Investigation Of The Attenuating Effects And Mechanisms Of DsbA-L Deficiency In Sepsis-associated Acute Kidney Injury

Objective Sepsis is a life-threatening syndrome characterized by a dysregulated host response to infection,resulting in organ dysfunction.Among the organs affected,the kidneys are commonly involved,leading to sepsis-associated acute kidney injury（SA-AKI）.The driving factors of SA-AKI encompass three main aspects:inflammatory response,microcirculatory dysfunction,and metabolic reprogramming.However,the specific impact of DsbA-L on immune cells and its influence on renal metabolic function in SA-AKI remain undisclosed.Consequently,our study focuses on three key areas.Firstly,we aim to establish an SA-AKI model using DsbA-L global knockout(DsbA-L^-/-)mice to clarify the effects of DsbA-L on immune cell phenotype and function in SA-AKI.Secondly,we seek to unravel the underlying mechanisms by which DsbA-L regulates immune cell alterations.Lastly,we aim to elucidate the consequences of DsbA-L deficiency on renal metabolic function in the context of SA-AKI.Methods1.A sepsis-associated acute kidney injury（SA-AKI）model will be established in 6-8-week-old DsbA-L^-/-mice and WT mice by intraperitoneal injection of lipopolysaccharide（LPS）at a dose of 10 mg/kg.The mice will be divided into four groups:na（?）ve-WT,na（?）ve-DsbA-L^-/-,LPS-WT,and LPS-DsbA-L^-/-,with six mice in each group.After 24 hours,serum and kidney samples will be collected from each group.The levels of creatinine and blood urea nitrogen will be measured,along with the assessment of immune cell proportions.Excess fat and connective tissue in the kidneys will be removed,and the remaining tissues will be preserved in liquid nitrogen for transcriptomic and metabolomic analysis.2.Bone marrow cells from DsbA-L^-/-mice and WT mice will be extracted and cultured in vitro.These cells will be differentiated into immature dendritic cells（DCs）and monocytes/macrophages.After seven days,DCs will be stimulated with or without LPS for 24 hours,divided into PBS-WT,PBS-DsbA-L^-/-,LPS-WT,and LPS-DsbA-L^-/-groups,with PBS as the negative control.Macrophages will be induced to differentiate into M1 phenotype using LPS stimulation and into M2 phenotype using IL-4 stimulation.The groups will include PBS-WT,PBS-DsbA-L^-/-,LPS-WT,LPS-DsbA-L^-/-,IL4-WT,and IL4-DsbA-L^-/-,and the phenotypic differences of macrophages and DCs will be assessed using flow cytometry.The functionality of DCs will be evaluated through mixed lymphocyte reaction.Results1.Following 24 hours of LPS injection,the DsbA-L^-/-group exhibited significantly lower levels of creatinine compared to the WT group（18.40±3.77 vs.40.75±2.56,P<0.001）.Flow cytometry analysis of macrophages and dendritic cells revealed a decrease in the proportion of DCs（MHC-II⁺CD11c⁺）in the LPS-DsbA-L^-/-group compared to the LPS-WT group（12.56±1.24 vs.18.08±0.71,P<0.001）.Additionally,the LPS-DsbA-L^-/-group displayed higher proportions and mean fluorescence intensity（MFI）of CD206⁺macrophages,while the proportion and MFI of MHC-II⁺macrophages were lower in the LPS-DsbA-L^-/-group compared to the LPS-WT group.2.WT and DsbA-L^-/-mice-derived bone marrow dendritic cells and macrophages were cultured in vitro.Flow cytometry analysis revealed no significant differences in DC phenotype.Co-culture experiments with T cells demonstrated that DsbA-L deficiency did not impair the antigen presentation capacity of DCs.However,in the LPS-stimulated DsbA-L^-/-group,the mean fluorescence intensity of CD14 was significantly lower compared to the LPS-WT group（276.0±4.04 vs.427.0±25.06,P<0.001）.Immunoblotting and RT-q PCR analysis unveiled reduced expression levels of c-FOS and NF-κB in the kidneys of the LPS-DsbA-L^-/-group,accompanied by decreased transcriptional levels of IFN-βand TNF-α.3.Transcriptomic and metabolomic sequencing of the kidneys from mice after LPS injection revealed a significant difference between the LPS-WT and LPS-DsbA-L^-/-groups,primarily in arginine metabolism.Specifically,the LPS-DsbA-L^-/-group exhibited decreased expression of NOS2 and a subsequent reduction in NO levels.Conclusion In this study,we made several notable findings regarding LPS-induced SA-AKI.Firstly,DsbA-L^-/-mice showed a significant decrease in creatinine levels compared to the WT group.Additionally,macrophages in the LPS-DsbA-L^-/-group exhibited elevated CD206expression and decreased MHC-II expression.Moreover,there was a notable reduction in the proportion of infiltrating dendritic cells.Mechanistically,the absence of DsbA-L led to downregulated CD14expression on macrophages,resulting in suppressed activation of c-FOS,NF-κB,and NOS2.This altered macrophage polarization and subsequently led to decreased secretion of inflammatory factors.