| BackgroundMany studies have shown that excessive production and inadequate degradation of extracellular matrix are the main events during the process of tubular basement membrane thickening, but underlying mechanism is not fully understood. Matrix metalloproteinase-2 (MMP-2) is a key enzyme that could degradate Ⅳ type collagen of basement membrane. In vitro experiments have proved that MMP-2 activity is inhibited by hypoxia in renal tubular epithelial cells, however, the corresponding regulation mechanism is unclear.Methods1. The analysis of the whole transcriptom pattern in the hypoxic HK-2 cells.The expression pattern of the whole transcriptom in the hypoxic HK-2 cells was detected by RNA sequencing technology.2.Under hypoxia, influence of RAB7 expression, inhibitors and key proteins of autophagy and endocytosis on MMP-2 activity,MMP-2 expression and regulatory protein.Lentiviral expression vectors were estabished to up-regulate or down-regulate RAB7 expression. Inhibitors,3-methyladenine (3-MA) and filipin were applied to inhibit autophagy and endocytosis, respectively. Established lentiviral interference vector of Beclin-1(Bec-1) and Caveolin-1 (Cav-1) were introduced to decrease the gene expression of Bec-1 and Cav-1, respectively. MMP-2 activity was examined by Zymography assay, MMP-2 mRNA level was detected by quantative real time PCR (qRT-PCR), The protein concentrations of MMP-2 and collagen IV (Col-IV) were performed using ELISA method, and the expression of MMP-14, RECK, and TIMP-2 proteins was analyzed by Western blotting.Results1. The pattern analysis of the whole transcriptom in the hypoxic HK-2 cells.The criteria for the differently expressed genes in this study was the absolute value of fold-change>2 and a higher statistical significance value (P value<0.05). A total of 279 genes were observed to be differentially expressed between hypoxic cells and normoxic cells based on RNA sequencing technologies and analyses. Concerning kidney diseases, up-regulated genes were associated with ECM-receptor interaction, renal cell carcinoma (RCC), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), while down-regulated genes were enriched in antigen processing and presentation, protein processing in endoplasmic reticulum, arginine and proline metabolism, and glutathione metabolism.51387 and 49144 alternative splicing events were identified in hypoxic group and normoxic group, respectively. A total of 9394 novel transcripts and 69706 SNPs were detected in the two samples.2.Under hypoxia, influence of up-regulated and down-regulated RAB7 in autophagy and endocytosis on MMP-2 active,MMP-2 expression and regulatory protein.After hypoxia, the MMP-2 activity in up-regulated RAB7 gene expression group significantly decreased relative to hypoxia group in the supernatant of the cells (hypoxia+up-regulated RAB7 gene group,0.10±0.06, hypoxia group, 0.48±0.07,P<0.01). In up-regulated RAB7 gene group, MMP-2 mRNA expression level significantly decreased (hypoxia+up-regulated RAB7 gene group, 0.81±0.08, hypoxia group,1.24±0.16,P<0.01),the content of MMP-2 protein decreased significantly the supernatant of the cells (hypoxia+up-regulated RAB7 gene group,2.52±0.26μg/L, hypoxia group,3.81±0.13μg/L,P<0.01),the content of Col-IV protein increased the supernatant of the cells (hypoxia+up-regulated RAB7 gene group,5.65±0.20μg/L, hypoxia group,5.43±0.17μg/L,P>0.05). In down-regulated RAB7 gene expression group,the MMP-2 activity significantly was higher than that of hypoxic group (hypoxia+down-regulated RAB7 gene group,1.00±0.08, hypoxia group,0.48±0.07,P<0.01), the MMP-2 mRNA expression significantly was higher than that of hypoxia group (hypoxia+down-regulated RAB7 gene group,3.80±0.23, hypoxia group,1.24±0.16,P<0.01), the content of MMP-2 protein increased significantly (hypoxia+down-regulated RAB7 gene group,4.31±0.30μg/L.hypoxia group,3.81±0.13μg/L,P<0.05),the content of Col-IV protein decreased (hypoxia+ down-regulated RAB7 gene group,5.01±0.20μg/L, hypoxia group,5.43±0.17μg/L, P<0.01).In up-regulated RAB7 gene group, the expression of TIMP-2 and MMP-14 decreased, In down-regulated RAB7 gene expression group TIMP-2 and MMP14 protein expression increased. Compared with the hypoxia group, in up-and down-regulated RAB7 gene expression groups, the expression of RECK protein decreased,in down-regulated RAB7 gene expression group RECK expression was significantly decreased (P<0.01).3.Under hypoxic condition, inhibitors of autophagy and