Mechanism Of Pyroptosis On Renal Injury In Brain Death Rat

Part 1Effect of Caspase-1 as Key Protein of Pyroptosis on Renal Injury in Brain Death RatObjectiveThis study aimed to investigate the role of caspase-1-mediated pyroptosis in renal injury induced by brain death.MethodsSprague-Dawley(SD)rats(male,weight 250-300 g)were randomly divided into four experimental groups as follows:Brain death group(BD group,n=30),Sham operation group(Sham group,n=6),Caspase-1-specific inhibitor pretreatment brain death group(BD+Z-YVAD-FMK group,n=30),and caspasel-specific inhibitor Z-YVAD-FMK pretreatment and sham operation group(Sham+Z-YVAD-FMK group,n=6).Brain death was induced in male Sprague-Dawley rats by inflating a subdurally placed balloon catheter.The rats were intravenously injected with a caspase-1 inhibitor(Z-YVAD-FMK)1 h before BD,and sham-operated rats served as controls.The blood samples and kidney tissue were collected at 0 h,1 h,2 h,4 h,and 6 h after BD.Blood urea nitrogen and serum creatinine levels were measured.Renal injury were assessed using immunohistochemistry and transmission electron microscopy(TEM).Renal expression of NLRP3,caspase-1,caspase-11，gasdermin D(GSDMD),IL-1β,and IL-18 were assessed using quantitative reverse transcriptase-polymerase chain reaction and western blotting.ResultsThe expression of NLRP3,caspase-1,GSDMD,IL-1β,and IL-18 in the BD group was upregulated,as indicated by Western Blot,IHC and real-time PCR,and there was a significant difference between the BD group and the sham operation group.Transmission electron microscopy(TEM)showed that the renal tubular epithelial cells were swollen and round,the cell membrane ruptured,the brush border of the renal tubules fell off,the number of mitochondria in the cells increased and some mitochondria were swollen,the structure of the mitochondria membrane disappeared,the nuclear was not uniform,and the nucleus was concentrated in the BD group.Treatment with Z-YVAD-FMK reduced the mRNA levels of caspase-1,GSDMD,NLRP3,IL-1β and IL-18 compared with those of the BD group,while the expression levels of caspase-11 was not changed After inhibiting the caspase-1,the protein expression of caspase-1,GSDMD,IL-1β and IL-18 decreased in the Group Z-YVAD-FMK.Furthermore,Z-YVAD-FMK effectively reduced pyroptosis in the brain-dead rats and significantly alleviated kidney injury.However,Z-YVAD-FMK did not affect caspase-11 expression.ConclusionThese results suggested that brain death induced caspase-1-dependent pyroptosis in renal tubular epithelial cells.Z-YVAD-FMK effectively reduced pyroptosis in the kidney in brain-dead rats.Part 2Regulation of Pyroptosis in Renal Tubular Epithelial Cells in Hypoxia/Reoxygenation by Caspase-1 InhibitorObjectiveThis study was aimed to investigate the mechanism of hypoxia/reoxygenation-induced pyroptosis in renal tubular epithelial cells.MethodsRenal tubular epithelial cells(NRK-52E)were subjected to 3 hours of hypoxia followed by 6 h of reoxygenation(H/R).Caspase-1 inhibitor(Z-YVAD-FMK)with different concentrations was incubated in NRK-52E cells before hypoxia and reoxygenation.Caspase-11 was knocked down by siRNA with nontarget siRNA serving as negative controls.CCK-8 was used to detect the cell viability of NRK-52E cells.The mRNA expression levels of NLRP3,caspase-1,caspase-11,GSDMD,IL-1 β and IL-18 were detected by Real-time PCR and the protein expression levels were detected by Western Blot.ResultsCCK-8 results revealed that NRK-52E cell viability was significantly decreased in the H/R environment and significantly increased upon Z-YVAD-FMK treatment.However,caspase-11 knockdown with siRNA did not exhibit a protective effect on cell viability after H/R by inhibiting the non-classical pathway of pyroptosis.Real-time qPCR and western blotting results showed that the expression of NLRP3,caspase-1,caspase-11,IL-1β,IL-18,and GSDMD in NRK-52E cells was upregulated under H/R conditions.Expression of IL-1β,IL-18,caspase-1,and GSDMD was lower in the Z-YVAD-FMK group than in the H/R group(P<0.01);however,there was no significant change in NLRP3 or caspase-11 expression.Further,mRNA and protein expression of GSDMD and caspase-11 were significantly lower in the siRNA group than in the H/R group(P<0.05);however,there was no significant change in NLRP3,caspase-1,IL-1β,and IL-18 in the siRNA group.ConclusionHypoxia/Reoxygenation induced caspase-1-dependent pyroptosis in NRK-52E cells.Pyroptosis participate in the regulation of brain death-related renal injury.Conclusion of articleBoth in vitro and in vivo results indicated that BD induced caspase-1-dependent pyroptosis,which was effectively reduced by Z-YVAD-FMK.Pyroptosis may occur in BD donors,promote inflammation,and induce kidney injury.Thus,it could be considered as a therapeutic target for BD-induced kidney injury.