The Role Of CXCR3 In High Glucose-Induced Podocyte Apoptosis And Its Mechanism

Objective: Diabetic nephropathy(DN)is a serious diabetic microvascular complication.Renal biopsy data analysis revealed renal damage including visceral epithelial cell and supportive cell dysfunction in diabetic patients.Podocytes,as end-differentiated cells,are important functional cells of the glomerulus.Once damaged,they cannot regenerate.The injury and apoptosis of podocytes will lead to dysfunction of glomerular filtration membrane and will be a disadvantageous factor to induce DN.CXCR3 is a common receptor of chemokine CXCL9/CXCL10/CXCL11,which is closely related to inflammatory response.CXCR3 distributed in the surface of a variety of immune cells,such as natural killer cells,plasmacytoid cells and dendritic cells,especially the activated T cells.The occurrence of DN is associated with the increased apoptosis and reduced proliferation of podocyte,and apoptosis of podocytes is closely related to these inflammatory cells and their cytokines.Therefore,the expression of CXCR3 in podocytes and its relation with apoptosis and inflammatory reactions under high glucose environment are worthy studying.To this end,we observed the proliferative activity of podocytes cultured in vitro and the expression of CXCR3 in podocytes at different glucose concentrations,studied the effect of CXCR3 on podocyte apoptosis in vitro,and further explored the effects of CXCR3 on the expression of podocyte apoptosis-related proteins Bax,Bcl-2,Caspase-3,podocin and nephrin,and explored the relationship between CXCR3 and reactive oxygen species(ROS).The relationship between the expression of inflammatory factors such as TNF-a?IL-6?IL-1? and the role of CXCR3 in the occurrence and development of DN was clarified.Methods: Firstly,mouse podocytes were cultured in vitro and were divided into five groups after differentiation and maturation,and then cultured the mouse podocytes in the glucose concentration of 5.5mM,15 mM,25mM,35 mM and 50 mM respectively.MTT colorimetric assay was used to detect cell proliferation activity.Quantitative real-time PCR(Q-rtPCR)was used to detect podocyte CXCR3 mRNA and Western blots(WB)were used to detect the expression of CXCR3 protein.Secondly,podocytes cultured and differentiated in vitro were divided into four groups:model group(30Mm glucose);interference group(30Mm glucose + CXCR3siRNA);empty carrier group(30Mm glucose+empty carrier);normal control group(5.5Mm glucose).Cell viability was measured by CCK-8 kit at 12 h,24h,48 h and 72 h,respectively.The Q-rtPCR were used to detect CXCR3 mRNA,and WB to detect the expression of CXCR3 protein,mitochondrial membrane potential(??m)was detected by JC-1 fluorescent probe,ROS content was measured by DCFH-DA,the contents of TNF-a,IL-6 and IL-1? were detected by ELISA,and WB was used to detect the protein content of podocin,nephrin,Bax,Bcl-2 and Caspase-3.Results: The activity of mouse podocytes cultured in vitro decreased with the increase of glucose concentration in a dose-and time-dependent manner.Meanwhile,the expression of CXCR3 mRNA and CXCR3 protein increased with the increase of glucose concentration in a dose-and time-dependent manner.The activity of podocytes in model group was lower than that in normal group,the content of CXCR3 mRNA and CXCR3 protein was higher than that in normal group,the mitochondrial membrane potential(??m)was lower than that in normal group,the apoptotic rate was higher than that in normal group,cell cycle arrest was in S and G2/M phase.The contents of ROS,TNF-a,IL-6 and IL-1? in podocytes of model group were significantly higher than those of normal group;the SD component proteins podocin and nephrin of podocytes of model group were significantly lower than those of normal group,the apoptotic proteins Bax and caspase-3 were higher than those of normal group,and the anti-apoptotic proteins Bcl-2 were lower than those of normal group.CXCR3 siRNA increased podocyte activity and ??M in high glucose environment,decreased apoptosis and ROS,TNF-a,IL-6,IL-1? levels,increased the expression of podocin,nephrin,Bcl-2 protein,and decreased the expression of Bax and Caspase-3.Conclusion: High glucose increased the expression of CXCR3 in mouse podocytes,decreased the proliferation activity of podocytes,caused cell cycle arrest and apoptosis,increased the level of ROS and inflammatory cytokines,and decreased the SD component protein expression.Inhibition of CXCR3 expression can enhance podocyte proliferation activity,reduce apoptosis,ROS and inflammatory cytokine,and increase SD component protein expression.CXCR3 may play an important role in podocyte apoptosis,which is related to the induction of ROS and inflammatory response.CXCR3 may be a potential therapeutic target for DN.