The Role Of IRF-4 In Inflammatory Response And Renal Fibrosis In The Last Stage Of Folic Acid-induced Acute Kidney Injury

Background:Acute kidney injury(AKI)is a common perioperative complication with high mortality.Survivors of severe AKI are prone to Chronic Kidney Injury(CKD),resulting in reduced quality of life and a high risk of cardiovascular events.Therefore,it is of great significance to prevent the transformation of AKI into CKD.At present,there is no ideal way to prevent AKI from converting to CKD.The mechanism of chronic transformation of AKI is closely related to inflammatory response and fibrosis.Interferon regulatory factor 4(IRF-4)is a key factor in regulating inflammatory response and fibrosis.It has been reported that knocking out the IRF-4 gene can reduce the inflammatory response of peritonitis in mice,specifically knocking out the IRF-4 gene in bone marrow cells can reduce renal fibrosis after ischemic injury,and down-regulation of IRF-4 can exacerbate ischemia/reperfusion-induced renal fibrosis.The role of IRF-4 in the model of folicacid kidney injury is unclear,and and it is of great clinical significance to study its role in the inflammatory response and renal fibrosis in the late stage of folic acidinduced AKI.Objective:In this study,IRF-4 knockout mice were used to explore the effect of IRF-4 in regulating inflammatory response and renal fibrosis in the late stage of folic acid-induced AKI in a mouse model of folic-acid induced AKI,so as to provide a reference for the prevention and treatment of chronic transformation of AKI.Methods:In this study,C57BL/6 wild(WT)mice and IRF-4 knockout(IRF-4 KO)mice were used to construct folic acid(FA)-induced AKI models.The experiment was divided into four parts,the first part was modeled by WT mice and taken on the 3rd,7th and 14th days,and the tubular injury scores and the proportion of extracellular collagen deposition area were observed by HE staining and Sirius staining.In the second part,WT mice were modeled and taken on the 14th day,and the IRF-4 positive cells count and IRF-4 protein expression levels were detected by immunohistochemistry and western blotting.In the third part,WT mice and IRF-4 KO mice were modeled and taken on 14th day,and HE staining,immunohistochemistry,real-time PCR were used to detect tubular injury scores,CD3positive cells and F4/80-positive cells count,pro-inflammatory factors CXCL16,IL18,IL-6 mRNA level indexes.In the fourth part,WT mice and IRF-4 KO mice were modeled and taken on the 14th day,and Sirius staining,real-time PCR were used to detect the proportion of extracellular collagen deposition area,TGF-β1 mRNA level,immunofluorescent single staining were used to to detect the proportion of positive area of α-SMA,Fibronectin and Collagen I,while immunofluorescence double staining were used to to detect CD45-α-SMA,F4/80-α-SMA,CD206-α-SMA doublepositive cells count index.IBM SPSS Statistics 25.0 software was used for data analysis,and the normal distribution data counting data was expressed as mean ±standard error(mean±SEM).The differences between groups were analyzed by oneway analysis of variance(ANOVA),LSD method was used for equal variance,while Tamhane T2 method was used for unequal variance,and P<0.05 was statistically significant.Results:Kidneys in FA-induced AKI mouse models exhibited progressive tubular damage and fibrosis;IRF-4 expression were upregulated in kidneys in the FAinduced AKI models of WT mouse;Compared with WT mice,the tubular injury scores,CD3 positive cells and F4/80 positive cells count in FA-induced AKI models after knocking out the IRF-4 gene were reduced,and the levels of pro-inflammatory factors CXCL16,IL-18 and IL-6 mRNA were down-regulated.Compared with WT mice,the proportion of extracellular collagen deposition area and TGF-β1 mRNA level in FA-induced AKI model after knocking out the IRF-4 gene were downregulated,the proportion of positive area of α-SMA,Fibronectin and Collagen I decreased,and the double-positive cells count of CD45-α-SMA,F4/80-α-SMA and CD206-α-SMA decreased.Conclusion:Progression of FA-induced AKI is accompanied by inflammatory responses and renal fibrosis;IRF4 promotes infiltration of macrophages and T lymphocytes in the kidneys,increases the expression of pro-inflammatory factors,aggravates the inflammatory response;At the same time,IRF-4 mediates the activation of myofibroblasts,promotes the transformation of macrophages to myofibroblasts,and promotes renal fibrosis.