects of Smad ubiquitin regulatory factor 2(Smurf2)on renal interstitial fibrosis, and investigated Smad-mediated TGF-βsignaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in rats, and explored the effects of glycyrrhizin acid(GA) on (RIF). Adult male Wistar rats were undergoing in unilateral ureteral obstruction (UUO) kidneys in rats, a model of progressive tubulointerstitial fibrosis. According to different time as 7days, 14days, 21days, 28days after the operated and their left kidneys were taken out to undergo light microscopy by PAS and Masson staining to observe the index of renal interstitial cell infiltration and index of renal interstitial fibrosis index.At the different time point,the fibrosis index and interstitial cell infiltration of the UUO group were significantly increased with a significant difference between the two groups.Smurf2 and Smad2 were increased time-dependently in the UUO group, TGF-β1mRNA began to increase in the UUO group and peaked 7 days after, but expression was increased significantly in UUO kidneys compared with sham-operated kidneys on days 7, 14, 21, and 28 in UUO.The expression of Smurf2mRNA and protein was significantly positively correlated with that of Smad2, index of interstitial fibrosis during the progressive phase of renal interstitial fibrosis(RIF).Smurf2 may be involved in the mechanism of the progression of RIF, and its effect may result from the induction of actived of TGF-β/Smad pathway.We also investigate the effect of Inhibition of the ubiquitin-proteasome MG132 on the expression of Smurf2 , Smad7 and CollagenⅣin cultured human proximal tubular epithelial cell line HK—2 induced by TGF-β1(5ng/ml) . We could conclude that TGF-β1(5ng/ml) could up—regulate the expression of Smurf2 mRNA and protein , up—regulate the expression of CollagenⅣmRNA and protein , decrease Smad7 mRNA and protein in vitro . MG132 (2.5uM)could significantly inhibit enhanced Smurf2 mRNA and protein , CollagenⅣmRNA and protein , increased Smad7 mRNA and protein expression in HK—2 cell line stimulated by TGF-β1 . Thus MG132 might being a potential antifibrotic drug in kidney diseases. To investigate the effects of glycyrrhizin acid(GA) on Smurf2 in unilateral ureteral obstruction (UUO) kidneys in rats and its mechanism. According to different interfered time as 7days,14days,28days after the intervention and their left kidneys were taken out to undergo light microscopy by Masson staining to observe the renal interstitial fibrosis index.At the different time point,twenty-eighth days after the intervention,the fibrosis index of the UUO group were significantly increased and the fibrosis index values of UUO+GA groups were significantly lower than that of the UUO group (both P<0.01)with a significant diference between the two groups.Smurf2 mRNA and protein , the protein of TGF-β1,Smad2,and FN were increased time-dependently in the UUO group, however the Smurf2mRNA and protein , the protein of TGF-β1,Smad2,and FN expression in UUO+GA group was significantly inhibited than that of the UUO group.Smad7mRNA had no significant diference between the three groups; Significant decreases of Smad7 protein were noted in UUO kidneys compared with sham-operated kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis. Smurf2,which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. GA might play a protective role of the kidney in UUO by inhibiting the overexpression of TGF-β/Smads pathway and decreasing the expression of Smurf2, the deposition of FN, and increasing the expression of Smad7, which may consequently result in a decreased production of extracellular matrix, thus blocking the renal interstitial fibrosis lesion. PartⅠEffect of interstitial lesion on Expression of Smad Ubiquitin Regulatory Factor 2 in rats with unilateral ureteral obstruction0bjective To investigate the effects of Smad ubiquitin regulatory factor 2(Smurf2) on renal interstitial lesion and its mechanism. Methods Adult male Wistar rats were randomly divided into two groups: Sham operated group(Sham),rats after sham operated; UUO group,undergoing unilateral left ureteral obstruction. UUO group was subdivided into sub—groups(6 for each) according to different time as 7days,14days,21days,28days after the operated and their left kidneys were taken out to undergo light microscopy by PAS and Masson staining to observe the index of renal interstitial cell infiltration and index of renal interstitial fibrosis index . At the different time point , Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of