Role And Its Mechanism Of Exosomes From High-glucose Treated Macrophages On Glomerular Mesangial Cells

Background and purpose: Diabetic nephropathy is one of the most common and serious complications of diabetes mellitus.Approximately 400 million diabetics and 20-30% of these patients in China will progress to DN,and 20-40% of patients with DN will progress to end-stage renal disease,which is seriously detrimental to human health.Therefore,the study of the pathogenesis and treatment of DN has been the close focus to scholars.Related researchers have shown that the pathogenesis of DN is mainly caused by non-enzymatic glycation,polyol pathway activation,protein kinase C activation,dyslipidemia,hypertension,renal hemodynamic changes,oxidative stress,vasoactive substances,cytokines and several other genetic factors.At the same time chronic micro-inflammatory state also played an important role in the occurrence and development of DN.Macrophages are the main regulatory cells of inflammatory response.In the early stages of DN,macrophage infiltration can be found.In addition to the macrophage infiltration,activated macrophages can cause the production of damaged media,whether it further aggravate renal damage by other mechanisms has drawn great attention in recent years.In diabetes,there is an abnormal interaction between kidney-infiltrating macrophages and renal mesangial cells,which is considered as an important aspect of the pathogenesis of DN.Recently,a new way of cell-cell communication,the exosome,has drawn people’s attention.Exosomes are membrane-bound vesicles produced and released by cells that contain a variety of molecules such as proteins,lipids,DNA,mRNA,and microRNAs.It has been reported that exosomes play an important role in the pathogenesis of DN.Related studies have shown that mesangial cells can overexpress inflammatory cytokines and fibrosis factors in the context of high glucose co-cultured macrophages and mesangial cells.This suggests that hyperglycemia may promote the activation of mesangial cells by some substance secreted by macrophages and participate in the process of fibrosis of diabetic nephropathy.Hence,we doubt whether high glucose regulate the function of mesangial cells by regulating the secretion of exosomes ? In this study,exosomes produced by macrophages were stimulated by high glucose in vitro and we observed how the exosomes play a mediator role between macrophages and mesangial cells in vivo,in vitro,in cells and at molecular level.Methods(1)Choose 30mmol/L high glucose as high glucose group(HG group),and set up normal glucose concentration group(NC group)and mannitol control group(MC group).Twenty-four hours after stimulation of high glucose on macrophages,exosomes were extracted using a kit.After the extraction,the morphology of exosomes was identified by transmission electron microscopy and the expression of exosome labeled proteins were detected by Western Blot.(2)After identification,the exosomes were co-cultured with mesangial cells as an irritant.Prior to co-culture we need determine the stimulating concentration and stimulating time.Exosomes secreted by macrophages(HG-Exo group)were selected as the observation object,and were observed in four groups of 10 μg/ml,20 μg/ml,30 μg/ ml and 40 μg/ml for 24 hours.The proteins were extracted from mesangial cells.The expressions of Col-IV,FN and α-SMA in mesangial cells were detected by Western Blot.According to the results,the final concentration of 30μg/ml was determined.The concentration of 30μg/ml was used as the stimulus,and then at the time of 0h,6h,12 h,24h and 48 h,the expressions of Col-IV,FN and α-SMA were detected.We finally choose 24 h as the stimulation time according to the analysis results.(3)After determining the final stimulating concentration and time,the macrophages derived from exosomes labeled with PKH67 were co-cultured with mesangial cells for 24 hours,then observed by laser scanning confocal microscopy.(4)Exosomes from NC group,MC group and HG group were stimulated with mesangial cells at a concentration of 30μg/ml for 24 hours,and were respectively labeled as NC-Exo group,MC-Exo group and HG-Exo group.The effects of exosomes on the proliferation of mesangial cells,secretion of extracellular matrix,secretion of inflammatory cytokines and transdifferentiation were observed 24 hours later.(5)The proliferation of mesangial cells was observed by CCK8 test and Edu test.(6)The secretion of extracellular matrix of mesangial cells was detected by Quantitative Real-time PCR method,Western Blot method,laser confocal method and ELISA method.(7)The secretion of inflammatory cytokines in mesangial cells was detected by Quantitative Real-time PCR method,Western Blot method and ELISA method.(8)The transdifferentiation of mesangial cells was detected by Western Blot method and confocal laser scanning microscope.(9)Real-time fluorescence quantitative PCR was used to detect the dynamic changes of TGF-β1 mRNA in macrophages,mesangial cells and exosomes.(10)Choose western blot method to detect whether TGF-β1/Smad3 pathway is involved in the interaction between macrophages and mesangial cells.(11)The effect of TGF-β1 mRNA on the interaction between macrophages and mesangial cells was verified by co-culture of exosomes extracted from macrophages transfected with TGF-β1 siRNA with mesangial cells.(12)The changes in the secretion of extracellular matrix,secretion of inflammatory factors and transdifferentiation were observed after the TGF-β1 siRNA transfection.(13)The protein expression of TGF-β1/Smad3 pathway was detected by Western Blot after the TGF-β1 siRNA transfection.(14)The secretion of extracellular matrix of mesangial cells was detected by Western Blot and confocal laser scanning microscope after the TGF-β1 siRNA transfection.