The Role Of NLRP3 Inflammasome In Acute Kidney Injury To Chronic Kidney Disease Transition

AIMS Acute kidney injury(AKI)is a severe disease with high incidence,and increased the risk of chronic kidney disease(CKD),resulting in an increased societal burden.It has been a focus of recent studies to slow the AKI to CKD transition.NLRP3(nucleotidebinding oligomerization domain [NOD]-like receptor protein 3)inflammasome,a critical component of innate immunity,plays an important role in both AKI and CKD.The role of NLRP3 inflammasome in AKI to CKD transition remains to be explored.The present study aims to investigate,1.the involvement of NLRP3 inflammasome in AKI to CKD transition;2.the regulation of mitochondrial damage and mitophagy by NLRP3 inflammasome in AKI to CKD transition.METHODS A mouse model of cisplatin-induced acute kidney disease(CP-AKD)was induced by a series of three low-dose cisplatin injections to C57BL/6J mice.Wild-type(WT)and NLRP3 knockout mice were used.Each genotype was divided into two groups as follows.Group1,the control group.The mice in this group recieved repeated injections of phosphate buffer saline(PBS),at a dosage of 7.5mg/kg/injection,on day 0,day 7,and day 21,for a total of 3 times.Group 2,the CP-AKD group.The mice in this group received repeated injections of cisplatin,at a dosage of 7.5mg/kg/injection,on day 0,day 7,and day 21,for a total of 3 times.To analyze the effects of MCC950,wild-type mice were given daily intraperitoneal injections for 7 days,starting from day 21,after the last dose of cisplatin injection,and sacrificed on day 28.To analyze NLRP3 inflammasome activation,fibrotic markers,and histological damage throughout the time course,a portion of mice were sacrificed on day 7,day 14,and day 28,that is,7 days after each injection of cisplatin.Otherwise,mice were sacrificed on day 28.Serum and kidney tissues were collected immediately after the mice were sacrificed.Renal function was analyzed by serum creatinine and blood urea nitrogen.Renal histologic injury was assessed by staining with hematoxylin and eosin,periodic acid-Schiff,Masson’s trichrome and Sirius red.The expressions of NLRP3 inflammasome-related proteins and mitophagy related proteins were assessed by western blotting.The m RNA levels of NLRP3,ASC(apoptosis-associated speck-like protein containing a caspase recruitment domain),caspase-1,and IL-1β were measured by quantitative PCR.The expression and localization of TGFβ1,Collagen III,vimentin and FSP-1(fibroblast-specific protein 1)were measured by immunohistochemistry.Staining of IL-1β and co-staining of E-cadherin and α-smooth muscle actin was performed by immunofluorescence.Mitochondrial damage was detected by transmission electron microscopy.Tissure oxidative stress were assessed by superoxide dismutase(SOD)activity,malondialdehyde(MDA)levels and DHE staining.RESULTS 1.Repeated low-dose cisplatin injections caused the AKI to CKD transition.BUN and serum creatinine levels were increased time-dependently due to repeated injections of cisplatin.HE staining showed that tubular epithelial cell necrosis,vacuolization,and tubular atrophy occurred after repeated injections of low-dose cisplatin.Masson’ s staining indicated collagen deposition in the interstitial area,and PAS staining showed accumulation of glycogen and mesangial expansion.2.Repeated injections of cisplatin induced the activation of NLRP3 inflammasome.The protein levels and RNA levels of NLRP3,ASC,caspase-1 and IL-1β was upregulated,as well as the maturation of caspase-1 and IL-1β.The increased expression of IL-1β was mostly seen in renal tubular epithelial cells.3.Renal fibrosis and epithelial-mesenchymal transiton in cisplatin-induced AKD was ameliorated by the inhibition of NLRP3 inflammasome.PAS staining indicated that cisplatin-induced mesangial expansion was significantly decreased in NLRP3-/-mice.Collagen deposition in interstitial areas induced by cisplatin injections was markedly alleviated in NLRP3 deficient mice.This was consistent with m RNA levels of COL1A1 and COL3A1,which encodes collagen I and collagen III,respectively.The protein levels of the two fibrotic markers were also substantially decreased in NLRP3-/-mice.The colocalization of E-cadherin and α-SMA in tubular epithelial cells was also attenuated by NLRP3 deletion.The increased expression of TGFβ1 and phosphorylation of Smad2/3 were attenuated in cisplatin-treated NLRP3-/-mice.The intervention of MCC950 also showed similar effect of NLRP3 deletion.The inhibition of NLRP3 inflammasome by MCC950 resulted in significant improvement in renal dysfunction,structrural damage and interstitial fibrosis.4.Reapted cisplatin injection caused mitochondrial damage and mitophagy in tubular epithelial cells.Repeated cisplatin injection increased the oxidative stress and mitochondrila damage in tubular epithelial cells.The release of cytochrome c was increased.The expression of mitochondrial dynamic related proteins,Mfn2 and Drp1,was changed after repeated cisplatin injections.PINK1/Parkin mediated mitophagy and BNIP3 mediated mitophagy was activated in kidneys of mice with CP-AKD.5.Inhition of NLRP3 inflammasome attenuated mitochondrial damage by increasing mitophagy.PINK1/Parkin mediated mitophagy was enhanced by NLRP3 deletion and MCC950 treatment,resulting in the reduction of oxidative stress and release of cytochrome c,and protection of mitochondrial structure.CONCLUSIONS 1.Repeated cisplatin injections resulted in acute kidney disease,a mouse model mimicking the AKI to CKD transition.2.NLRP3 inflammasome was involved in the AKI to CKD transition induced by cisplatin.3.Inhibition of NLRP3 inflammasome attenuated renal fibrosis and epithelialmesenchymal transition induced by cisplatin injections.4.Inhibition of NLRP3 inflammasome decreased mitochondrial damage by enhancing mitophagy.