Effect And Mechanism Of Fgl2 On Rhabdomyolysis-induced Acute Kidney Injury

BackgroundRhabdomyolysis is a life-threatening severe syndrome,mainly caused by trauma(crush syndrome),muscle hypoxia,genetic defects,drug or drug abuse,and so on.It involves the destruction of skeletal muscle cells,resulting in the leakage of intracellular contents into the extracellular and intravascular compartments when skeletal muscle is damaged.Recent studies have confirmed that acute kidney injury(AKI)is the major and most serious complication of rhabdomyolysis.Some studies have indicated patients with rhabdomyolysis combined with AKI have higher mortality and poor prognosis.At present,the mechanisms of rhabdomyolysis-induced acute kidney injury(RM-AKI)mainly include the renal vasoconstriction and hypoperfusion caused by the activation of renin-angiotensin,sympathetic nerve and vasopressin,damage to the renal tubular epithelial cells by myoglobin-derived free radicals,myoglobin casts’ blocking to distal renal tubular,inflammation,and apoptosis.It has been found that supportive treatment such as fluid resuscitation and hemodialysis can alleviate the pain of patients to some extent,but it’s not enough.Increasing evidence suggests that mechanisms such as inflammation and apoptosis are indispensable in RM-AKI,so exploring the role of inflammation and apoptosis in RM-AKI may provide new theoretical basis and therapeutic targets for clinical prevention and treatment.Fibrinogen-like protein 2(Fgl2),a member of the fibrinogen superfamily,is a glycoprotein with functions of coagulation activity and immunosuppression.It is expressed by macrophages,T cells,and tumor cells and other kinds of cells.Many studies show Fgl2,an immunoregulatory protein,is involved in the occurrence and progression of a variety of diseases,such as autoimmune glomerulonephritis,tumors,viral hepatitis.In our previous studies,we found that Fgl2 was involved in the occurrence and development of cisplatin-induced AKI in mice and renal fibrosis caused by ureteral ligation.However,it is still unclear whether Fgl2 plays a role in RM-AKI.Objective1.To identify the correlation between Fgl2 and RM-AKI2.To explore the role and mechanism of Fgl2 in RM-AKIMethods1.To detect the expression of Fgl2 in RM-AKI50% glycerol was injected into the hind leg of wild-type(WT)mice intramuscularly to establish a RM-AKI model.The expression of Fgl2 in renal tissue was detected through Quantitative real-time polymerase chain reaction(q RT-PCR),western blot and Immunohistochemistry(IHC).2.To study the effect of Fgl2 in RM-AKIA RM-AKI model was established in Fgl2 knockout(Fgl2 KO)mice of the same background.The serum and kidney tissues were collected after 48 hours.2.1 To measure Serum creatinine(Scr)and blood urea nitrogen(BUN)2.2 To observe and score the renal histopathology of WT and Fgl2 KO mice by HE and PAS staining2.3 To detect NGAL(Neutrophil gelatase-associate lipocalin)and KIM-1(kidney injury molecule 1)in kidney tissues of WT and Fgl2 KO mice by q RT-PCR3.To investigate the possible mechanism of Fgl2 in RM-AKI3.1 To detect the m RNA levels of IL-6,MCP-1,TNF-α,IL-1β and IL-10 and the F4/80 positive cells between WT mice and Fgl2 KO mice after modeling3.2 The apoptosis of renal tubular epithelial cells between WT mice and Fgl2 KO mice was detected by Tunel.The expression of apoptosis-related proteins such as Bcl2,Bax,cleaved caspase-3,cleaved caspase-9 were detected by western blot.Results1.Compared with the saline groups,the expression of Fgl2 in the renal tissue of WT mice was significantly increased in RM-AKI groups.2.Fgl2 gene KO alleviates RM-AKI.2.1 The degree of renal dysfunction was significantly mitigated in Fgl2 KO mice when happened RM-AKI.The levels of Scr and NGAL were significantly mitigated in Fgl2 KO mice compared with WT mice with RM-AKI but the BUN and KIM-1 showed no statistical significance between two groups.2.2 In RM-AKI groups,the pathological injuries of kidney was alleviated in Fgl2 KO mice compared with WT mice.After glycerol treatment,necrosis,swelling,cast formation and tubular dilatation of kidney tissues can be observed by HE and PAS staining in both WT and Fgl2 KO mice.But the injury was milder and the pathological injury score was lower in the Fgl2 KO mice than in the WT mice.3.In RM-AKI groups,the inflammation and apoptosis decreased in Fgl2 KO mice compared with WT mice.3.1 The inflammatory response in renal tissue was alleviated in Fgl2 KO mice compared with WT mice in RM-AKI groups.Compared with WT mice,the levels of pro-inflammatory factors TNF-α,IL-1β,IL-6,and MCP-1 were decreased and macrophage infiltration in renal tissue was reduced,while the expression of anti-inflammatory cytokine IL-10 increased in Fgl2 KO mice.3.2 Apoptosis of renal tubular epithelial cells in Fgl2 KO was decreased compared with WT mice.In RM-AKI groups,there was no significant difference of Bcl2 in WT mice and Fgl2 KO mice.However,compared with WT mice,Bax,cleaved caspase-3 and cleaved caspase-9 were down-regulated and Tunel staining showed that apoptosis was significantly alleviated in Fgl2 KO mice.Conclusion1.Fgl2 gene deletion plays a protective role in RM-AKI.2.Fgl2 gene deletion may alleviate RM-AKI by inhibiting inflammation and reducing apoptosis.