Effects of autophagic flux regulation on the adhesion and migration of macrophages

Objective Macrophage infiltration is an important histopathological feature of many chronic renal diseases including diabetic nephropathy (DN). By modulating the autophagic flux stages of macrophages (RAW264.7), this study investigated their impacts on the adhesion and migration capacities of macrophages. Methods DN rat models were established with streptozotocin. The rats were sacrificed at the end of 12 weeks after treatment. Pathological staining was made to observe renal pathological changes, and renal macrophage markers and autophagy-related markers were detected. Under the normal and high glucose (30 mM) conditions, the number of autophagosomes in RAW264.7 macrophages, the expressions of LC3, Beclin-1 (autophagy-associated marker), and P62 (autophagosome clearance marker), and the number of macrophages with adhesion and migration were measured. The autophagy lysosomal degradation inhibitor chloroquine (CQ) and the autophagosome-production-activating agent rapamycin (RAPA) were added, respectively, to observe the effects on expressions of autophagy markers as well as adhesion and migration of macrophages. The electron microscopy method was used to observe the number of autophagosomes in macrophages and the changes of autophagy lysosomes. Results DN rats showed obvious renal damage: increased glomerular volume, thickened basement membrane, increased mesangial matrix, increased renal expressions of CD68 (macrophage marker), and P62 (t=3.35, t=16.27, P<0.05), and decreased expression of LC3 (t=51.12, P<0.05). In the high glucose group of in vitro experiments, the electron microscopy showed that the number of autophagosomes was lower than that of the normal group; Western blot and immunofluorescence showed that the expressions of autophagy-related proteins LC3 and Beclin-1 were both decreased, while the expression of P62 was increased (t=27.02, t=45.56, t= 32.71, P<0.05), and the number of macrophages with adhesion and migration was increased (t=6.87, t=8.76, P<0.05); However, after treatment with CQ, the electron microscopic observation showed that the lysosomal degradation of autophagosomes in macrophages was inhibited; Western blot and immunofluorescence showed that the expressions of autophagy-related proteins LC3 and Beclin-1 were decreased, while and the expression of P62 was increased (t=14.64, t=12.45, t=8.57, P<0.05); Besides, CQ further promoted the high glucose-induced increases of adhesion and migration of macrophages (t=4.37, t=7.27, P<0.05); RAPA increased the number of macrophage autophagosomes, and Western blot and immunofluorescence showed that RAPA also increased the level of autophagy in macrophages, ie, the expressions of LC3 and Beclin-1 were increased, while the expression of P62 was decreased (t=9.37, t=11.53, t=8.73; P<0.05), and the high glucose-induced increase of the number of macrophages with adhesion and migration were reduced (t=4.16, t=5.74, P<0.05). Conclusions High glucose inhibited the level of autophagy, and promoted the adhesion and migration of macrophages. Inhibition of the degradation of autophagosomes could reduce the level of autophagy, and promoted the adhesion and migration of macrophages. Activation of autophagosomes production could increase the level of autophagy and reduce the adhesion and migration of macrophages. Key words: Macrophages; High glucose; Autophagic flux; Adhesion; Migration