| Background and Objective:Nephrolithiasis,also known as kidney stones,is a common urinary system disease,and the prevalence of adults was over 6% in China and is increasing gradually.The recurrence rate of nephrolithiasis exceeded 50% within 5-10 years since the initial onset.The current treatments consist of hydration therapy,medication of thiazide diuretics and potassium citrate,and surgical treatment including shockwave lithotripsy,ureteroscopic retrieval and percutaneous nephrolithotomy.The stone composition is complex,and about 80% of it is calcium-containing.Calcium oxalate monohydrate(COM)is the main form of calciumcontaining stones.Crystalline nephropathy(CN)is defined as nephropathy caused by crystal formation in the kidney.During the deposition of calcium oxalate crystals in the kidney,the pathophysiological damage process is called calcium oxalate crystal-induced renal injury.The molecular mechanisms of COM crystal-induced renal injury were complicated.Reactive oxygen species(ROS),epithelial-mesenchymal transition(EMT),micro RNA(miRNA),and inflammasome activation,played important roles in the pathogenesis and progression of calcium oxalate crystal-induced renal injury.Firstly,COM crystals could be attached to the tubular epithelial cells and then be phagocyted,and induced the upregulated ROS generation,and activated the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)inflammasome followed by the release of interleukin-1?(IL-1?)and pro-IL-18 to the extracellular space.Secondly,COM crystals caused significant changes in the expression profile of miRNA,and the adhesion between crystals and cells was inhibited by regulating specific miRNAs.Besides,the formation and removal of stones were related to the phenotype and function of macrophages.M1 macrophages promoted the formation of kidney crystals,while M2 macrophages inhibited the growth of kidney crystals,and the macrophage phagocytic ability of M2 is more potent than M1.A previous study performed a miRNA microarray detection on the exosomes from human renal proximal tubular epithelial cells(HK-2)treated with sodium oxalate and found many changed miRNAs.But it's uncertain that which miRNA is critical in nephrolithiasis.Based on this,our preliminary experiment examined those altered miRNAs and found that miR-28-5p in HK-2 cells was significantly downregulated by COM crystal.Relevant studies have found that miR-28-5p affects the proliferation,migration,invasion,and EMT by interacting with target genes,which implied that miR-28-5p might participate in nephrolithiasis.The inflammasome studies involving other diseases showed that caspase-1/4/5 with proteolytic properties cleaves gasdermin D(GSDMD),and the N-terminus of GSDMD formed a plasma membrane pore to trigger pyroptosis,which allowed small molecules such as mature IL-1? and mature IL-18 to pass through.However,the role of GSDMD-related pyroptosis in nephrolithiasis is unclear yet.Therefore,we aimed to clarify the expression,function,and target genes of miR-28-5p in HK-2 cells induced by COM crystals through cell experiments.Furthermore,we aimed to determine the role and mechanism of sphingosine kinase 1(SPHK1),sphingosine-1-phosphate(S1P),and S1 P receptors(S1PRs)mediating GSDMD in renal cells.And verify the effects of S1 PRs on the polarization and function of macrophages.Given the relation of ROS and S1 P together with the connection of ROS and nephrolithiasis,we aimed to evaluate the effects of vitexin on nephrolithiasis and the expressions of SPHK1?S1PR2 and GSDMD.To sum up,the study aimed to explore the function and mechanism of SPHK1/S1P/S1PR2 and GSDMD involved in nephrolithiasis,and provide scientific basis and data support for finding therapeutic targets and evaluation markers.Methods:Part ? The function and mechanism study of miR-28-5p in COM crystal-induced renal injury through negatively regulating SPHK11.The Cell Counting Kit-8(CCK-8)assay was used to detect the cell viability of HK-2 treated by COM crystals;the real-time quantitative polymerase chain reaction(RT-qPCR)assay was used to detect the miR-28-5p level of HK-2 cells.2.The miR-28-5p effect on cell migration of HK-2 was detected by the damagehealing experiment and Transwell