Sphk2 Promotes Diabetic Tubulointerstitial Fibrosis Through Regulation Of NLRP3 Inflammasome Activation

Objective:Tubulointerstitial fibrosis（TIF）is the main pathological basis for diabetic kidney disease（DKD）to develop into end-stage renal failure.Renal tubule injury directly induces TIF through multiple mechanisms,which is considered to be the driving force and key therapeutic target of DKD.Activation of NLRP3 inflammasome in renal tubular epithelial cells（TEC）can trigger inflammatory responses that promote tubular injury and ultimately lead to TIF in DKD.Sphingosine kinase 2（Sph K2）,an important signal transduction enzyme in renal sphingolipid signaling pathway,has been reported to be involved in the regulation of TIF in acute kidney injury,but whether it is involved in the regulation of diabetic TIF is still unclear.It has been reported that Sph K2 promotes the activation of NLRP3 inflammasome in mouse macrophages,but it remains unclear whether Sph K2 mediates the activation of NLRP3 inflammasome in TECs.The purpose of this study is to investigate whether Sph K2 can promote TIF in DKD by regulating NLRP3inflammasome activation in TECs.Methods:1.The activation of NLRP3 inflammasome in rat renal tubular epithelial cells（NRK52E cells）was inhibited by transfection with caspase-1 in vitro,and then the expressions of proteins related to renal tubular injury and fibrosis were detected to determine the effects of NLRP3 inflammasome activation on renal tubular injury and TIF in high glucose environment.2.In vivo experiments,the expressions of Sph K2 and NLRP3inflammasome-related proteins in the kidney tissues of diabetic mice(Lepr^db/dbmice)were detected to investigate whether the activation of renal NLRP3inflammasome is related to the renal protein expression of Sph K2 in diabetic environment.3.In vitro experiments,Sph K2 gene in NRK52E cells was silenced or overexpressed by transfection experiments,and the transfected cells were treated with high glucose medium.Molecular biological detection methods,cell immunofluorescence experiment and mitochondrial pressure test experiment were used to explore the regulation and the molecular mechanisms of Sph K2 on NLRP3 inflammasome activation in transfected cells under high glucose environment.4.The above in vitro results were verified in vivo.TEC-specific Sph K2 knockout（Sph K2 TEC-KO）diabetic mice were obtained by mating and being induced with streptozotocin（STZ）.Immunofluorescence,molecular biological detection methods were performed to detect related indexes on the mouse kidney tissues to further verify the regulation and mechanism of TEC-specific Sph K2 gene knockout on NLRP3 inflammasome activation in renal tubules of diabetic mice.Results:1.In vitro experiments showed that high glucose-induced injury and fibrosis of TECs could be improved by inhibiting NLRP3 inflammasome activation in NRK52E cells.2.In the kidney tissues of Lepr^db/db diabetic mice,the protein expressions of Sph K2 and inflammasome-related proteins increased with age and are positively correlated,which suggested that the renal protein expression of Sph K2 may be related to the activation of inflammasome.3.In vitro experiments showed that under high glucose environment,silencing Sph K2 gene in NRK52E cells could reduce the m RNA levels and protein expressions of inflammasome-related genes or proteins,which suggested that silencing Sph K2 gene in TECs could inhibit inflammasome activation to improve TEC injury and fibrosis.4.In further molecular mechanism studies,we confirmed that Sph K2 in NRK52E cells mediates mitochondrial damage by promoting mitochondrial dynamic imbalance,mitochondrial membrane potential decline and mitochondrial oxidative phosphorylation dysfunction induced by high glucose,leading to the abnormal generation of mitochondrial ROS（mt ROS）in cytoplasm that promotes the binding of TXNIP to NLRP3 and activates inflammasome.5.The results of in vivo validation showed that TEC-specific Sph K2 gene knockout can reduce tubular mitochondrial damage and inhibit the activation of NLRP3 inflammasome in DKD mouse kidneys,which was consistent with the results of in vitro study.Conclusions:Sph K2 promotes the activation of NLRP3 inflammasome in TECs by promoting mitochondrial injury and increasing mt ROS production under high glucose environment,thus promotes the progression of renal tubular injury and TIF in DKD.