ARG1 Expression In Macrophages Mitigates Early Acute Kidney Injury

Background:Macrophages play an important role in the process of renal injury.Macrophages help tissue repair or further aggravate tissue damage by phagocytosis or activation of T cells to clear pathogens,which depends on the function and phenotypic plasticity of macrophages.The phenotype of macrophages changes dynamically under the influence of local microenvironment.With the development and maturity of single cell sequencing technology,the typing of macrophages in vivo tends to be accurate.Studies have shown that arginase 1（ARG1）is a specific marker of macrophages,and ARG1 expression in macrophages promotes tumor progression and tumor related inflammation.Previous studies of our team have found that ARG1⁺macrophages play an important role in the progression from acute renal injury（AKI）to chronic kidney disease（CKD）.As the diversity of renal macrophage subsets and their plasticity explain their different functional responses in distinct renal diseases,weather ARG1 expression in macrophages palying a different role in the early stage after AKI,compared to late stage,is unclear.Therefore,this study mainly discussed the changes of ARG1 expression in renal macrophages in the early stage of AKI and the role of ARG1⁺macrophages in it,so as to provide a theoretical basis for the prevention and treatment of renal diseases based on ARG1⁺macrophages.Methods and results:First of all,we used the public database of single cell sequencing to analyze the expression of ARG1 in different stages of renal injury,and found that ARG1 was mainly expressed in macrophages in the injured kidney.The infiltration of ARG1⁺macrophages in the kidney after renal ischemia-reperfusion（IR）injury increased in the early stage and decreased gradually in the late stage.By constructing the model of renal ischemia-reperfusion（IR）at different time points to observe the changes of the number of total macrophages and ARG1⁺macrophages,we found that the number of total macrophages and ARG1⁺macrophages increased early after injury,and then gradually decreased,which was consistent with the data from public single cell transcriptome sequencing database.Subsequently,in order to explore whether ARG1 affected the progress of AKI by consuming arginine,mice were fed with arginine of different concentrations（1%and 3%）to 5 and 28 days after IR operation.Pathological staining,immunofluorescence and real time polymerase chain reaction（RT-PCR）were used to verify that there was no significant statistical difference in renal injury among different concentration groups at different stages of injury.Above all,arginine supplementation did not change the expression of ARG1 and the number of renal ARG1+macrophage infiltration after AKI,and had no effect on renal injury.Therefore,this study further discussed the effect of knocking down ARG1 expression in macrophage after IR injury.Our team’s previous studies have verified that knocking down ARG1 in macrophages inhibits the progression of mid-and late-stage（d5-d28）AKI.However,wheather knocking down ARG1 in macrophage influences early renal injury after IR is not clear.So we hybridized C-X3-C chemokine receptor 1（CX3CR1）^{Cre ERT2}mice with ARG1^flox/flox(ARG1^f/f)mice by gene editing technique to construct the mice that can induce ARG1 knockout in macrophages.The expression of ARG1 in macrophages was knocked out by intraperitoneal injection of tamoxifen.After the elution of two weeks,we established the IR injury model,and the mice were sacrificed 12 hours later.Through pathological staining,it was found that early knockout of ARG1 in macrophage aggravated AKI compared with the control group.In order to further explore the effect of knockout ARG1 in macrophage on the early injury of AKI,we constructed liposome-encapsulated small interference RNA（si RNA）knockdown ARG1 expression in vitro.After 24 hours of co-culture of lipid-encapsulated si RNA（ARG1-si RNA）with ARG1+macrophages,compared with the control group,the expression of ARG1 in ARG1-si RNA liposome treated group was significantly decreased,which confirmed that ARG1-si RNA liposome knock down the expression of ARG1 in macrophages.By injecting ARG1-si RNA liposome via tail vein one day before IR and after operation,and then sacrificing the mice after 12h and 24h,we found that knockdown of ARG1 in macrophage aggravated early AKI.These results suggest that ARG1 expression in macrophages contribute to the renal regeneration or injury resistance in the early stage of AKI.Conclusion:Macrophages expressing ARG1 play different roles in different stages of renal injury.Knocking down ARG1 in macrophages inhibits the process of inflammation and renal fibrosis in the middle and late stage after AKI,but aggravates AKI in the early stage,indicating that ARG1 expression in macrophages improves renal repair and regeneration in the early stage after IR injury.The treatment of renal disease by targeting ARG1⁺macrophages should be timely intervened according to the disease progression after IR.