chamber.Cell proliferation was detected by a 5-Ethynyl-2'-deoxyuridine(Ed U)assay.Potential target genes of miR-28-5p were predicted by Target Scan7.2,and were detected using the RT-qPCR method when HK-2 cells were overexpressed of miR-28-5p or treated by COM.RT-qPCR and Western Blot methods were used to examine the transcription and protein expression of SPHK1 in HK-2 cells treated by different concentrations of COM crystals for various time courses.3.Si-SPHK1 combined with COM was supplemented to HK-2 cells: ROS production was detected;the Ed U method was used to detect cell proliferation;the cell migration was detected by a Transwell chamber;the terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)method was used to detect cell apoptosis;Western Blot was conducted to detect the expression of NLRP3,mature IL-1?,GSDMD and cleaved GSDMD.Part ? The mechanism of SPHK1/S1P/S1PR2 regulating pyroptosis induced by COM crystal in renal tubular epithelial cells4.Searching for changed molecules related to sphingolipid metabolism was conducted in the renal transcriptome sequencing of glyoxylate-induced mouse models from a previous study of us.Intraperitoneal injection of glyoxylate was undertaken to construct a mouse model of crystal-induced renal injury,and von Kossa was used to detect calcium-containing crystal deposits in kidney tissue,blood biochemistry assay was conducted to detect serum creatinine and urea levels,immunohistochemistry(IHC)and Western Blot were used to detect the proteins localization and expression of renal SPHK1 and S1PR2.5.The effect of COM on the transcriptional expression of SPNS2 in HK-2 cells was detected by RT-qPCR.The level of intracell and extracellular S1 P was detected by enzyme linked immunosorbent assay(ELISA)when cells were treated by COM or si-SPHK1;RTqPCR and Western Blot were used to detect the effect of COM at different concentrations on the transcription and protein levels of S1PR2 in HK-2 cells.In addition,Western Blot was used to detect the effect of COM on the expression of S1PR2 protein in tubule epithelial cells of rat(NRK-52E)and mouse(TCMK-1).6.An S1PR2 antagonist(JTE-013)was used to inhibit the S1PR2 activity of the HK-2 cell.CCK-8 assay was used to detect cell viability,and the damage-healing experiment was used to detect cell migration.Ed U assay and Hoechst 33258 dye were used to detect cell proliferation and apoptosis,respectively.7.JTE-013 combined with COM was supplemented to HK-2 cells,and Western Blot was used to detect EMT-related proteins(E-Cadherin,Pan-cytokeratin(Pan-ck)and Vimentin),NLRP3 inflammasome activation and pyroptosis-related proteins(NLRP3,Cleaved Caspase-1,IL-1?,Mature IL-1?,GSDMD,Cleaved GSDMD)and signaling pathway proteins(serine/threonine kinase(Akt),P-Akt,NF-?B p65 and P-p65).After HK-2cells were stimulated with different concentrations of S1 P,Western Blot was used to detect pyroptosis-related proteins and signal pathway proteins.8.HK-2 cells were overexpressed with SPHK1,and then treated by JTE-013.Western Blot was used to detect the classical pathway proteins activated by S1PR2(extracellular regulated protein kinases 1/2(Erk1/2)and P-Erk1/2),the inflammasome and pyroptosis signaling pathway proteins(phosphoinositide 3-kinase(PI3K),Akt,P-Akt,NF-?B p65 and P-p65).Part ? The function and mechanism of S1P/S1PR2 regulating the pyroptosis and polarization induced by COM crystal in macrophages9.The human leukemia monocytic cell line(THP-1)was induced into M0 by phorbol12-myristate 13-acetate(PMA),and then induced into M1 or M2 by lipopolysaccharide(LPS)+ interferon ?(IFN-?)or IL-4+IL-13,respectively.M0 was also treated by COM crystal,and RT-qPCR was used to detect the levels of tumor necrosis factor-?