| ObjectivesThe aim of this study was to investigate the protective effect of apelin on angiotensin?Ang??-induced hypertensive human tubular epithelial cell line human kidney 2?HK-2?,and human mesangial cell?HMC?.Furthermore,we verified if apelin had a protective effect on hypertensive renal fibrosis by binding to the APJ receptor and inhibiting the transforming growth factor 1?TGF-?1?/connective tissue growth factor?CTGF?signaling pathway.Methods1.The expression of putative receptor protein related to angiotensin type 1receptor?APJ?in renal tissues from hypertensive patients and non-hypertensive patients was explored by immunohistochemistry method.2.A HK-2/HMC model was established for Ang?induced hypertensive damage and apelin drug intervention experiment was conducted.Apelin-13 and Angiotensin II medium containing 10-8mol/L and 10-5mol/L were respectively given to incubate the cells for 24-48h.Four experimental groups?n=3 for each?were established:control group,apelin-13 group,Ang II group and apelin-13+Ang II group.The protein expression of TGF-?1/CTGF signaling pathway was detected by Western blot.Expression levels of TGF-?1,CTGF,collagen?mRNA were measured by RT-PCR.3.It was verified that the protective effect of apelin on Ang II-induced hypertension environment was achieved by inhibiting the TGF-?1/CTGF signaling pathway after binding to the APJ receptor.Hk-2/HMC cell models with high APJ expression and low APJ expression were established by lentivirus cell transfection respectively.After the successful construction of the model,the HK-2/HMC cell models were also divided into control group,apelin-13 group,Ang II group and apelin-13+Ang II group for the above intervention.The protein expression of TGF-?1/CTGF signaling pathway in cells was detected by Western blot.Expression levels of TGF-?1,CTGF,collagen?mRNA were measured by RT-PCR.4.Gene prediction analysis system tests the downstream of the candidates for key node and target Genes of Apelin/APJ system and TGF-?1/CTGF signaling pathways by the Bioinformatics software,and uses the Gene Ontology?GO?database,kyoko Encyclopedia of Genes and Genomes?KEGG?database for the genetic analysis of signaling pathways.String online database was used to construct the candidate Gene encoding protein interaction network.Results1.Immunohistochemical results suggested that APJ receptor was expressed in HK-2 and HMC cells in renal tissue,but there was no significant difference between hypertensive and non-hypertensive patients.2.In the HK-2 and HMC models with normal expression of APJ receptor,the mRNA expression levels of TGF-?1 and CTGF and the protein expression levels of TGF-?1,CTGF and collagen?were significantly increased in the Ang II group compared with the control group?P<0.05?.Compared with Ang II group,the mRNA expression levels of TGF-?1 and CTGF and the expression levels of TGF-?1,CTGF and collagen?in apelin+Ang II group were significantly decreased?P<0.05?.3.In the HK-2 and HMC models with low APJ expression,the mRNA expression levels of TGF-?1,CTGF and the protein expression levels of TGF-?1,CTGF and collagen?in the Ang II group were significantly higher than those in the control group?P<0.05?.Compared with Ang II group,the mRNA expression levels of TGF-?1,CTGF+Ang II group and the protein expression levels of TGF-?1,CTGF and collagen?were not significantly decreased in apelin+Ang II group?P>0.05?.4.Comparison between normal HK-2 cells and HK-2 cells with low APJ expression:compared with normal HK-2 cells,the decreased levels of TGF-?1,CTGF and collagen?protein in the apelin+Ang II group were significantly reduced in the HK-2 cells with low APJ expression,and the difference was statistically significant?P<0.05?.5.Comparison between normal HMC cells and HMC cells with low APJ expression:compared with normal HMC cells,the decreased expression levels of TGF-?1,CTGF and collagen?in the apelin+Ang II group were significantly reduced in the HMC with low APJ expression,and the difference was statistically significant?P<0.05?.6.Signal pathway analysis by bioinformatics GO,KEGG database and String software showed that Apelin/APJ system had a close relationship with TGF-?1/CTGF signaling pathway,which was consistent with the results of this study.Meanwhile,multiple key target proteins still existed in the downstream of TGF-?1/CTGF signaling pathway and cross-linked with multiple fibrosis-related signaling pathways.Conclusions1.Apelin has a protective effect on HK-2/HMC cell damage induced by Ang II in the hypertensive environment.2.The protective function of Apelin needs APJ receptor to exert its biological effects.3.The anti-fibrosis effect of apelin/APJ system may be related to the reduction of collagen?and fibrillar junction protein production by inhibiting the TGF-?1/CTGF signaling pathway. |
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