The Effect And Mechanism Of LIGHT-HVEM/LT?R Pathway In The Pathogenesis Of Sepsis Associated Acute Kidney Injury

BackgroundSepsis-associated acute kidney injury(SA-AKI)may be a common clinical disorder characterized by a sudden decline in kidney function and an unexpected decrease in glomerular filtration rate.It constitutes almost 50% of cases diagnosed with acute kidney injury in intensive care units(ICU).Furthermore,SA-AKI increases the risk of chronic kidney disease and is a leading independent cause of high mortality,which is a major drain on public health resources.Therefore,it has been currently a hot and ticky issue to understand the mechanisms underlying the pathogenesis of SA-AKI and look for some innovative strategies.LIGHT(Homologous to Lymphotoxins,exhibits inducible expression and competes with HSV Glycoprotein D for HVEM,a receptor expressed by T lymphocytes),the 14 th member of the TNF superfamily,has received lots of attention as a novel immunomodulatory molecule since it was found 20 years ago.By interacting with Herpesvirus entry mediator(HVEM),LIGHT signal can generate a costimulatory signal for the activation and proliferation of T cells and cause the production of cytokines to promote pro-inflammatory responses.By binding with Lymphotoxin ? receptor(LT?R),LIGHT pathway may also increase the release of chemokines and adhesion molecules to induce the recruitment of immune cells to accelerate the immune response.As a bidirectional immunomodulatory molecule,LIGHT pathway regulates the inflammatory response and plays an important role in the pathophysiology of immune and inflammatory diseases such as rheumatoid arthritis and autoimmune hepatitis,inflammatory bowel disease.SA-AKI is essentially a hyperactive systemic inflammatory response to an infectious insult,and it leads to renal dysfunction.However,the contribution and mechanism by which LIGHT mediates SA-AKI remain elusive.The uncontrolled inflammatory response is the starting point of its waterfall effect.To the end,we speculate that the LIGHT pathway may play an important role in the pathophysiology of SA-AKI by regulating inflammation and act as an innovative intervention target in the treatement of SA-AKI.Toll-like receptors(TLRs)are one of the most important pattern recognition receptors(PRRs)in the natural immune system,which can recognize pathogen-related molecular patterns(PAMPs)or be recognized by danger-related molecular patterns(DAMPs).When TLRs are activated,the transcription factor nuclear factor-kappa B(NF-?B)is released translocate to the nucleus,resulting in the increase of inflammatory mediators such as IL-1?,IL-2,IL-6,IL-12,and TNF-?.In addition,TLRs can promote the activation of the adaptive immune system by antigen-presenting cells(APC),which promotes the differentiation of CD4 helper cells,the activation of B cells,and the production of antibodies.Among them,TLR4,as an important member of the TLRs family,has been confirmed to be involved in the development of ischemia-reperfusion injury,cisplatin-induced nephrotoxicity,and crescentic glomerulonephritis.As one important member of the TLRs,TLR4 has been demonstrated to be involved in the pathogenesis of ischemia-reperfusion injury,cisplatin-induced nephrotoxicity,and crescentic glomerulonephritis.The activation of TLR4-NF-?B pathway is considered to be a key starting point in the cascade amplification of inflammation in LPS-induced SA-AKI.However,the contribution and mechanism by which LIGHT mediates SA-AKI remains elusive.Accordingly,further intensive studies are warranted to elucidate its role and relationship with TLR4-NF-?B pathway.Objectives1.To study the expression of LIGHT and its receptor HVEM/LT?R in the SA-AKI model in vivo and in vitro.2.To investigate the contribution of LIGHT-HVEM/LT?R pathway in the pathogenesis of SA-AKI in vivo and in vitro.3.To explore the mechanism of LIGHT-HVEM/LT?R pathway mediating SA-AKI.Methods1.Expression of LIGHT-HVEM/LT?R pathway in SA-AKI(1)Mice with an intraperitoneal injection of Lipopolysaccharide(LPS)was employed as a model of SA-AKI in vivo.Western Blot(WB),real-time polymerase chain reaction(RT-PCR),and immunohistochemistry(IHC)were employed to detect the expression of LIGHT and the receptors HVEM,and LT?R after establishing the model.In addition,the co-localized expressions of LIGHT-LT?R/HVEM and CK-18(a marker of the renal tubular epithelial cell)were detected by immunofluorescence(IF)and confocal laser scanning microscopy.(2)Human kidney-2(HK-2)cells challenged with LPS were employed as a model of SA-AKI in vitro.WB,RT-PCR,IF were employed to detect the expression of LIGHT and its receptors HVEM and LT?R after LPS treatment.2.The effect of LIGHT-HVEM/LT?R pathway on SA-AKI mice(1)LIGHT KO mice and WT mice were injected with LPS(40 mg/kg,i.p.)or saline to establish a lethal model,and the survival rate was monitored every 6 h for a total of 96 h.