The Role And Mechanism Of Sphingosine Kinase 2 In Regulating The Occurrence Of Renal Interstitial Fibrosis

Part 1 TGF-?1 promotes renal interstitial fibrosis via sphingosine kinase 2-dependent activation of Fyn,STAT3,and AKTSphingosine-1-phosphate(S1P),and its cognate receptors play potentially important roles in different types of nephropathies.Sphingosine kinases(Sphks)are the rate-limiting enzymes in the conversion of sphingosine to biologically active S1 P.However,the role of Sphks in renal interstitial fibrosis is unclear.The present study aimed to determine the role of Sphk2 in renal interstitial fibrosis and the downstream targets of this enzyme.In the renal interstitium of patients with renal fibrosis,Sphk2 highexpressing cells(mainly interstitial fibroblasts)were significantly elevated and highly correlated with disease progression.In a murine model of renal interstitial fibrosis,Sphk2 was upregulated in the kidney of wild-type mice in response to disease progression.Importantly,Sphk2-knockout(KO)mice exhibited significantly lower levels of extracellular matrix(ECM)production,inflammatory cytokines,and immune cell recruitment in kidney tissues,compared to their wild-type counterparts,whereas the expression of pro-fibrotic cytokines,such as transforming growth factor-?1(TGF-?1),was unaffected.TGF-?1 effectively upregulated Sphk2 in the renal interstitial fibroblast cell line,NRK-49 F,independent of canonical Smad signaling activation.Furthermore,si RNA-mediated Sphk2 knockdown or suppression of Sphk2 activity via ABC294640 exposure effectively attenuated the activation of key signaling molecules such as AKT and STAT3,and the production of fibrosis products,but showed no effects on Smad2 and Smad3 activation.Sphk2 binds and phosphorylates Fyn,a member of the Src family,to activate downstream STAT3 and AKT,thereby promoting ECMsynthesis.Therefore,our findings indicated that targeting the Sphk2-Fyn-STAT3/AKT signaling pathway may provide a novel therapeutic approach for TGF-?1-mediated renal fibrosis.Part 2 Stablization of HDAC4 by Sphk2 in the development of renal interstitial fibrosisRenal fibrosis is the common final outcomes of chronic kidney diseases,usually leading to renal failure.Sphingosine kinases(Sphks)deliver their functions mainly through catalyzing sphingosine to bioactive sphingosine-1-phosphate(S1P).Our previous study has proved that Sphk2 induces renal fibrosis by activating renal fibroblasts.Histone deacetylases(HDACs)control gene transcription by removing the acetyl group from the acetyl lysine residue of histone.HDAC4 shuttles between nucleus and cytoplasm by dephosphorylation and phosphorylation.In the presenet study,we observed TGF-?1could effectively elevate the protein level of HDAC4 in NRK-49 F cells.Meanwhile,si RNA-mediated HDAC4 knockdown or suppression of HDAC4 activity via LMK 235 exposure effectively attenuated the production of fibrosis products,fibronectin and ?-SMA,induced by TGF-?1.In murine model of renal interstitial fibrosis,mice underwent unilateral ureteral obstruction were intraperitoneally administrated LMK 235 for consecutive 7 days,and LMK 235 were found significantly suppressed the production of fibrotic products fibronectin and ?-SMA,injury of renal tubules and glomerulus,infiltration of inflammatory cells and deposition of ECM.Sphk2 knock-out or blockade by ABC294640 sharply inhibited protein level of HDAC4 in vivo and in vitro,in contrast,ABC294640 obviously increased the level of HDAC4 m RNA in NRK-49 F.Sphk2 was proved to directly interact with HDAC4 by co-immunoprecipitation.In conclusion,HDAC4 interacted with Sphk2 and stablized HDAC4,then participated in the development of renal interstitial fibrosis.