Study Of The Crosstalk Between Mesangial Cells And Glomerular Endothelial Cells In Mesangial Proliferative Glomerulonephritis

Backgrounds: PGN is one of the most important reasons for CKD.Ms PGN is the most common pathological type of PGN,which is characterized by mesangial cells proliferation and accumulation of extracellular matrix.Exploring the specific pathogenesis of Ms PGN and having a deeper understanding of the pathological changes of PGN can provide new targets for disease treatment and reduce the incidence of CKD.Experimental Ms PGN studies showed that when the mesangial cells were damaged,the glomerular endothelial cells were also pathologically altered.It can be seen that there is a close interaction between mesangial cells and endothelial cells.However,the specific molecular mechanisms involved in signal transduction between the two cells are still unclear.VEGFA affects VEGFR2 causing activation of endothelial cells.Experimental Ms PGN studies showed that the expression of VEGFA was significantly increased in activated mesangial cells.Angpt2 binds to TEK Tyrosine Kinase(TEK Tyrosine Kinase,Tie2)receptor,which can also cause endothelial activation.In the glomerulus,Angpt2 is mainly expressed by endothelial cells.The expression of VEGFA and Angpt2 in serum of tumor patients increased simultaneously,and anti-VEGFA treatment can effectively reduce the expression of Angpt2,suggesting that the two play a synergistic role in tumor disease.However,the expression changes and their roles in Ms PGN still need to be further explored.Objectives: 1.Establish anti-Thy-1 nephritis rat model to find the pathological changes of mesangial cells and glomerular endothelial cells.2.Co-culturing of mesangial cells and endothelial cells in vitro reveals the interaction between the two cells and explores the specific mechanism.3.Explore the effect of promoting endothelial cells surface receptor Tie2 in vivo against the pathological changes of anti-Thy-1 nephritis,which provides new ideas for the treatment of Ms PGN.Methods: 1.Establish rat mesangial proliferative glomerulonephritis model(anti-Thy-1 nephritis)by injecting anti-Thy-1 antibody through tail vein,and collect urine samples on the 3rd,5th and 7th day after model establishment.And collect glomerular tissue and serum samples on 7th day.Serum creatinine and serum urea nitrogen were detected by serum creatinine and urea nitrogen kit.UACR was detected with urine protein and creatinine kit.The pathological changes of glomeruli were observed by pathological PAS staining,and glomerular nuclei were counted.Immunohistochemical and immunofluorescence staining were used to detect the expression of PCNA and RECA-1 in the glomeruli.The expression of CD34,VEGFA and Angpt2 in glomerulus was detected by Western Blot.VEGFA expression was localized by immunofluorescence co-staining with Thy-1 and VEGFA.The expression of Angpt2 was localized by RECA-1 immunofluorescence co-staining with Angpt2.The concentration of Angpt2 in serum of rats was determined by ELISA.2.To explore the function of mesangial cells on endothelial cells,RT-qPCR was first used to detect the production of cytokines related to the activation of endothelial cells by activated mesangial cells.Then,we used Transwell to co-culture them.Ed U uptake method detects cell proliferation.Crystal violet staining detects cell migration.Cellular immunofluorescence and Western Blot were used to detect the expression of ?-SMA.3.To explore the function of endothelial cells on mesangial cells,RT-qPCR was first used to detect the production of cytokines related to the activation of mesangial cells by activated endothelial cells.Then,we used Transwell to co-culture them.Ed U uptake method detects cell proliferation.Crystal violet staining detects cell migration.Cellular immunofluorescence and Western Blot were used to detect the expression of ?-SMA.4.Co-culture mesangial cells and endothelial cells in vitro Transwell,using VEGFA neutralizing antibody to block VEGFA;Angpt1 to block Angpt2.The expression of VEGFA in mesangial cells and Angpt2 in endothelial cells were detected by Western Blot and immunofluorescence staining.The changes of VEGFR2,Tie2 and Erk1/2 expression were detected by Western Blot.Cell proliferation was detected by Ed U.5.After establishing the anti-Thy-1 nephritis model,intraperitoneal injection of Vasculotide was continued on the 4th,5th and 6th day,and glomerular tissue and serum samples were collected on the 7th day.The pathological changes of glomeruli were observed by pathological PAS staining,and pathological scores were performed.Immunohistochemical staining was performed to detect the expression of PCNA and RECA-1 in glomeruli.Western Blot detects the expression of Tie2 and CD34 in glomeruli.Results: 1.Anti-Thy-1 nephritis rat glomerular cells proliferate significantly on the 7th day.The number of PCNA or RECA-1 positive cells increased in the model group.The expression of endothelial cell marker protein CD34 increased in the glomerulus,indicating that glomerular