The Effects Of Autophagic Flux Regulation On Macrophages Adhesion And Migration Induced By High Glucose

Objectives:Macrophage infiltration is an important histopathological feature of many chronic renal diseases including diabetic nephropathy.This process is closely related to the adhesion and migration of macrophages in renal tissue and the changes of phenotype.Autophagy maintains normal physiological functions of cells by degrading misfolded proteins and dysfunctional organelles.It is a highly dynamic,multi-phase process,and also known as autophagic flow that mainly includes two phases of autophagosomes and autolysosome.However,the relationships between autophagy and macrophage function and phenotype are unknown.This study aims to investigate the influence of different stages of autophagy flux on macrophage autophagy level and its adhesion and migration function by regulating autophagosome formation and the process of degradation and fusion of autolysosome.Methods:(1)In vivo,rats were randomly distributed into control(NC)and DN groups.The pathological changes in renal tissue were assessed,and expression of autophagy markers(CD68,LC3,P62)were analysed.(2)In vitro,RAW264.7 cells were divided into normal and high glucose(HG,30 m M)groups.The capacity for macrophage adhesion and migration and the expression of autophagy markers were observed with and without autophagy modulators(rapamycin,3-methyladenine,bafilomycin,A1 and chloroquine for RAPA,3-MA,BAFA,CQ).The numbers of autophagosomes and the process of fusion and degradation of autolysosome were observed by electron microscopy.Results:(1)(1)In vivo,compared with the NC group,the blood glucose,renal weight/weight ratio(KW/BW),Scr,BUN,and 24 urine protein levels were increased in the DN group at the end of the 12 th week(P<0.05),and the weight was decreased in the DN group(P<0.05),indicating that the DN model was successfully established.(2)The volume of the glomeruli were increased,the basement membrane was thickened,and the mesangial matrix was increased in the kidney tissues of rats in the DN group.(3)Macrophage infiltration was increased in renal tissue of DN group showed by CD68,the macrophage maker.(4)The expression levels of P62 of renal tissues was increased in the DN group,and the expression level of LC3 was decreased(P<0.05).(2)In vitro,(1)HG or 3-MA reduced the numbers of autophagosomes with less expression of LC3 and Beclin-1,but increased expression of P62.HG or 3-MA promote the adhesion and migration capacity of macrophages(P<0.05).(2)Moreover,BAFA and CQ inhibited the process of fusion and degradation of the autolysosome as well as the expression of LC3 and Beclin-1.We noticed an increase expression of P62 by BAFA and CQ stimulation(P<0.05).These effects further facilitated the adhesion and migration capacity of macrophages.(3)However,RAPA increased the numbers of macrophage autophagosomes,resulting in an increase expression of LC3 and Beclin-1,whereas a reduction expression of P62,which lead to inhibition of adhesion and migration of macrophages induced by HG(P<0.05).Conclusions:Modulating the stage of autophagy flux(autophagosome formation and autolysosome fusion and degradation)can regulate the autophagy level,which alter the capacity of macrophage adhesion and migration:1.HG and 3-MA inhibite formation of macrophage autophagosomes and reduces the autophagy level,which promote the macrophages adhesion and migration.2.RAPA can activate macrophage autophagosome formation and improve the autophagy level under high glucose concentration,but leading a reduce of the macrophages adhesion and migration.3.BAFA and CQ inhibit the process of autolysosome fusion and degradation,suppress the autophagy level,which promote the macrophages adhesion and migration.