Effects And Mechanism Of Fgl2 During The Pathogenesis Of Renal Fibrosis

BackgroundChronic kidney disease?CKD?is defined as impaired structure or function caused by various reasons more than 3 months.The prevalence of CKD is going to increase every year with the aging of China's population and the increasing metabolic diseases,bringing heavy economic burden on the families and society.End stage renal disease?ESRD?is defined as the terminal event of CKD,but there is no specific drug for this disease at present.Renal fibrosis is a common final feature of various CKDs that leads to ESRD,and is one of the most important reasons of renal function loss.Despite that our knowledge about renal fibrosis has improved greatly over the past few years,the exact underlying mechanism of the disease process remains unknown.So it is of great significance to study the pathogenesis and specific targets of renal fibrosis for the prevention and treatment of this disease.Studies have shown that inflammation plays an important role in the pathogenesis of renal fibrosis,whereas macrophages,a significant member in immune system,also play a vital role in inflammation regulation.Macrophages exhibit two main phenotypes in response to different stimuli,namely,the classically activated M1 macrophages,and the alternatively activated M2 macrophages.Current studies have shown that there are a large number of macrophages in the fibrotic renal tissues,and its polarization plays an important role during the pathogenesis of renal fibrosis.M1 macrophages accelerate the progression of fibrosis by promoting tissue damage,whereas M2 macrophages promote the development of renal fibrosis by increasing the formation of the extracellular matrix and the secretion of profibrotic factors.Fibrinogen-like protein 2?Fgl2?is a prothrombinase,and shows both coagulation activity and immunomodulatory effects.Recent studies have shown that Fgl2 is involved in the pathogenesis of numerous diseases by regulating the inflammatory response.Inflammation is the core of renal fibrosis,so we speculate that Fgl2 may be involved in the development of this disease.In this study,we established UUO-induced renal fibrosis model and then investigated the effects of Fgl2 on macrophage polarization by using immunohistochemistry,western Bolt,flowcytometry,quantitative real time polymerase chain reaction.Objective1.To investigate the effects of Fgl2 deficiency on the pathogenesis of renal fibrosis.2.To investigate the roles of Fgl2 in M1 and M2 macrophage polarization during renal fibrosis development.Methods1.Effects of Fgl2 deficiency on the pathogenesis of renal fibrosis.1.1 Renal tissue samples from CKD patients were collected and the expression of Fgl2 in renal tissues was measured by IHC.1.2 Renal tissues were collected from the Fgl2^+/+mice on days 3,7,or 14 after UUO surgery and the expression of Fgl2 in renal tissues was detected by IHC,WB,and qRT-PCR1.3 Renal tissues were collected from the Fgl2^+/+mice on day 7 after UUO surgery,and the following experiments were measured.?1?IHC,WB,and qRT-PCR were used to detected the expression of?-SMA,Cola?and fibronectin.?2?The deposition of collagen was measured by Masson staining.?3?The mRNA levels of TGF-?1 were detected by qRT-PCR.2.Roles of Fgl2 in M1 and M2 macrophage polarization during renal fibrosis development.2.1 Renal tissues were collected from the Fgl2^+/+mice on day 14 after UUO surgery,immunofluorescence was used to detect the expression of Fgl2 in macrophages?F4/80⁺cells?that infiltrated in fibrotic kidney tissues.2.2 Renal tissues were collected from the Fgl2^+/+mice on days 7 or 14 after UUO surgery,and the following experiments were measured.?1?The percentage of macrophages?F4/80⁺CD11b⁺cells?,M1 macrophages?F4/80⁺CD11b⁺MHC-?⁺cells?,and M2 macrophages?F4/80⁺CD11b⁺CD206⁺cells?in fibrotic kidney tissues was detected by FCM.?2?The mRNA levels of M1 macrophage-associated molecules?iNOS,IL-12p40,IL-6,IL-1?,and TNF-??and M2 macrophage-associated molecules?MR,Arg-1,IL-10,Fizz-1,YM-1,and TGF-?1?in fibrotic kidney tissues were detected by qRT-PCR.2.3 BMDM were induced from BM cells of the Fgl2^+/+and Fgl2^–/–mice by incubating with M-CSF,and then stimulated with combination of IFN-?and LPS or IL-4 to induced the polarization of M1 macrophages or M2 macrophages,respectively.?1?FCM was used to detecte the percentage of M1 macrophages and M2 macropahages in Fgl2^+/+and Fgl2^–/–derived-BMDM.?2?qRT-PCR was used to measure the mRNA levels of M1 macrophage-associated molecules?iNOS,IL-12p40,IL-6,IL-1?,and TNF-??and M2 macrophage-associated molecules?MR,Arg-1,Fizz-1,and YM-1?in Fgl2^+/+and Fgl2^–/–derived-BMDM.Results1.Effects of Fgl2 deficiency on the pathogenesis of renal fibrosis.1.1 Compared with normal kidney tissues,the expression of Fgl2 was increased in the kidney tissues from patients with CKD.1.2 Compared with Sham group mice,Fgl2 expression was increased in the kidney tissues from UUO-treated mice.1.3 Compared with Fgl2^+/+mice,Fgl2^-/-mice showed more severe renal fibrosis on day 7after UUO surgery,as evidenced by the increased expression of?-SMA,collagen?,and fibronectin,TGF-?1 secretion,and collagen deposition?shown by Masson's trichrome staining?.2.Roles of Fgl2 in M1 and M2 macrophage polarization during renal fibrosis development.2.1 Fgl2 was highly expressed in the macrophages that infiltrated in the fibrotic kidney tissues on day 14 after UUO surgery.2.2 On days 7 and 14 after UUO surgery,there was no difference in the percentage of total macrophages?F4/80⁺CD11b⁺cells?and M1 macrophages?F4/80⁺CD11b⁺MHC-?⁺cells?in the kidney tissues between Fgl2^–/–and Fgl2^+/+mice,but Fgl2 deficiency led to a higher percentage of M2 macrophages?F4/80⁺CD11b⁺CD206⁺cells?in the kidney tissues.2.3 On day 7 after UUO surgery,there was no difference in the expression of M1macrophage-associated molecules in the kidney tissues between Fgl2^–/–and Fgl2^+/+mice,but Fgl2 deficiency led to a higher expression of some M2 macrophage-associated molecules?MR,TGF-?1?in the kidney tissues.2.4 On day 14 after UUO surgery,compared with that in Fgl2^+/+mice,some M1macrophage-associated molecules?IL-12p40,TNF-??and part of M2 macrophage-associated molecules?MR,TGF-?1,IL-10,Fizz-1?were increased in the kidney tissues of Fgl2^–/–mice.2.5 During the process that BMDM polarized towards M1 macrophages,compared with that in Fgl2^+/+mice,the percentage of M1 macrophages and the expression of M1macrophage-associated molecules?iNOS,IL-12p40,IL-6,IL-1?,TNF-??were increased in Fgl2^-/-mice-derived BMDM.2.6 During the process that BMDM polarized towards M2 macrophages,compared with that in Fgl2^+/+mice,the percentage of M2 macrophages and the expression of M2macrophage-associated molecules?MR,Arg-1,Fizz-1?were increased in Fgl2^-/-mice-derived BMDM.Concusion1.Fgl2 highly expressed in the fibrotic kidney tissues.2.Fgl2 deficiency aggravates UUO-induced renal fibrosis in mice.3.Fgl2 deficiency mediate pathogensis of renal fibrosis by facilating macrophage polarization.