Molecular Mechanism Of Pyroptosis-Mediated Septic-Acute Kidney Injury In Zebrafish

Sepsis is a kind of clinical disease with high morbidity and mortality.The pathogenesis of sepsis is host immune system dysregulation,which provokes overwhelming inflammation to induce organ dysfunction,even organism death.Septic-acute kidney injury(S-AKI)is a common complication of sepsis,which is considered as faster progression and more severe damage on kidney compared with non-septic AKI.Currently,there are no effective therapies for S-AKI because the mechanisms of sepsis remain to be elucidated.Thus,establishing an ideal in vivo infection model to illuminate the molecular mechanisms of S-AKI will contribute to the clinical treatment of severe disease.Bacterial lipopolysaccharide(LPS),an important component of endotoxin,is the main cause of sepsis.Recent studies have revealed that inflammatory caspase in host cells could recognize the LPS and activate downstream signaling cascades to induce pyroptosis,mediating the invading pathogens clearance.However,excessive pyroptosis would cause auto-immune diseases and even multi-organ failure.Thus,whether the progress of pyroptosis plays a role in mediating S-AKI remains largely unknown.Herein,we utilized a lethal-dose LPS immersion infection model in zebrafish larvae and determined the pericardial edema and tail fin fester as the pathological phenotypes of sepsis.Through a CRISPR/Cas9-based rapid knockout method(Crispants),we knocked-out the inflammasome related genes in GO zebrafish larvae and screened the key signal molecules related to the process of sepsis.We found that the pathological phenotypes of sepsis were relieved in caspy2-/-and gsdmeb-/-larvae,compared to that in wild type,caspy-/-pycard-/-,casp3a-/-and gsdmea-/-larvae,and the survival rate was also rescued in corresponding zebrafish larvae.Consistent with our previous studies in understanding the caspy2/GSDMEb axis-mediated pyroptosis,our results suggest that pyroptosis signaling might play important roles in the pathogenesis of sepsis in zebrafish.To further identify the molecular mechanisms of pyroptosis-mediated sepsis in zebrafish,we developed an in vivo PI staining model in zebrafish larvae,and found that the area of kidney proximal tubule in wild type larvae showed obvious positive fluorescence signal,whereas no such phenomenon was observed in caspy2-/-and gsdmeb-/-larvae.Meanwhile,through the proximal tubular cells-labeled transgenic zebrafish infection model,we found that the fluorescence of proximal renal tubule was weak expressed and dispersed,consistent with the disappeared structure of proximal renal tubule in wild type larvae,but not in caspy2-/-or gsdmeb-/-larvae.These results suggest that caspy2/GSDMEb axis plays critical role in mediating the pyroptosis in proximal renal tubule cells.Besides,we also generated the Crispants larvae in neutrophils-labeled transgenic zebrafish larvae,and found that neutrophils were significantly recruited to the proximal renal tubule in wild type zebrafish larvae,but not in caspy2-/-or gsdmeb-/-larvae during LPS infection.The result preliminary suggests lethal LPS infection can result into tissue injury in proximal renal tubule during sepsis.To better understand the role of pyroptosis in mediating S-AKI in zebrafish larvae,we stepped forward to perform the histopathological analysis in LPS infected zebrafish larvae.We found that the proximal renal tubule showed an obvious cavitation with inflammatory infiltration and a significant loss of brush border epithelium in wild type larvae.Besides,the expressions of AKI marker genes(e.g.kim-1,timp2,and igfbp7)were significantly increased.However,these S-AKI phenotypes were alleviated in caspy2-/-and gsdmeb-/-larvae.These results suggest that S-AKI is triggered by caspy2/GSDMEb mediated pyroptosis during LPS-induced sepsis.Accordingly,we further designed a specific GSDMEb competitive peptide inhibitor to pretreat the larvae,and found that the zebrafish larvae showed significantly resistance to lethal LPS-induced sepsis,and the symptoms of S-AKI were alleviated as well.These results suggest that the pyroptosis activation plays crucial role in mediating S-AKI in zebrafish,and also provide a theoretical basis for developing clinical therapeutic targets for S-AKI.Based on the zebrafish sepsis model above,we further confirmed the concurrent pathways by utilizing lethal LPS-infection in mouse model.We found that both Casp11-/-and GSDMD-/-mice could resist to lethal LPS-induced sepsis,and the production of cytokines and alarmins(including IL-1?,IL-18,TNF-?,HMGB1 and LDH)in serum were lower than that in wild type mice,as well as the production of AKI indicator BUN,CK and NGAL.Besides,through histopathological analysis,we found an enlargement of proximal renal tubules in wild type,but not Casp11-/-or GSDMD-/-mice.These findings reveal a central role of non-canonical inflammasome signaling-gated pyroptosis in mediating S-AKI in mice.Meanwhile,this also proves that utilizing zebrafish as an ideal complementary model is reasonable,which will contribute to better understand the process of sepsis in vivo.Finally,to explore the sepsis and organ damage caused by bacterial infection,this work ultilized a gram-negative bacteria E.piscicida which could induce hemorrhagic septicemia in fish.We established an immersion infection model in zebrafish larvae and found that sepsis could not be induced under this model.However,neutrophils were obviously recruited to the bacterial colonization area.Besides,the number of neutrophils were unexpected decreased at late stage of infection.Moreover,more motality and bacterial burden were found in neutrophil depletion zebrafish larvae.These results suggest that the important roles of zebrafish neutrophils in anti-bacterial infection,and lay the foundation for further understanding the molecular mechanism of bacterial infection induced host sepsis and organ damage.In conclusion,this work established a lethal LPS infection-induced sepsis model in zebrafish larvae,and revealed that pyroptosis of renal tubular cells is essential for S-AKI process.This work developed an ideal model for in vivo sepsis analysis,providing a theoretical basis for anti-sepsis drug screening and small molecule drug target design in clinical treatment.