Smurf2,Smad2 and TGF-βl and the correlation between them were analyzed.The correlation between the expression level of Smurf2 and interstitial fibrosis markers was also studied. Results the fibrosis index and interstitial cell infiltration of the UUO group were significantly increased (both P<0.01)with a significant difference between the two groups.Smurf2 and Smad2 were increased time-dependently in the UUO group, TGF-β1mRNA began to increase in the UUO group and peaked 7 days after, but expression was increased significantly in UUO kidneys compared with sham-operated kidneys on days 7, 14,21, and 28 in UUO(P<0.01 ).The expression of Smurf2mRNA and protein was significantly positively correlated with that of Smad2(r=0.975,P<0.01;r=0.979,P<0.01 ), index of interstitial fibrosis(r=0.911,P<0.01;r=0.964, P<0.01)during the progressive phase of renal interstitial fibrosis(RIF). Conclusion Smurf2 may be involved in the mechanism of the progression of RIF, and its effect may result from the induction of actived of TGF-β1/Smad pathway. PartⅡEffect of MG132 on Expression of Smad Ubiquitin Regulatory Factor 2 and Smad7 Induced by TGF-β1 in HK—2 Cell LineObjective To investigate the effect of MG132 on the expression of Smurf2 , Smad7 and CollagenⅣin cultured human proximal tubular epithelial cell line HK—2 induced by TGF-β1(5ng/ml) . Methods The mRNA expression was tested by reverse transcript tase-polymerase chain reaction(RT—PCR) . The protein expression was examined by Western blot . Results (1)The basic levels of Smurf2 mRNA and protein ,Smad7 mRNA and protein, CollagenⅣmRNA and protein expression were observed in HK—2 cell line . Smurf2 mRNA and protein were up—regulated by TGF-β1 (5ng/ml) (increased 57.4%,75.56%)(P<0.01) . CollagenⅣmRNA and protein also were up—regulated by TGF-β1 (5ng/ml) (increased 46.4%,97.95%)(P<0.01) . Smad7 mRNA and protein were down—regulated by TGF-β1 (5ng/ml) (decreased 43%,62.03%)(P<0.01) . (2)The MG132 (2.5uM)significantly inhibited enhanced Smurf2 mRNA and protein, CollagenⅣmRNA and protein, increased Smad7 mRNA and protein expression in HK—2 cell line stimulated by TGF-β1 (P<0.01) . Conclusion TGF-β1(5ng/ml) could up—regulate the expression of Smurf2 mRNA and protein , CollagenⅣmRNA and protein , decreased Smad7 mRNA and protein in vitro . MG132 could inhibit enhanced Smurf2 mRNA and protein , CollagenⅣmRNA and protein , increased Smad7 mRNA and protein expression in HK—2 cell line stimulated by TGF-β1 .PartⅢEffect of glycyrrhizin acid on Expression of Smad Ubiquitin Regulatory Factor 2 in rats with unilateral ureteral obstructionObjective To investigate the effects of glycyrrhizin acid(GA) on renal interstitial fibrosis lesion and its mechanism. Methods Adult male Wistar rats were randomly divided into three groups: Sham operated group(Sham):rats after sham operated; UUO group,undergoing unilateral left ureteral obstruction ; UUO and treated with GA (20 mg?kg- 1·d - 1 group(UUO+GA) .Each group was subdivided into sub—groups(6for each)according to different interfered time as 7days,14days,28days after the intervention and their left kidneys were taken out to undergo light microscopy by Masson staining to observe the renal interstitial fibrosis index.At the different time point,Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of Smurf2 ,Smad7and TGF-βl; Western blotting methods was used to detect the protein expression of Smurf2 and Smad7. Results Twenty-eighth days after the intervention,the fibrosis index of the UUO group were significantly increased and the fibrosis index values of UUO+GA groups were significantly lower than that of the UUO group (both P<0.01)with a significant diference between the two groups.Smurf2 mRNA and protein were increased time-dependently in the UUO group, however the Smurf2mRNA and protein,Smad2 and FN protein expression of the UUO+GA group was significantly inhibited than that of the UUO group (all P<0.01).Smad7mRNA had no significant diference between the three groups; Significant decreases of Smad7 protein were noted in UUO kidneys compared with sham-operated kidneys, TGF-β1 mRNA began to increase in the UUO group and peaked 7 days after, but expression was increased significantly in UUO kidneys compared with sham-operated kidneys on days 7, 14, and 28 in UUO, respectively, compared with sham-operated kidneys. 0.05). Conclusion GA inhibits the renal interstitial fibrosis lesion,downregulates the expression of the Smurf2 ,TGF-β1,Smad2,FN , and up-regulate the Smad7 expression, thus blocking the renal fibrosis.