Effect And Mechanism Of Cysteine-rich Protein 61 On Renal Fibrosis And Renal Fibroblasts

Objective To detect the changes of cysteine-rich protein 61（Cyr61）expression in renal fibrosis after ischemia-reperfusion acute renal injury（IR-AKI）and in renal fibroblasts,and to investigate the effects and the related mechanisms of Cyr61 on renal fibroblasts.Methods（1）The rat model of renal fibrosis was established after IR-AKI.We detected the renal function by biochemical test,assessed the fibrosis by Masson staining,and revealed the level of Cyr61 protein by Semi-quantitative analysis of Western blotting.（2）We used bioinformatics technique to reveal the mRNA level of Cyr61 in activated fibroblasts of vivo.（3）TGF-β1（increasing concentration of 0,0.5,1,2,5μg/L）induced the activation of normal rat kidney fibroblasts（NRK-49F cells）for 48h.Cell proliferation was measured by CCK8 method.The level of Cyr61 protein was semi-quantitatively analyzed by Western blotting.（4）NRK-49F cells after transfected by plasmids,were divided into the following groups:blank group（none treatment by plasmid）,control group（null plasmid transfection）,Cyr61⁺/Cyr61^--group（over-expression/low-expression plasmid transfection）.After transfected by over-expression plasmid after 24,48 and 72 h,NRK-49F cells’proliferation was determined by CCK8 assay.（5）Further,part of cells were activated by 5μg/L TGF-β1 and were separated into control group,activated group,Cyr61⁺/Cyr61^--group and Cyr61⁺/Cyr61^--activated group.Cell proliferation was measured by CCK8 method.NRK-49F cells was classified with standard of cell cycle by flow cytometry.The transcription of Cyr61,fibrosis-related factors（collagen Col1α1 and Col3α1,matrix metalloproteinase MMP9 and MMP13）and aging signaling pathways（p53/p21 and p16/Rb1 pathways）-related factors were analyzed by PCR assay.And the protein changes of Cyr61,Col3 and MMP9 were semi-quantitatively analyzed by Western blotting.ResultsIn the process of fibrosis after IR-AKI,the area of collagen fiber was most obviously at AKI 1W,while the Cyr61 protein was at the lowest level at AKI 1W（P<0.05）.（2）Gene chip analysis show that the expression of Cyr61 in renal fibroblasts was decreased at 3days after IR（P<0.05）.（3）The proliferation activity of NRK-49F activated by TGF-β1 in an increasing concentrations was all increased（all P<0.05）.Simultaneously,the protein level of Cyr61 was declined in 1μg/L TGF-β1 group while improved in 5μg/L TGF-β1group compared with control group（P<0.05）.（4）After transfected by Cyr61⁺plasmid for24,48 and 72 hours,the proliferative ability of cells decreased with a time-dependent manner（all P<0.05）.（5）Cyr61 could reduce the transcription of collagen fibers Col1α1and Col3α1 in NRK-49F cells,and decrease the level of Col3 protein.Cyr61 could improve the transcription of matrix metalloproteinase MMP9 and MMP13,as well as the expression of MMP9 protein（all P<0.05）.（6）Cyr61 could inhibit the proliferation of NRK-49F cells,promote them in cell cycle of G1,and increase the transcription of p53,p21 and Rb（all P<0.05）.Conclusions Cyr61 may not only inhibit the fibrotic phenotype in vitro,but also inhibit proliferation by promoting fibroblasts arrest in G1 phase through the p53/p21/Rb interrelated cell aging signal pathway,subsequently affecting the fibrosis after IR-AKI.