Effect And Mechanism Of NLRP3 Inflammasome Activation In Acute And Chronic Kidney Disease

AIM: Inflammation is common feature in both acute and chronic kidney disease,while sustained inflammation contributes to the progression of tubulointerstitial fibrosis.NLRP3 is an important member of the NLR family,which is constituted of a leucine-rich repeat region,a conserved nucleotide-binding oligomerization domain and an N-terminal effector domain.Previous studies showed that NLRP3 could be activated by danger-associated molecular patterns(DAMPs)and pathogen-associated molecular patterns(PAMPs),such as mitochondrial dysfunction and extracellular ATP.NLRP3 inflammasome activation will activate effector protease caspase-1 to promote the maturation and secretion of IL-1? and IL-18.The aims of our study were to explore the effect and mechanism of the NLRP3 inflammasome in acute and chronic kidney injury.METHOD:1.To generate the renal ischemia/reperfusion mice model,the right renal pedicle of WT and NLRP3 knock out mice were clamped for 25 min or sham operated under anesthesia.At the end of 3 days,mice were sacrificed.Blood and urine were collected for renal function analysis.Kidney tissues were harvested for pathologic and immunohistochemistry staining.NLRP3 inflammasome activation and downstream cytokines expression were analyzed by Realtime PCR and Western Blot.In vitro,HK-2 cells were stimulated with ischemia/reperfusion injury for different time durations.Cell lysates were collected for Real-time PCR and Western Blot analysis.The expression and location of NLRP3,Mito Tracker and Mito SOX were detected by confocal microscopy.The silencing of NLRP3 and TXNIP,or Mito TEMPO were also used to explore the mechanism of NLRP3 inflammasome activation.2.To generate the Ang ? infused mice model,WT,AT1 R KO and NLRP3 KO mice underwent left nephrectomy and then recovered for 7 days.The mice were continuously infused with Ang ? or saline using osmotic mini-pumps.At the end of 28 days,mice were sacrificed.Blood and urine were collected for renal function analysis.Kidney tissues were harvested for pathologic and immunohistochemistry staining.NLRP3 inflammasome activation and downstream cytokines expression were analyzed by Real-time PCR and Western Blot.In vitro,HK-2 cells were stimulated with different doses of Ang ? over a range of time periods.Cell lysates were collected for Real-time PCR and Western Blot analysis.The expression and location of NLRP3,Mito Tracker and Mito SOX were detected by confocal microscopy.The silencing of AT1 R,AT2R and NLRP3,or Mito TEMPO were also used to explore the mechanism of NLRP3 inflammasome activation.3.Generate Ang ? infused model with Lysm cre TNFf/f or CD4 cre TNFf/f mice,and harvest kidney tissue for Real-time PCR analysis.RESULTS:1.It was found that NLRP3 inflammasome activation was induced by I/R injury,peaking at day 3 after reperfusion.Consistent with this observation,NLRP3 deletion significantly attenuated I/R-induced kidney damage and expression for markers of inflammasome activation.Then,we observed mitochondrial dysfunction,characterized by ultrastructural changes and cytochrome C(Cyt c)redistribution.Mitochondria-targeted antioxidant Mito TEMPO prevented m ROS overproduction and the decline in mitochondrial membrane potential(MMP)in vitro.Mito TEMPO treatment also inhibited NLRP3 inflammasome activation and co-localization of NLRP3 and TXNIP after simulated ischemia/reperfusion(SI/R)injury.Finally,we transfected HK-2 cells with TXNIP si RNA to explore the role of TXNIP in m ROS-induced NLRP3 inflammasome activation.We found that TXNIP si RNA significantly inhibited NLRP3 inflammasome activation.2.In vitro,Ang ? triggered NLRP3 inflammasome activation in a dose-and time-dependent manner,and this effect is mediated by AT1 receptor rather than AT2 receptor.Mito TEMPO,a mitochondrial targeted antioxidant,attenuated Ang ? induced mitochondrial reactive oxygen species(m ROS)production and NLRP3 inflammation activation.Following chronic Ang ? infusion for 28 days,we observed remarkable tubular epithelial cells(TECs)injury,mitochondrial damage,and albuminuria in WT mice.However,these abnormalities were significantly attenuated in AT1 receptor KO mice.Then,we examined the role of mitochondria in Ang ?-infused mice with or without mito TEMPO treatment.As expected,Ang ?-induced mitochondrial dysfunction and NLRP3 inflammasome activation were markedly inhibited by mito TEMPO.Notably,NLRP3 deletion significantly protected TECs from Ang ?-triggered mitochondrial dysfunction and NLRP3 inflammasome activation.3.Macrophage specific TNF deletion did not affect Ang ?-induced NLRP3 inflammasome activatin.T cells specific TNF deletion did not affect Ang ?indcued NLRP3 inflammasome activation.CONCLUSION: NLRP3 inflammasome plays an important role in acute and chronic kidney injury.Damaged mitochondira in tubular epithelial cells are major sourc of ROS contributing to NLRP3 inflammasome activation.There findings identify potential targets for clinic treatment of acute and chronic kidney injury.