(15)Mesangial cell transdifferentiation was detected by Western Blot and confocal laser scanning after the TGF-β1 siRNA transfection.(16)The secretion of inflammatory factor by mesangial cell were detected by Western Blot method after the TGF-β1 siRNA transfection.(17)Exosomes extracted from NC,MC and HG groups were injected into the mice via the tail vein once every other day for eight weeks.Eight weeks later,the hematuria samples of mice were collected for the determination of biochemical markers.The kidneys of rats were collected to observe the pathological changes of kidney,the secretion of matrix in mesangial area was detected by immunohistochemistry and the expression of TGF-β1/Smad3 pathway proteins and extracellular matrix of mesangial cells were detected by Western Blot.Results(1)The exosomes observed by transmission electron microscopy are bilayer membrane-like structures with shapes of round or circle and 40nm-100 nm in diameter,which are relatively uniform in size and uniformly walk in the field of vision.This is in agreement with the literature about the typical exosome morphology.(2)The expressions of CD63 and TSG101 in the exosomes extracted from each group showed no expression of Calnexin,which indicates that the exosomes extracted from each group had no cell components.(3)Confocal laser scanning microscopy showed that PKH67-labeled exosomes could be taken up by mesangial cells.(4)CCK8 test and Edu test showed that the proliferation of mesangial cells in HG-Exo group was significantly higher than that in NC-Exo group,the difference was statistically significant(P <0.05).(5)Fluorescent quantitative PCR showed that there was a significant increase in the extracellular matrix Col-IV mRNA and FN mRNA in HG-Exo group compared with NC-Exo group(P<0.05).Western Blot showed that the numbers of extracellular matrix Col-IV and FN in HG-Exo group were significantly higher than those in NC-Exo group(P<0.05).Confocal laser scanning showed that the fluorescence intensity of Col-IV and FN in HG-Exo group increased compared with NC-Exo group.ELISA showed that there was an increase in extracellular matrix Col-IV and FN in HG-Exo mesangial cells compared with NC-Exo group,with statistical significance(P<0.05).(6)Fluorescence quantitative PCR showed that mesangial cells in HG-Exo group had more inflammatory cytokines TNF-α,IL-1β and MCP-1 mRNA than those in NC-Exo group(P<0.05).Western Blot showed that the mesangial cells in HG-Exo group had more inflammatory cytokines of TNF-α,IL-1β and MCP-1 than those in NC-Exo group(P<0.05).ELISA showed that there was an increase in TNF-α,IL-1β and MCP-1 in HG-Exo group compared with NC-Exo group,the difference was statistically significant(P<0.05).(7)Western Blot showed α-SMA in HG-Exo group increased than that in NC-Exo group,with statistical significance(P<0.05).Laser confocal results showed that the fluorescence intensity of α-SMA in HG-Exo group increased compared with NC-Exo group.(8)The mRNA of TGF-β1 in macrophages and mesangial cells increased dynamically,the difference was statistically significant(P<0.05).The difference between the HG group and the NC group of the expressions of TGF-β1/Smad3 pathway was statistically significant(P<0.05).The difference between the HG-Exo group and the NC-Exo group of the expressions of TGF-β1/Smad3 pathway was statistically significant(P<0.05).(9)Western Blot showed that the expressions of the proteins in the TGF-β1/Smad3 pathway in transfected group were lower than those in HG-Exo group after the TGF-β1 siRNA transfection,the difference was statistically significant(P<0.05).(10)Western Blot showed that the expression of Col-IV and FN in transfected group were lower than those in HG-Exo group after TGF-β1 siRNA transfection,the difference was statistically significant(P<0.05).Confocal laser scanning showed that the fluorescence intensity of Col-IV and FN in transfection group was weaker than that in HG-Exo group after TGF-β1 siRNA transfection.(11)Western Blot showed that the expressions of inflammatory cytokines TNF-α and TGF-β1 in HG-Exo group were significantly lower than those in HG-Exo group(P<0.05).(12)Western Blot showed that the expression of α-SMA in TGF-β1 siRNA transfection group decreased compared with HG-Exo group,the difference was statistically significant(P<0.05).Confocal laser scanning showed that the fluorescence intensity of α-SMA in transfection group was weaker than that in HG-Exo group after TGF-β1 siRNA transfection.(13)HbA1c and renal weight/body weight in HG-Exo group had no significant difference compared with NC-Exo group(P>0.05).Excretion rate of 24-hour urinary protein in HG-Exo group was significantly higher than that in NC-Exo group(P<0.05).(14)Hematoxylin-eosin staining and PAS staining showed that mesangial cells increased,stroma increased and some basement membrane thicken in HG-Exo group.(15)Immunohistochemistry showed that there was matrix Col-IV and FN deposition in the mesangial area of HG-Exo group.(16)Western Blot showed that the expressions of Col-IV and FN and TGF-β1/Smad3 pathway proteins increased in HG-Exo group,which were significantly different from NC-Exo group(P <0.05).Conclusion(1)In vitro high glucose stimulation can cause increasing excretion of exosome from macrophages;(2)Exosome from macrophages in vitro can be taken up by mesangial cells;(3)The uptake of exosomes can cause its proliferation,activation,secretion of extracellular matrix and secretion of inflammatory cytokines in mesangial cells;(4)TGF-β1 mRNA is involved in the connection between macrophages and mesangial cells through exosomes;(5)TGF-β1 mRNA may induce mesangial cell morphology and function changes by activating TGF-β1/Smad3 pathway in mesangial cells;(6)High glucose stimulated macrophage exosomes in mice can cause renal mesangial fibrosis and the TGF-β1/Smad3 pathway activation.