The Expression And Significance Of STC1 In Septic Acute Kidney Injury

Objective:Lipopolysaccharide(LPS)-induced Epithelial injury plays a critical role in the pathogenesis of acute kidney injury(AKI).Stanniocalcin-1(STC1),a pleiotropic glycoprotein,has been reported to protect ischemic renal injury by reducing redox oxygen species,modulating inflammatory release,and inhibiting cell apoptosis.However,regulators of STC1 expression as well as its physiologic function in kidneys were unknown.We sought to define the functional role of STC1 expression in LPSinduced kidney injury in vitro and in vivo.Acute Kidney Injury(AKI)is a common clinical syndrome that occurs due to rapid decline in renal function caused by a variety of causes.Among many factors that cause AKI,sepsis is the main cause of AKI,especially in critically ill patients.Sepsis has a high incidence of acute kidney injury and a poor prognosis.It has become a global public health problem.Lipopolysaccharide(LPS)is the main component of the outer membrane of all gram-negative bacteria and the main pathogenic component of gram-negative bacteria.In sepsis,pathogens that invade the human body release a variety of pathogen-associated molecular patterns(Pathogen-associated Molecular Patterns,PAMPs),such as lipopolysaccharide(LPS),etc.,recognized by the Toll Like Receptor(TLR)on the surface of RTECs,activates TGFβ/Smad2/3.TLR are expressed in Renal Tubular Epithelial Cells(Renal Tubular Epithelial Cells).Then,the immune response are further activated and induced the release of cytokines,inflammatory mediators,reactive oxygen free radicals(ROS)and other substances,causing necrosis or apoptosis of tissue cells,and ultimately inevitably leading to LPSinduced acute kidney injury(LPS-AKI).Stanniocalcin-1(Stanniocalcin 1,STC 1)is expressed in kidneys and other organs.85%of the nucleotide sequences in the coding region of human and mouse STC1 genes are identical,and both can encode one A protein monomer composed of 247 amino acid residues,of which 238 residues are identical.Previous studies have shown that STC1,as an extracellular signal protein that functions in the cell,is involved in kidney damage.At present,the research of STC1 in kidney disease mainly focuses on ischemia-reperfusion acute kidney injury(I/R AKI)and contrast-induced acute kidney injury(CI-AKI),anti-glomerular basement membrane nephropathy,there is rare study on the role and specific molecular mechanism of STC1 LPS-AKI.Based on this,we propose the hypothesis that STC1 is involved in LPS-induced acute kidney injury in sepsis(LPS-AKI):when sepsis occurs,pathogens that invade the human body release a variety of PAMP s,such as LPS,which are expressed in TLR4 recognition by renal tubular epithelial cells activates the STC1TGF β/Smad pathway,causing subsequent inflammation,apoptosis,and fibrosis,leading to renal pathological changes.Therefore,in this study,C57BL/6 wild-type mice and STC1 KO(STC1 knockout)mice were used to establish LPSinduced acute kidney injury animal models and primary mouse renal tubular epithelial cell.The role of STC1 in LPS-induced sepsis acute kidney injury(LPS-AKI),and preliminary study of its molecular mechanism can provide new biological markers for the early diagnosis of LPS-AKI,and provide new treatments.Materials and Methods:C57BL/6 mice,STC1-/-(C57BL/6 background)mice were randomly divided into blank control group,experimental control group.A mouse model of AKI was established.Primary mouse renal tubular epithelial cells(mRTECs)isolated from wide type mice and STC 1-/-mice were cultured.We detected changes in serum creatinine(Scr)and blood urea nitrogen(BUN)before and after model establishment,observed and scored renal tubular injury,and measure the expression of signal pathway proteins and downstream inflammatory factors via Real-time quantitative PCR,Flow cytometry(FCM),immunohistochemistry(IHC)and so on.All statistical analysis was performed by using IBM SPSS Statistics 26.0 and Graphpad Prism 7.0.Statistical significance was determined as p<0.05.Results:In vivo,after LPS injection,the levels Scr and BUN in the experimental group were significantly increased,and that of STC 1 KO group were higher in comparison with LPS-AKI group of wide type mice(p<0.05).However,no significant difference was detected between the control groups without LPS on the Scr and BUN levels(p>0.05).Also,LPS caused higher elevation of morphological injury and tubular apoptosis,enhanced TNF a,Icam 1,NLRP3,aSMA,Col 1,Bcl2,Bax and Caspase 3 expression,and increased expression of TGF β,Smad2/3 and STC 1(P<0.05)in STC 1-/-group both via analysis of quantitative PCR Western Blot and IHC.In primary mouse renal tubular epithelial cells,LPS significantly increased expression level of TNF a,Icam 1,NLRP3,aSMA,Col 1,TGF β and STC 1 according to quantitative PCR and Western Blot(P<0.05),and that of primary mouse renal tubular epithelial cells from STC 1-/-mice were much higher(P<0.05).Moreover,whether in WT mRTECs cells or STC 1 KO mRTECs cells,the proportion of apoptosis was increased with the increase of stimulation time(all P<0.05).In addition,the apoptotic ratio in STC1 KO mRTECs cells was significantly higher than that in WT mRTECs cells.at the same time points(2 h,12 h,24 h).Conclusion:Our study reveals a novel TGF β/Smad/STC1 pathway that has homeostatic as well as LPS-induced cytoprotective functions in renal tubular epithelium.STC 1 have protective effects on LPS-induced acute renal tubular injury in mice,possibly by targeting TGF β,regulating the TGF β/Smad,and inhibiting the expression of downstream inflammatory factors.This study reveals that STC1 is involved in the occurrence and progression of LPS-induced septic acute kidney injury(LPS-AKI),and preliminarily clarifies that STC1 may inhibit the inflammation,apoptosis,and fibrosis factors of renal tubular epithelial cells through the TGFβ/Smad2/3 signaling pathway.However,STC1 transgenic mice(STC1 high expression)are still needed to construct an LPS-AKI model to further demonstrate the specific molecular mechanism of the STC1/TGFβ/Smad2/3 pathway in LPS-AKI.At the same time,recombinant human stanniocalcin-1(rhSTC1),an exogenous protein,will be used to intervene in the acute kidney injury(LPS-AKI)model of sepsis to explore the treatment of STC1 in LPSAKI The effect provides theoretical basis and laboratory evidence for STC1 as a potential target for the treatment of LPS-AKI in the future.It has been included in the next experiment plan.At the same time,STC1 is expected to become a diagnostic marker for acute kidney injury,but it still needs clinical verification.