The Mechanism Of M2 Macrophages Infusion Ameliorating Diabetic Nephropathy In Db/db Mice

Chronic kidney disease(CKD)is a major cause of global morbidity and mortality.Diabetic nephropathy(DN)is one of the main causes of CKD,and it is also a major cause of end-stage renal disease(ESRD).The pathogenesis of DN is complex,and more and more studies have shown that immune regulation and inflammatory processes are largely involved in the occurrence and progression of DN.Recent studies have demonstrated that macrophages infiltration of renal tissues is one of the characteristic features of the inflammatory response in DN and plays an important role in the development of DN.The accumulation of macrophages is closely related to the damage of renal structural and renal dysfunction.Phenotypically,macrophages that can secrete pro-inflammatory cytokines are defined as classically activated macrophages(M1),and those that are capable of modulating immune responses through anti-inflammatory or resolution mechanisms are termed alternatively activated M2macrophages(M2).Previous studies have shown that the macrophages infiltrating into the kidney tissue of patients are mainly M1 macrophages,and the ratio of M1 macrophages to M2macrophages is seriously imbalanced.Therefore,we hypothesize that M2 macrophages infusion can reserve the macrophages balance and decrease chronic inflammation in the kidney of DN mice.The aim of this study was to investigate whether the infusion of M2 macrophages could improve the pathological structure of glomeruli and ameliorate renal function.And we aimed to investigate whether the infusion of M2 macrophages can reverse the macrophages balance in kidney,reduce the inflammation of glomerular mesangial cell and hinder the progression of DN.Finally to explore the mechanism of M2 macrophages in alleviating the inflammation of glomerular mesangial cell and decreasing the glomerular extracellular matrix(ECM)deposition.Bone marrow-derived macrophages were isolated from the tibias and femurs of male C57BL/6 mice(6 weeks old),and were stimulated with 20 ng/ml IL-4 for 24 h to obtain M2macrophages.In this study,we used 10-week-old db/db mice to induce a DN model and then performed 1×10~6M2 macrophages infusion suspended in 0.2 ml of phosphate buffered solution(PBS)once per week for 4 weeks through tail vein.DN group were treated with an infusion of 0.2 ml PBS alone.Blood glucose and weight were monitored every week.The insulin sensitivity were evaluated one week after infusion.Blood was collected to detect the level of interleukin-1β(IL-1β),monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),serum creatinine,blood urea nitrogen,high density lipoprotein cholesterol,low density lipoprotein cholesterol,total cholesterol and triglyceride.Urine were collected to measure the level of urine microalbumin and urine creatinine.Immunofluorescence and quantitative real-time polymerase chain reaction(q RT-PCR)were used to evaluate the level of IL-1β,MCP-1,TNF-α,transforming growth factor-β(TGF-β),IL-6,collagen IV(Col-4),fibronectin(FN)and alpha-smooth muscle actin(α-SMA).Western blotting was performed to explore the changes of Janus kinase 2(JAK2)/signal transducers and activators of transcription 3(STAT3)pathway.CM-Dil was used to track M2macrophages.In vitro,SV40-MES-13 cells that stimulated by high glucose were used to verify the effect of M2 macrophages and detect the changes of JAK2/STAT3 pathway.A JAK2 mimic lentivirus was used to examine the possible mechanism.Results showed that the blood glucose and body weight of 10-week-old db/db mice were significantly higher than those of db/m mice and the urinary albumin to creatinine ratio(ACR)level was 4.7 times higher than that of db/m mice(p<0.01).Hematoxylin-eosin staining and periodic acid-schiff staining of tissue from 10-week-old db/db mice revealed glomerular hypertrophy,slight hyperplasia of mesangial cells,mesangial matrix expansion and glycogen deposition compared with those in db/m mice.The level of urinary ACR,serum creatinine and blood urea nitrogen were 10.61±1.35 ng/mg,35.44±1.50 mg/dl and 23.33±5.63μmol/l,respectively.M2 macrophages infusion ameliorated the structure of glomerulus and kidney function.After multiple infusions of M2 macrophages,the ratio of the co-expressed positive area of Col-4 and mesangial cells decreased to 10.33±1.53%,and the ratio of the co-expressed positive area of FN and mesangial cells decreased to 10.00±1.00%.The results of q RT-PCR showed that the level of Col-4 and FN in the DN group significantly increased and decreased after infusing with M2 macrophages.The positive rate of IL-1βand MCP-1 in the group of DN were 34.67±3.51%and 49.00±3.00%,respectively.While those in M2 group were 13.67±5.13%and 13.00±4.24%,respectively.Results of q RT-PCR showed that the level of IL-1β,MCP-1,TNF-α,TGF-βand IL-6 in the DN group significantly increased,while significantly decreased in the M2 group.In the peripheral blood,the levels of IL-1β,MCP-1,TNF-αand IL-6 increased significantly in the DN group,and significantly decreased in the M2 group mice.We also found that the infused M2 macrophages homed to the kidney,reversing the disturbances in M1/M2 homeostasis in DN mice.While the improvement of body weight,blood glucose,lipids and insulin sensitivity were not obvious.In vitro,the immunofluorescence staining of Col-4,FN andα-SMA decreased in SV40-MES-13 cells that was stimulated by high glucose and co-cultured with M2 macrophages.The secretion of IL-1β,MCP-1,TNF-α,TGF-βand IL-6 in cell supernatant also reduced.M2 macrophages also can downregulate the protein level of phosphorylated JAK2 and phosphorylated STAT3.After transduction with a JAK2 overexpression virus,the therapeutic effects of M2 macrophages on ameliorating inflammation and decreasing ECM expression were inhibited.In conclusion,our findings suggested that multiple infusions of M2 macrophages can reverse macrophages imbalance,significantly alleviated inflammation and decreased ECM deposition in glomerulus and hindered the progression of DN at least partially by downregulating the JAK2/STAT3 pathway in glomerular mesangial cells.M2 macrophages infusion may be a new therapeutic strategy for DN treatment.