The Role And Mechanism Of Macrophage-derived Exosomes In Endothelial Cell Dysfunction In Sepsis Acute Kidney Injury

BackgroundAcute kidney disease(AKI)is a common clinical syndrome,mainly manifested by rapid decline in renal function and accumulation of metabolic wastes.The causes of AKI include infection,drugs,ischemia-reperfusion,etc.,and sepsis caused by lipopolysaccharide(LPS)is a common cause of AKI.In ICU patients,AKI caused by sepsis accounts for about half of the incidence of AKI.The main pathological manifestations of septic AKI are vacuolar degeneration and apoptosis of renal tubular epithelial cells,renal interstitial inflammatory cell infiltration,and endothelial cell damage.The activation of macrophages and the damage of endothelial cells are involved in the progression of septic AKI,but the specific mechanism is still unclear.Exosomes are cell-derived membrane-like vesicles with a diameter of about 30100nm,which can contain lipids,polypeptides,RNAs,etc.Exosomes can transport functional components to recipient cells,regulate the functions of recipient cells,and are an important medium for cell-to-cell communication.Studies have confirmed that exosomes produced in renal tubular epithelial cells induced by albumin can activate macrophages into M1 type and participate in the development of renal interstitial inflammation.Other studies have confirmed that exosomes produced by macrophages can promote the phagocytosis of macrophages and the migration of monocytes in crystal nephropathy.However,the role of macrophage-derived exosomes in endothelial homeostasis in septic AKI is still unclear.In this study,we explored whether and how macrophage-derived exosomes are involved in endothelial cell damage in septic AKI.AimWhether exosomes derived from macrophages are involved in the damage of endothelial cells in septic AKI and its mechanism.Method1.In vitro experiments1)Cultivate RAW264.7 and treat the macrophages with PBS and LPS.Respectively,extract the culture supernatant after 24 hours,and obtain purified exosomes by centrifuging.Methods such as electron microscopy,nanoparticle tracking analysis(NTA)and western blot(WB)were used to identify exosomes.2)Purify the macrophage-derived exosomes and stain them with PKH26.The exosomes produced by RAW264.7 with PBS and LPS(labeled with PKH26)were added to rat glomerular endothelial cells(RGECs).After 12 hours of culture,the cytoskeleton was stained with phalloidin,the nucleus was stained with DAPI.Whether the PKH26-labeled exosomes could be internalized by RGECs was detected under confocal microscope.3)The exosomes produced by RAW264.7 stimulated with PBS or LPS were added to RGECs in vitro.After 24 hours,the cell viability was measured by CCK8,and the expression levels of VCAM-1,NLRP3,Caspase-1,ASC and IL-1β were measured by WB.4)The macrophages were pretreated with Acid sphingomyelinase(ASM)inhibitor amitrityline and stimulated with LPS.After 24 hours,the macrophage exosomes were extracted to detect the expression of CD63 and CD92.In vivo experiment1)LPS-AKI model was established in C57 BL/6 mice.The wild-type C57 BL/6 mice were divided into 3 groups(control group,experimental 12h group,and experimental 16h group),each group were 8 mice.LPS were prepared with PBS at a concentration of 2mg/mL.The control group was injected with PBS and the experimental group was injected with LPS at a concentration of 20mg/kg.The samples were taken at 12h and 16h.Blood urea nitrogen(BUN)and creatinine(Cr)were detected from plasma.Part of kidney tissue was subjected to immunohistochemistry(Immunohistochemistry,IHC)and pathological staining,and a part was used for WB detection.Kidney tissues were subjected to IHC to detect the expression level of macrophage marker F4/80.Renal cortex tissues were subjected to WB to detect the expression of neutrophil gelatinase related lipocalin(Neutropil gelatinase-associated lipocalin,NGAL),endothelial cell injury marker VC AM-1,and the expression of exosome marker CD63.2)The exosomes produced by LPS-stimulated macrophages were injected into C57 BL/6 mice by tail vein injection in vitro.Plasma was taken to detect the change of Cr and BUN,and kidney tissue was taken for analysis pathological staining.Using WB to detect the expression of kidney injury marker NGAL and endothelial cell injury marker VCAM-1.3)Divide ASM+/-mice and the same batch of wild-type C57 BL/6 mice into 2 groups,the control group(PBS)and the experimental group(LPS 20mg/Kg).After intraperitoneal injection for 16h,plasma was taken for detection of Cr and BUN.Kidney tissue was taken for pathological staining,and WB was used to detect the change of kidney damage maker NGAL and the expression level of endothelial cell damage marker molecule VCAM-1.Result1.In vitro experiments1)The secretion of exosomes from macrophage increases after LPS stimulated.2)The exosomes produced by LPS stimulation of RAW264.7 can cause damage to RGECs,including decreased cell viability,increased expression of VCAM-1,and activation of NLRP3 inflammasomes.3)Inhibition of ASM can inhibit macrophage exosome secretion.2.In vivo experiments1)In LPS-AKI,the infiltration of macrophages is increased and the secretion of exosomes is increased.2)The exosomes produced by LPS stimulation of macrophage cause the expression levels of NGAL and VCAM-1 increased significantly.3)In the LPS-AKI model,the expression levels of NGAL and VCAM-1 of ASM+/-mice were significantly reduced.These results indicate that inhibition of ASM activity in AKI can reduce endothelial cell injury and renal injury.ConclusionIn LPS-AKI,macrophage infiltration and exosome secretion increased.These exosomes internalized by glomerular endothelial cells and aggravate the damage of glomerular endothelial cells,which plays an important role in occurrence and development of sepsis AKI.