The Role And Mechanism Of Macrophage Activation Induced By Exosomes From High Glucose-treated Renal Tubular Epithelial Cells

Background and purpose Diabetic nephropathy（DN）,a severe microvascular complication of diabetes,is the leading cause of end-stage renal disease（ESRD）worldwide^[1].Numerous studies have confirmed that DN is an inflammatory disease and infiltrating macrophages are believed to play essential roles in the pathogenesis of DN^[2].Exosomes are membranous nanovesicles of 30–100 nm in size,which are secreted from a variety of cell types.Exosomes migrate between body fluids and play an important role in cell-to-cell communication.Recent sudies indicate that renal innate cells can communicate with each other through the release of exosomes,thus alleviating or aggravating DN^[3-6].Currently,studies on the activation of macrophages by cell-derived exosomes are mainly focused on the stem cells and adipocytes.For example,Zhao’s research confirmed that exosomes from adipose-derived stem cells attenuate adipose inflammation and obesity through polarizing M2 macrophages^[7].The connection between the activation of macrophages and the exosomes secretion from renal innate cells is unclear.This paper taking exosomes from high glucose-treated renal tubular epithelial cells as the research object to investigate it’s role in the macrophage activation.It’s a new way of looking at the interaction between renal innate cells and inflammatory cells in DN,so as to provide a new way for the prevention and treatment of DN.Methods 1.The supernatant of renal tubular epithelial cells which were cultured in normal control group（5.5mmol/L D×glucose）or high glucose group（30mmol/L D×glucose）for48h were collected and ultracentrifuged to harvest exosomes;Exosomes were identified by transmission electron microscope and Western blotting;2.THP-1 were stimulated by PMA to be THP-1 macrophage and detected by flow cytometry;Grouping:blanck control（Control）;exosomes from normal glucose concentration group（NC-Exo）;exosomes from high glucose concentration group（HG-Exo）;3.Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage;4.Observing the morphology of THP-1 macrophage microscopically after co-cultured with exosomes;5.Western blot was used to detect the expression of iNOS,a marker protein of activated macrophages,and to explore the optimal time and concentration of exosome stimulation;6.Laser scanning confocal microscope was also used to detect the expression of iNOS;7.Detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes;8.The expression of inducible nitric oxide synthase（iNOS）,tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）,monocyte chemotactic protein-1（MCP-1）in THP-1macrophages were detected by quantitative Real-time PCR（qRT-PCR）;9.The expression of TNF-α,IL-1βand MCP-1 in supernatants were detected by enzyme linked immunosorbent assay（ELISA）;10.The expression of p-c-Jun NH2-terminal kinase（p-JNK）,phoshpo-p38mitogen-activated protein kinase（p-p38MAPK）and nuclear factorκB p65（NF-κB p65）in THP-1 macrophages were detected by Western blotting.Results 1.Vesicles were extracted from the supernatants of HK2 cells cultured in normal and high glucose by ultracentrifugation;Vesiclesthat harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63,TSG101 but absence of calnexin which is a marker of endoplasmic reticulum;.2.After stimulated by PMA,the expression of CD68 of THP-1 cells were significantly increased;3.Laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages;4.The morphological change of THP-1 macrophages were observed microscopically after stimulated by each group exosomes,especially the HG-Exo THP-1 macrophages;5.Western blot showed that:Compared with normal control group,HG-Exo had increased the expression of iNOS in THP-1 macrophages significantly;6.Laser scanning confocal microscope also showed that high glucose exosomes had increased the expression of iNOS in THP-1 macrophages;7.Transwell results suggest that high glucose exosome can improve the chemotaxis of THP-1 macrophages significantly;8.RT-PCR showed that:Compared with normal control group,HG-Exo had increased the expression of iNOS,TNF-α,IL-1βand MCP-1 in THP-1 macrophages（all P<0.01）;9.ELISA showed that:The expression of TNF-α,IL-1βand MCP-1 in supernatants of HG-Exo THP-1 macrophages were increased significantly（all P<0.01）;10.Western blot showed that:The expression of p-JNK,p-p38 MAPK and NF-κB p65proteins in the HG-Exo THP-1 macrophages alsoincreased significantly（all P<0.01）.Conclusions 1.Both groups of exosomes can be internalized by THP-1 macrophages.2.Compared with normal control group,HG-Exo could activated THP-1 macrophages significantly.3.MAPK/NF-κB signaling pathway was involved in the mechanism of THP-1macrophage activation induced by HG-Exo.