endocytosis effected MMP-2 activity,MMP-2 expression and regulatory protein.When the HK-2 cells were treated with 3-MA, active MMP-2 was reduced in comparison with hypoxia group in the supernatants (hypoxia+3-MA group,0.25±0.04, hypoxia group 0.36±0.07, P>0.05), the level of MMP-2 mRNA dramatically increased (hypoxia+3-MA group,15.53±2.12, hypoxia group,1.24±0.16, P<0.01), while the content of MMP-2 protein was parallel, and the level of Col-IV protein was increased in the supernatants (hypoxia+3-MA group,5.46±0.06μg/L, hypoxia group, 5.33±0.14μg/L, P>0.05). When the HK-2 cells were treated with filipin, the MMP-2 activity was significantly enhanced (hypoxia+filipin group,0.60±0.08, hypoxia group, 0.36±0.07, P<0.01), the expression of MMP-2 mRNA was dramatically increased (hypoxia+filipin group,6.40±2.22, hypoxia group,1.24±0.16, P<0.01), the content of MMP-2 protein was augmented (hypoxia+filipin group,4.44±0.31μg/L, hypoxia group,3.71±0.12μg/L, P<0.05), and the level of Col-IV protein was slightly diminished (hypoxia+filipin group,5.18±0.02μg/L, hypoxia group,5.33±0.14μg/L, P>0.05).The results of Western blotting showed that the expression of TIMP-2 was inhibited by 3-MA and filipin, respectively, but the effect of 3-MA was stronger than that of filipin in hypoxic HK-2 cells. Both 3-MA and filipin inhibited the expression of RECK, and filipin obviously increased MMP-14 protein expression. The data of these regulatory proteins were further confirmed by immunofluorescence staining.4.Under the hypoxic condition,down-upregulated key proteins of autophagy and endocytosis affected MMP-2 activity,MMP-2 expression and regulatory protein,respectively.After interference Bec-1 gene expression under hypoxia, the MMP-2 relative activity was lower than that in hypoxia group in the supernants (hypoxia+Bec-1shRNA group,0.35±0.17, hypoxia group,0.49±0.02, P>0.05), the expression of MMP-2 mRNA was increased (hypoxia+Bec-1shRNA group,2.00±0.03, hypoxia group,1.24±0.16, P<0.05), the content of MMP-2 protein was no significant difference in the supernatants (hypoxia+Bec-1shRNA group,3.30±0.18μg/L, hypoxia group,3.54±0.20μg/L, P>0.05), the level of Col-IV protein was approximately equal in the supernatants (hypoxia+Bec-1shRNA group,5.45±0.28μg/L, hypoxia group, 5.30±0.20μg/L, P>0.05). After interference Cav-1 gene expression, the MMP-2 relative activity was significantly higher than that of hypoxia group in cell culture media (hypoxia+Cav-1shRNA group,0.66±0.05, hypoxia group,0.49±0.02, P<0.01), the expression of MMP-2 mRNA was increased (hypoxia+Cav-1shRNA group,15.80±3.11, hypoxia group,1.24±0.16, P<0.01), the content of MMP-2 protein presented the same trend (hypoxia+Cav-1shRNA group,4.15±0.26μg/L,hypoxia group,3.54±0.20μg/L, P<0.01), while the level of Col-IV protein was decreased slightly (hypoxia+Cav-lshRNA group,4.90±0.60μg/L, hypoxia group,5.30±0.20μg /L,P>0.05).The western blotting results of regulatory proteins demonstrated that the expression of TIMP-2 and MMP-14 was inhibited by down-regulated Bec-1 gene and Cav-1 gene in HK-2 cells under hypoxia, respectively, but Bec-1 gene interference was more effective. Down-regulating expression of Bec-1 gene and Cav-1 gene inhibited the expression of RECK, and the effect of Cav-1 gene down-regulation was more powerful.Conclusions1. The results demonstrated that hypoxia increased the expression of damage and repair genes associated with the generation of extracellular matrix, which could lead to tubular basement membrane thickening. The genes related to antigen processing and presentation, protein processing in endoplasmic reticulum, arginine and proline metabolism, and glutathione metabolism were down-regulated, which implied that hypoxia attenuated the anti-injury capability in HK-2 cells. Renal cell carcinoma relevant genes were up-regulated, which suggested that high incidence of renal cell carcinoma may be induced by renal hypoxia in patients with chronic kidney diseases2. Autophagy and endocytosis affected the expression or activity of MMP-2, which was proved by various analyses. Autophagy mainly inhibited the expression of MMP-2 mRNA, whereas endocytosis mainly down-regulated MMP-2 activity.3. Endocytosis mediated by Cav-1 increased the expression of RECK, and decreased the expression of TIMP-2 and MMP-14, which could diminish MMP-2 activity in HK-2 cells under hypoxia.4. Cav-1 and RAB7 may be potential targets to slower the development of chronic kidney disease. |
PDF Full Text Request