(2)The SA-AKI mice were sacrificed at 24 h after LPS intraperitoneal injection(20mg/kg,i.p.).(1)The serum was collected to perform biochemical tests,and then the contributions of LIGHT gene on renal function were detected.(2)Renal tissues were collected to performed paraffin embedding and pathological examinations.HE staining was employed to assess the degree of renal injury and scored.(3)Transmission electron microscopy was employed to observe the ultrastructural damage of renal injury in WT mice and LIGHT KO mice.(4)ELISA was employed to determine the changes of inflammatory mediators in serum.The expression of inflammatory factors(IL-6,TNF-?,IL-1?)and kidney injury biomarkers(KIM-1,NGAL)in kidney tissues was detected by RT-PCR.Immunofluorescence and MPO were employed to detect inflammatory cell infiltration in local kidney tissues.(3)Renal function and pathological damage were determined after the pretreatment with the soluble fusion proteins of the membrane-anchored receptors HVEM-Fc or LT?R-Fc to block LIGHT pathway at 24 h and 2h before LPS injection.3.The mechanism of LIGHT-HVEM/LT?R pathway regulating SA-AKI in vivo and in vitro(1)The expression of TLR4-Myd88-NF-?B in kidney tissues from WT or LIGHT KO mice at 24 h after LPS or 0.9% saline injection was determined using RT-PCR,IHC,WB in vivo.(2)Recombinant human LIGHT(rhLIGHT)and TAK242(a TLR4 inhibitor)were employed to block the effect of LIGHT on SA-AKI in vitro.HK-2 cells were divided into four groups before culturing for another 24 h,as follows :(1)vehicle group,(2)LPS group(50 ?g/m L),(3)LPS(50 ?g/ml)+ rhLIGHT(5 ?g/m L),(4)LPS(50 ?g/m L)+ rhLIGHT(5?g/m L)+ TAK242(0.5 ?M).CCK-8 assay and flow cytometry were employed to determine cell injury,and RT-PCR was utilized to measure inflammatory mediators(IL-6,TNF-?,IL-1?)secreted by HK-2 cells.(3)The expression of the TLR4-Myd88-NF-?B pathway was detected according to the group allocation.In addition,immunofluorescence and confocal laser scanning microscopy were used to detect the nucleation of NF-?B in HK-2 cells in vitro.Results1.Sepsis increased LIGHT-HVEM/LT?R expression in LPS-induced SA-AKI in vivo and vitro(1)In the vivo experiment,a remarkable increase in the protein and m RNA levels of LIGHT,HVEM,and LT?R in the kidney tissues in LPS-injected mice,compared with the saline-injected control mice.Additionally,the immunofluorescence results demonstrated that LIGHT-HVEM/LT?R expression was highly co-localized with the expression of CK-18,a marker of tubular epithelial cells.(2)In the vitro experiment,the expression of LIGHT-HVEM/LT?R in HK-2 cells increased significantly,compared with the vehicle group.2.Role of LIGHT-HVEM/LT?R pathway on SA-AKI mice(1)LIGHT deficiency significantly prolonged the survival of SA-AKI mice.(2)LIGHT deficiency significantly alleviated Bun,SCr and the expression of KIM-1 and NGAL in SA-AKI mice.(3)LIGHT deficiency significantly attenuated pathological damage and activated autophagy in SA-AKI mice.(4)LIGHT deficiency significantly inhibited the up-regulation of inflammatory factors(TNF-6,IL-1?,IL-6)and inflammatory cell infiltration in renal tissues.(5)Pretreatment with HVEM-Fc or LT?R-Fc to block LIGHT-HVEM or LIGHT-LT?R interaction in mice alleviated renal function and pathological damage in LPS-induced SA-AKI.3.The mechanism of LIGHT-HVEM/LT?R pathway regulating SA-AKI in vivo and vitro(1)In the vivo experiment,LIGHT deficiency significantly inhibited LPS-induced upregulation of the TLR4-MyD88-NF-?B pathway.(2)In the vitro experiment,exogenous rhLIGHT protein promoted cell injury in LPS-treated HK-2 cells,decreased cell viability and increased the percentage of apoptosis.(3)In the vitro experiment,exogenous rhLIGHT protein significantly upregulated the expression of the TLR4-MyD88-NF-?B pathway.(4)In the vitro experiment,TAK242 pretreatment inhibited the upregulation and translocation of LPS induced p-NF-?B and partly reversed the increase of the expression of inflammatory mediators.ConclusionsOur study demonstrates that the following conclusions from the in vitro and in vivo results:(1)The expression of LIGHT-HVEM/LT?R pathway is remarkably upregulated in LPS-induced SA-AKI model in vivo and in vitro.(2)LIGHT aggravates kidney injury and pathological damage in SA-AKI mice in vivo.(3)LIGHT increases inflammatory mediator production and inflammatory cell infiltration in SA-AKI in vivo.(4)LIGHT blocking with pretreatment of HVEM-Fc or LT?R-Fc relieves LPS-induced SA-AKI in vivo.(5)LIGHT promotes the expression of the TLR-Myd88-NF-?B pathway to mediate inflammation in vivo and in vitro,which eventually results in SA-AKI.