Study On The Role And Mechanism Of CD38 In LPS-Induced Renal Injury In Septicemia Mice

Objective:The acute kidney injury（AKI）that induced by sepsis,often leads to death due to multiple organ dysfunction syndrome（MODS）.LPS,the main ingredient of the cell walls of gram-negative bacteria,which is the incentive of sepsis.LPS can stimulate inflammatory cells to produce large number of pro-inflammatory factors,such as TNF-α,IL-1β,by binding to Toll-like Receptor 4（TLR4）specifically,a transmembrane glycoprotein in cells,that can induce sepsis and manifests as systemic inflammatory response.Acute kidney injury（AKI）,is one of the most common complication of sepsis,which is often aggravated by lack of effective treatment,leading to renal failure and increasing the mortality of sepsis.Therefore,it is urgent to find new targets that can effectively inhibit AKI to improve the prognosis of sepsis.Previous studies in our laboratory have reported that the expression of TLR4 in DCs of mice lacking Cluster of Differentiation（CD38）was significantly increased,while it inhibited collagen-induced arthritis.The expression of TLR4 in spleen B cells of CD38^-/-mice was also significantly increased,accompanied by B cell development arrest,and the development disorder of B cells was closely related to the decline of anti-infection ability.Therefore,we intend to explore whether the elevation of TLR4 induced by CD38 deletion exacerbate LPS-induced septicemic renal injury.The purpose of this study is to explore that:1.The effects of CD38 deficiency in LPS induced septicemia in mice and the role of TLR4 in it;2.To illuminate the role and key point of CD38 deficiency in aggravated LPS induced AKI of septicemia mice;3.To explore the mechanism of TLR4/NF-κB pathway in aggravated LPS induced AKI of septicemia mice caused by CD38deficiency.This study will broaden our understanding on the role of CD38 in LPS-induced AKI of septicemia mice,it will provide a new theoretical basis for the influence factors in LPS-induced AKI of septicemia mice,and will provide a new target for curing LPS-induced AKI of sepsis in clinic.Methods:1.20 male WT,CD38^-/-and CD38^-/-TLR4^mut mice in 8 weeks were selected respectively,and each genotype mice were divided into normal control group and LPS group equally,observe and record the changing of weight,coat color,mental state and survival rate.2.The kidneys of WT,CD38^-/-and CD38^-/-TLR4^mutmice before and after LPS stimulation were harvested,then sliced and stained with hematoxylin-eosin（HE）to compare their pathological changes.3.The serum of WT,CD38^-/-and CD38^-/-TLR4^mut mice stimulated by LPS for 2hours was analyzed by multi-factor analysis to compare the expression of related pro-inflammatory factors（IFN-γ、TNF-α、IL-1β、IL-6 et,al）.4.The kidney in mice were acquired to extract RNA and reversed transcription for c DNA,and then to detect the expression of mRNA level（including:CD38,TLR4,IL-1β,TNF-α,IL-6,IFN-γ,iNOS）in LPS induced WT,CD38^-/-and CD38^-/-TLR4^mut mice through real-time qPCR technology.5.The serum of WT,CD38^-/-and CD38^-/-TLR4^mutmice stimulated by LPS for 2hours collected and the related indexes of renal function,such as serum creatinine（SCr）,urea nitrogen（BUN）and uric acid（UA）,were detected by automatic biochemical analyzer according to standardized operating procedures（SOP）.6.The kidneys of WT,CD38^-/-and CD38^-/-TLR4^mut mice before and after LPS stimulation were taken and stained by immunohistochemistry（IHC）.The expression levels of TLR4,NF-κB p65 and NF-κB p105 in the kidneys were observed.7.The protein expression level of CD38,TLR4,NF-κB p65,NF-κB p105,p-NF-κB p65 and p-NF-κB p105 of kidney in LPS induced WT,CD38^-/-and CD38^-/-TLR4^mut mice were detected by Western blotting.Results:1.After stimulation with LPS,the weight of mice decreased significantly from 0to 72 hours,combined with tremble,chilly,dispirited and increased of white secretion in the canthus;And the survival of LPS-induced mice decreased compared with normal WT mice,and the mortality of CD38^-/-mice is higher than LPS-induced WT and CD38^-/-TLR4^mutmice.2.Compared with normal WT mice,the percentage of weight loss of mice was the most obvious after LPS stimulation for 2 hours.3.Compared with normal WT mice,the expression level of pro-inflammatory cytokines IFN-γ（p<0.01）、TNF-α（p<0.01）、IL-1β（p<0.01）and IL-6（p<0.01）were increased obviously in 2h after LPS induced mice.4.After 2h induced mice with LPS,the expression of pro-inflammatory cytokines IFN-γ（p<0.01）、TNF-α（p<0.05）and IL-1β（p<0.01）increased obviously compared with WT mice,while IL-6 has no obvious change;When compared with CD38^-/-mice,only the expression level of IFN-γ（p<0.05）decreased obviously in CD38^-/-TLR4^mut mice.5.After 2h induced with LPS,the mRNA expression of TLR4（p<0.05）and pro-inflammatory cytokines IFN-γ（p<0.05）、TNF-α（p<0.01）、IL-1β（p<0.01）、IL-6（p<0.01）and iNOS（p<0.01）were increased obviously;And when compared with CD38^-/-mice,the mRNA expression of TLR4（p<0.01）and all the pro-inflammatory cytokines above decreased obviously,the expression level of pro-inflammation cytokines almost uniformly with WT mice except for TLR4.6.Before LPS induced,the pathological has no change during WT、CD38^-/-and CD38^-/-TLR4^mut mice;The degree of kidney injury in mice was aggravated 2h after LPS induced mice,and showed narrowing of the glomerular capillary lumen、smaller of tubule lumen and the inflammatory cell infiltration increased;After 2h induced mice with LPS,when compared with WT mice,the above pathological changes even worse,while there has no obvious pathological changes in CD38^-/-TLR4^mut mice.7.Compared with normal WT mice,the level of SCr（p<0.05）and BUN（p<0.01）increased significantly after LPS stimulation for 2 hours,while the level of UA has no significant difference.8.After 2h induced mice with LPS,the whole levels of SCr（p<0.01）and BUN（p<0.01）and UA（p<0.01）increased obviously,and the expression in CD38^-/-mice is higher than WT and CD38^-/-TLR4^mut mice.9.Before LPS stimulation,the expression of TLR4 and NF-κB（p65 and p105）has no obvious difference,the expression of TLR4 decreased,while nuclear import of NF-κB（p65 and p105）has no obvious difference;the expression of TLR4 increased,accompany with nuclear import of NF-κB（p65 and p105）increased obviously after 2h induced with LPS,among which the expression of TLR4 and nuclear import of NF-κB（p65 and p105）is higher than kidney in WT and CD38^-/-TLR4^mut mice.10.After 2h induced mice with LPS,when compared with WT mice,the expression of TLR4（p<0.01）and phosphorylation of NF-κB p65（p<0.01）and NF-κB p105（p<0.05）increased obviously in CD38^-/-mice,while the expression of TLR4（p<0.01）and phosphorylation of NF-κB p65（p<0.01）and NF-κB p105（p<0.01）decreased obviously in CD38^-/-TLR4^mut mice,Conclusions:The deletion of CD38 can promote TLR4 expression,activate NF-κB signaling pathway and mediates the production of pro-inflammatory factors in renal tissue,thus aggravating the acute kidney injury in LPS-induced sepsis,and this study provides a new theoretical basis for CD38 as a potential therapeutic target for septic renal injury.