The Role Of Pyroptosis In Contrast-induced Acute Kidney Injury

Objective Contrast-induced acute kidney injury(CI-AKI)is now the third most common cause of hospital-acquired renal insufficiency,and its pathological features are mainly necrosis of renal tubules.Pyroptosis is a necrotic-type cell death,accompanied by the release of proinflammatory factors,such as IL-1 and IL-18,which depends on the cleavage mediated by inflammatory caspases(caspase-1/5/4/11).The aims of this study are: 1.to evaluate the role of caspase-4/5/11 mediated epithelial pyroptosis in CI-AKI;2.to evaluate the role of caspase-1 mediated epithelial pyroptosis in CI-AKI.Methods 1.The primary mouse renal tubular epithelial cells(TECs)were isolated from wild type(WT)and Casp11-/-,Nlrp3-/-and Casp1-/-mice and cultured under sterile conditions at 37 °C and 5% CO2 in epithelial cell medium(ECM).Primary human renal tubular epithelial cells(TECs)were purchased from Scien Cell and cultured in ECM supplemented with 2% fetal bovine serum(Scien Cell),1% epithelial cell growth supplement(Scien Cell),and 1% penicillin/streptomycin solution(Scien Cell).Adherent human TECs were then treated with iohexol and isosmotic mannitol in separate experiments.Experiments were performed in triplicate.Cells were divided into 12 groups: 1.Control group;2.Iohexol group;3.Mannitol group;4.Iohexol +pan caspase inhibitor(Z VAD FMK)5.Iohexol+Caspase-4 si RNA group;6.Mannitol+Caspase-4 si RNA group;7.Iohexol+Caspase-5 si RNA group;8.Mannitol+Caspase-5 si RNA group;9.Iohexol+Nlrp3 si RNA group;10.Mannitol+Nlrp3 si RNA group;11.Iohexol +Caspase-1 si RNA group;12.Mannitol+Caspase-1 si RNA group.The protein expression of Caspase-4/5/11,NLRP3,Caspase-1,IL-1 and Gsdmd in cells by assessed by Western Blot,respectively,and a special kit(FLICATM Caspase-1 Assay Kit,Immunochemistry Technologies)was used to detect the bioactivity of Caspase-1 in cells.Caspase-4/5,NLRP3,Caspase-1 and IL-1 m RNA expression in cells detected by real-time PCR.ELISA was used to detected the concentration of IL-1 in cell culture supernatants and a Cyto Tox 96 Non-Radioactive Cytotoxicity Assay(Promega)measured cell death according to the manufacturer's instructions.2.The model of CI-AKI mice was established.There were 4 types of mice: wild type,Caspase-11 gene knockout,NLRP3 gene knockout,and Caspase-1 gene knockout.Each mouse was divided into 4 groups,with 6 in each group.Group 1: the normal control group(no treatment in this group);Group 2: model group(3 weeks after right nephrectomy and water deprivation for 1 day,furosemide(10 ?l/g)was injected via tail vein);Group 3: model + iohexol group(3 weeks after right nephrectomy and water deprivation for 1 day,furosemide(10 ?l/g))was injected via tail vein and 20 minutes later,iohexol(10 ?l/g))was injected via tail vein);Group 4: model + normal saline(NS)group(3 weeks after right nephrectomy and water deprivation for 1 day,furosemide(10 ?l/g))was injected via tail vein,furosemide(10 ?l/g))was injected via tail vein and 20 minutes later,NS(10 ?l/g))was injected via tail vein).Mice in all groups were killed 24 hours after iohexol or other drugs injection,and blood and kidney tissue specimens of mice were collected.Serum creatinine(SCr)and blood urea nitrogen(BUN)was assessed by an automatic biochemical analyzer;the protein expression of Caspase-11,NLRP3,Caspase-1,IL-1 and Gsdmd in mouse kidney was assessed by Western Blot;Acute kidney injury biomarkers(KIM-1,IL-18)and inflammatory cytokines IL-6 m RNA expression was detected by real-time PCR.H&E staining and immunolabelling were used to evaluate degree of renal tubular injury(tubules dilation and necrosis,tubular epithelial cell shedding and vacuolization,tubular brush border injury)and expression of caspase-11,NLRP3,caspase-1and IL-1 in kidneys,respectively.Results 1.Contrast medium induces renal tubular epithelial cells pyroptosis via activation of inflammatory caspases.We observed that 72-hour iohexol exposure activated TECs to release IL-1? in a dose-dependent manner as low as 10 mg/ml(p<0.0001).Caspase-11,NLRP3 and caspase-1 in mouse TECs and caspase-4/5,NLRP3 and Caspase-1 in human TECs were markedly upregulated by iohexol incubation for 72 hours(p<0.0001);Accordingly,significant gene upregulation was also observed in human TECs by iohexol(p<0.0001).Pyroptosis,also known as a regulated cell death,is characterized by cell lysis.Human TECs showed significant cell lysis after iohexol treatment,whereas the effect was blocked by the pan-caspase inhibitor Z-VAD-FMK(p<0.0001).Consistent with