The Protective Effect And Mechanisms Of Abl Protein Tyrosine Kinase Inhibitor On Renal Interstitial Fibrosis In Experimental Obstructive Nephropathy In Mice

Section one The effects of Abl protein tyrosine kinase on renal interstitial fibrosis lesions in UUO miceOBJECTIVE: To evaluate the effect of Imatinib mesylate (ST571) on the process of renal interstitial fibrosis following unilateral ureteral obstruction in mice.METHODS: 48 male mice were randomly assigned to four groups: Shame operation group, UUO group, and UUO receiving STI571 treatment daily. The mice of treatment group were treated with ST571 (80,160mg/Kg/d) after operation through intragastric administration. The mice of shame operation group were just separated left ureter while the mice of UUO group and treatment group were performed operation by left ureter double ligature. Mice of each group were killed at 8,11 days after operation respectively. Morphological changes of renal tissue were observed by PAS and Masson stain and were investigated through light microscope. The method of percentage evaluation was used to evaluate the degree of renal fibrosis. The number of the inflammatory cells which accumulate in renal interstitial was calculated. The content of total collagen from each group was examined by acidolysis and shade selection. Immunohistochemistry method was performed to investigate the protein expression of fibronectin(FN) in kidney. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of FN and ColⅢmRNA.RESULTS:1 Compared with UUO group, the degree of swelling and hydrocele of the obstructive renal in the treatment group was milder. The color of latter was redder.2 The degree of renal fibrosis in treatment group was much lower than that in UUO group at 11 days after surgery (p<0.05). There was significant difference between the two treatment group. (p<0.05)3 In both UUO and treatment groups, the number of the inflammatory cells which accumulated in renal interstitial increased markedly in time-dependent manner from 8 to 11 days after operation. There was no significant difference between UUO group and treatment group.4 The expression of FN and ColⅢincreased in time-dependent manner in both UUO group and treatment group, while, it was significantly lower in treatment group at 11 days after surgery (p<0.05). There was significant difference between the two treatment groups. (p<0.05)CONCLUTION: Compared to UUO group, Imatinib mesylate significantly ameliorated the histological changes of the UUO renal tissue. The expression of FN were decreased significantly in treatment group.It could reduce the deposition of extracellular matrix in the interstitium.It played a protective role in the renal interstitial fibrosis. Section two The mechanisms of STI571 to ameliorate the degree of renal interstitial fibrosis in experimental obstructive nephropathy in miceObjective: To explore the mechanism of Abl protein tyrosine kinase inhibitor to ameliorate the degree of renal interstitial fibrosis.METHODS: 48 male mice were randomly assigned to four groups: Shame operation group, UUO group, and UUO receiving STI571 treatment daily. The mice of treatment group were treated with STI571 (80,160mg/Kg/d) after operation through intragastric administration. The mice of shame operation group were just separated left ureter while the mice of UUO group and treatment group were performed operation by left ureter double ligature. Mice of each group were killed at 8,11 days after operation respectively. Immunohistochemistry method was performed to investigate the protein expression of TGF-β1,PAI-1,α-SMAand PCNA in the interstitium. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression ofα-SMA and Vimentin mRNA. Western-blot was performed to examine the expression ofα-SMA and Vimentin protein.RESULTS:1 The results of immunohistochemistry showed that there was little expression of TGF-β1,PAI-1 in shame operation group .The expression of TGF-β1,PAI-1 increased in time-dependent manner in both UUO and treatment group. But there was no significant difference between UUO group and treatment group. (p>0.05).2 There was little expression ofα-SMA in shame operation group .The expression ofα-SMA increased in time-dependent manner in both UUO and treatment group while it was significantly less in the latter (p<0.05). There was significant difference between the two treatment group at 11 days after surgery. (p<0.05)3 The expression of PCNA increased in time-dependent manner in both UUO and treatment group while they were significantly less in the latter (p<0.05). 4 The results of RT-PCR and Western-blot showed that the expression ofα-SMA and vimentin mRNA or protein increased since operation. The expression ofα-SMA and vimentin mRNA or protein in treatment group markedly lower than UUO group (p<0.05 ).Conclusion: Imatinib mesylate could suppress the activation and proliferation of fibroblasts in the interstitium, downregulate the expression ofα-SMA in the interstitium, reduce the accumulation of myofibroblast and deposition of ECM, ameliorate renal interstitial fibrosis. Meanwhile it did not accompany with the change of expression of TGFβ1 and the fibronolysis system.Section three Effects of protein tyrosine kinase inhibitor STI571 on TGF-β1 induced activation and extracellular matrix expression in mouse fibroblastsObjective: To study the effects of protein tyrosine kinase inhibitor STI571 on TGF-β1 induced fibrotic response in the mouse fibroblasts, so as to investigate its effects on preventing tubulointerstitial fibrosis.Methodology: In cultured mouse fibroblasts cell line (3T3-swiss),the effects of PKI STI571 on activation and proliferation induced by TGF-β1 were observed by MTT assay. The effects of PTKI STI571 on the level of TGF-β1 inducedα-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type III m RNA expression was observed by reverse trancriptase–polymerase chain reaction (RT-PCR).the effects of PKI STI571 on the level ofα-SMA protein expression induced by TGF-β1 was observed by Western Blot.Results:TGF-β1 stimulated the NIH/3T3 cells proliferation and it can be blocked by STI571. In NIH/3T3 cells, TGF-β1 enhancedα-SMA, FN and ColⅢmRNA expression. The level of FN and ColⅢmRNA expression in STI571 treated groups were significantly decreased compared with TGF-β1 stimulated group. But level ofα-SMA mRNA andα-SMA protein expression in STI571 treated groups were not significantly decreased.Conclusion: Protein tyrosine kinase inhibitor STI571 may inhibit TGF-β1-induced cell proliferation and extracellular matrix sythesis in the mouse fibroblasts, but it can not block theα-SMA expression induced by TGF-β1.