endothelial cells proliferate in anti-Thy-1 nephritis.2.On the 7th day after the establishment of anti-Thy-1 nephritis,the expression of VEGFA was significantly increased in the mesangial area of the glomerulus.So,mesangial cell-derived VEGFA might play an important role in the anti-Thy-1 nephritis pathological changes.Western Blot and serum ELISA results showed that the expression of Angpt2 in glomeruli of model group was increased,and the concentration of serum Angpt2 was also significantly increased.At the same time,immunofluorescence co-staining showed that Angpt2 was mainly expressed by endothelial cells.These results indicate that endothelial cell derived Angpt2 might play an important role in the anti-Thy-1 nephritis pathological changes.3.RT-qPCR results showed that PDGF-BB activated mesangial cells expressed VEGFA and IL-6,cytokines related to endothelial cells activation.PDGF-BB activated mesangial cells were co-cultured with endothelial cells,and the proliferation and migration abilities of endothelial cells were improved.Immunofluorescence staining and Western Blot results showed that the expression of ?-SMA was increased in co-cultured endothelial cells.Thus,activated mesangial cells promote endothelial cells proliferation and migration.4.RT-qPCR results showed that VEGFA activated endothelial cells expressed PDGF-BB,a cytokine related to mesangial cell activation.VEGFA activated endothelial cells were co-cultured with mesangial cells.Ed U uptake showed that the proliferation ability of co-cultured mesangial cells was improved.Crystal violet staining showed that the migration ability of co-cultured mesangial cells was enhanced.Immunofluorescence staining and Western Blot results showed that the expression of ?-SMA was increased in co-cultured mesangial cells.Thus,activated endothelial cells promote mesangial cells proliferation and migration.5.The expression of VEGFA increased in PDGF-BB activated mesangial cells.PDGF-BB activated mesangial cells were co-cultured with endothelial cells and VEGFA was blocked in the co-culture system.Western Blot results showed that the phosphorylation level of VEGFR2 on the surface of endothelial cells decreased.Ed U results showed decreased proliferation of glomerular endothelial cells.In conclusion,mesangial cells affect endothelial cell proliferation through VEGFA/VEGFR2 signaling pathway.6.VEGFA promotes the expression of Angpt2 in endothelial cells.PDGF-BB activated mesangial cells were co-cultured with endothelial cells,and the expression of Angpt2 in endothelial cells increased.After adding VEGFA neutralizing antibody,the expression of Angpt2 was significantly inhibited.Thus,activated mesangial cells produce VEGFA to increase expression of Angpt2 in endothelial cells.7.When co-cultured with PDGF-BB activated mesangial cells,the phosphorylation level of Tie2 in endothelial cells was down-regulated.The phosphorylation level of Tie2 increased after the addition of VEGFA neutralizing antibody in the co-culture system.VEGFA inhibits Tie2 phosphorylation on endothelial cells.However,the inhibition of VEGFA on Tie2 phosphorylation was alleviated by knocking down the expression of Angpt2.Thus,activated mesangial cells produce VEGFA and inhibit the phosphorylation of Tie2 on endothelial cells by Angpt2.8.PDGF-BB activated mesangial cells were co-cultured with endothelial cells,and Angpt1 was added into the co-culture system.Ed U uptake experiment showed that endothelial cell proliferation was decreased,and Western Blot showed that the Erk1/2 signaling pathway of endothelial cells was inhibited.Angpt2(inhibiting Tie2 phosphorylation)promoted endothelial cell proliferation.U0126(specific inhibitor of Erk1/2 signaling pathway)effectively alleviated the pro-proliferation effect of Angpt2 on endothelial cells.In conclusion,Angpt2/Tie2 signaling pathway affects endothelial cell proliferation through the Erk1/2 signaling pathway.9.Intraperitoneal injection of Vasculotide in anti-Thy-1 nephritis rats increased the phosphorylation of Tie2 in the glomeruli.PAS staining and pathological score showed that the pathological changes of glomeruli were alleviated.The expression of PCNA,RECA-1 and CD34 in the glomerulus decreased.It can be seen that Vasculotide can inhibit Tie2 phosphorylation and effectively alleviate the proliferation of glomerular endothelial cells in anti-Thy-1 nephritis.Conclusions: 1.Anti-Thy-1 nephritis glomerular endothelial cells proliferate significantly,and the expression of VEGFA and Angpt2 in the glomeruli increases.2.Co-cultivation of mesangial cells and endothelial cells in vitro confirmed the existence of cell-cell communication between them.3.Activated mesangial cells promote endothelial cells proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 signaling pathways.4.Angpt2/Tie2 signaling pathway affects endothelial cells proliferation through Erk1/2 signaling pathway.5.Increasing the phosphorylation level of Tie2 can effectively alleviate the proliferation of glomerular endothelial cells in anti-Thy-1 nephritis.