inhibition of human TECs lysis due to iohexol-induced upregulation of inflammatory caspases,we also observed that the maturation and secretion of IL-1? in human TECs by iohexol was obviously reduced.2.Caspase-4/5/11 and NLRP3,Caspase-1 were required for contrast-induced IL-1 cleavage and pyroptosis in mouse and human TECs.TEC-specific deletion of caspase-11 prevented IL-1? cleavage following iohexol treatment for 72 hours(p<0.0001).Further,we used caspase-4 and-5,Nlrp3,caspase-1 si RNAs and observed that downregulation of caspase-4 or-5,or Nlrp3,caspase-1 by si RNAs prevented iohexol-induced cell lysis,maturation and secretion of IL-1 in human TECs(p<0.0001).3.Contrast-induced acute kidney injury involves tubule damage,renal inflammation,and failure.Iohexol injection by tail vein could result in renal tubular epithelial cells injury with swelling and vacuolization,and interstitial inflammation within the outer medulla as well as within the cortex,which were absent in the control,model mice and mice injected with normal saline(NS)(p<0.0001).The structural alterations of iohexol-induced acute kidney injury were associated with acute renal failure,as shown by a transient increase in SCr and BUN levels,compared with those in control,model mice and mice injected with normal saline(NS)(p<0.0001).Intrarenal m RNA expression of the acute kidney injury biomarkers KIM-1 and IL-18,as well as that of the proinflammatory cytokine IL-6,was increased after iohexol injection(p<0.0001).4.Contrast-induced acute kidney injury is associated with caspase-11 mediated,and NLRP3/Caspase-1 mediated pyroptosis.Immunological staining showed mice injected with iohexol showed a marked increase in caspase-11 and NLRP3,caspase-1 expression,which was absent in control,model mice and mice injected with NS(p<0.0001).Immunoblot analyses of caspase-11,NLRP3 and caspase-1 in the iohexol group also showed significant increases in the protein levels,respectively,compared with that in control,model and mice injected with NS groups(p<0.0001).In addition,immunological staining and Immunoblot analyses all showed that iohexol could significant increase the mature IL-1 expression in mice injected with iohexol,compared with that in control,model mice and mice injected with NS(p<0.0001).5.Caspase-11 and NLPR3/Caspase-1 were required for contrast-induced acute kidney injury.Here,we used Casp11–/–,NLRP3–/–,Casp1–/–mice to evaluate the tubular injury,intrarenal inflammation,and kidney function in contrast-induced acute kidney injury.Histologic features determined by H&E in Casp11–/–,NLRP3–/–,Casp1–/– mice following exposure to iohexol(10 ?l/g)demonstrated that deletion of caspase-11,NLRP3,caspase1 could fully prevent iohexol-induced acute kidney injury in contrast with WT mice(p<0.0001).Caspase-11,NLRP3,caspase-1 deletion also preserved the kidney function by significant decrease in SCr and BUN levels,compared with those in WT mice.Intrarenal m RNA expression of the acute kidney injury biomarkers KIM-1 and IL-18,as well as that of the proinflammatory cytokine IL-6,showed marked reduction following caspase-11,NLRP3,caspase-1 deletion,as compared with that in WT mice(p<0.0001).Further,analysis for levels of mature IL-1? in the kidney,released during pyroptosis,also showed marked reductions following caspase-11,NLRP3,caspase-1 deletion(p<0.0001).6.Renal tubular epithelial cell caspase-11 and NLRP3/Caspase-1 activation is required for Gsdmd cleavage.Immunofluorescence staining demonstrated specifically Gsdmd+ tubular epithelial cells were significantly increased by iohexol treatment,but the response was abrogated in Casp11–/–,NLRP3–/–,Casp1–/– mice(p<0.0001).Iohexol markedly increased formation of the active,cleaved Gsdmd p30 protein in TECs from WT mice(p<0.0001).Moreover,Gsdmd cleavage was inhibited in mouse TECs of Casp11–/–,NLRP3–/–,Casp1–/– mice following iohexol challenge(p<0.0001).Iohexol induced IL-1? secretion in renal TECs,whereas both ECs and MCs didn't respond to the stimulation(p<0.0001).Western blotting confirmed this finding and also showed that iohexol stimulation only induced pro–IL-1? and mature IL-1 expression in renal TECs(p<0.0001).Conclusions 1.Contrast media cause renal tubular epithelial pyroptosis;2.Renal tubular epithelial pyroptosis underlies CI-AKI;3.Renal tubular epithelial pyroptosis in CI-AKI is mediated by Caspase-4/5/11 and NLRP3/Caspase-1inflammasome pathways;4.Gsdmd activation is an important molecular mechanism of Caspase-4/5/11 mediated and NLRP3/Caspase-1 mediated renal tubular epithelial pyroptosis.