(TNF-?)and IL-1? in the above groups.10.RT-qPCR was used to detect the levels of S1 PRs in M0,M1,M2 and COM-treated macrophages.RT-qPCR and Western Blot were conducted to detect the effect of COM crystal on the transcription and protein expression of S1PR2 in macrophages.11.Macrophages were treated by JTE-013 and COM crystals.Cell viability and apoptosis were detected by CCK-8 and Hoechst 33258 staining,respectively.The effect of JTE-013 on the phagocytic ability of macrophages was detected by Cy3-labeled COM crystal or co-cultured HK-2 cells labeled by Dil.RT-qPCR was used to detect the effect of JTE-013 on the expression of phenotype-related molecules(M1: TNF-?,IL-1? and IL-6.M2: interleukin 1 receptor antagonist(IL-1RN),CD163 and CD206).Western Blot was used to detect the expression of pyroptosis-related proteins.CCK-8 assay and Western Blot were used to detect the effect of S1 P on cell viability and the expression of pyroptosis-related proteins.12.Western Blot was used to test the effect of JTE-013,COM and S1 P on the expression of macrophage phenotype-regulating transcription factors(P-p65 and P-Stat6).Part ? The role and mechanism of vitexin on crystal-induced renal injury via inhibiting GSDMD cell pyroptosis13.Four groups were designed including the control group(Ctrl),the glyoxylateinduced model group(Gly),the low-dose vitexin group(Gly+VTX 10),and the high-dose vitexin group(Gly+VTX 20).After 7 days of treatment,renal samples were harvested.Renal tissue sections were stained by H&E and von Kossa staining to observe renal pathological damage and crystal deposition,and biochemical detection kits were used to detect renal oxidative stress indexes(MDA,SOD,GSH and CAT),TUNEL and immunofluorescence assay were used to detect kidney tissue apoptosis and renal macrophages infiltration respectively.Immunohistochemistry assay was conducted to detect the expression of GSDMD,IL-1?,osteopontin(OPN),CD44 and monocyte chemotactic protein 1(MCP-1)in kidneys.Western Blot was used to detect the expression of SPHK1 and GSDMD pyroptosis-related proteins in kidneys.14.In HK-2 cells,the effect of vitexin on cell viability and apoptosis was detected by CCK-8 and TUNEL.Cell LDH release was detected by a ROS kit.Cell immunofluorescence was conducted to detect the expression of EMT-related proteins(Pan-ck,Vimentin and ?-Smooth muscle-actin(?-SMA)).Western Blot was conducted to detect the expression of EMT-related proteins,EMT-regulating proteins(Wnt,?-Catenin and phospho-?-Catenin)and GSDMD pyroptosis-related proteins.15.In THP-1-derived macrophages,the effect of vitexin on cell viability was detected by CCK-8.RT-qPCR was conducted to test the expression of S1PR2,TNF-?,IL-1?,IL-1RN and CD163.Cell LDH release was detected by a ROS kit.Western Blot was used to detect the expression of GSDMD pyroptosis-related proteins.Results:1.COM crystals inhibited HK-2 cell viability in a concentration-and time-dependent manner,and significantly downregulated the cell expression of miR-28-5p.2.Overexpressed miR-28-5p significantly inhibited the migration and proliferation of HK-2 cells.Mi R-28-5p negatively regulated the expression of SPHK1 in HK-2 cells.The transcription and protein of SPHK1 in HK-2 cells were significantly increased by COM.3.In the HK-2 cells,SPHK1-interfering significantly reduced the ROS generation induced by COM crystals,and inhibited the proliferation and migration,and decreased the protein expression of NLRP3,Mature IL-1?,GSDMD and Cleaved GSDMD.4.The renal transcriptome sequencing of glyoxylate-induced mice showed that several molecules including Sphk1 and S1pr2 were upregulated.The serum levels of creatinine and urea increased significantly in the mice injected with glyoxylate.Besides,the proteins expression of SPHK1 and S1PR2 were upregulated significantly in the model mouse,and located at the epithelial cells which was embedded in the dilated renal tubules.5.In the HK-2 cells,COM crystals caused the up-regulation of SPNS2 transcription and increased extracellular S1 P secretion,but had no noticeable effect on the intracellular S1 P level.The increasing extracellular S1 P release was inhibited by SPHK1-interfering.COM crystals significantly increased the transcription and protein expression of S1PR2 in HK-2 cells,and also increased the protein expression of S1PR2 in the renal tubular epithelial cells of rats and mice.6.In the HK-2 cells,the S1PR2 antagonist(JTE-013)attenuated the inhibition effect of COM on cell proliferation,and inhibited cell apoptosis and migration.7.In the HK-2 cells,JTE-013 increased the protein expression of E-Cadherin and Panck,and reduced the protein expression of Vimentin.It also reduced the protein expression of NLRP3,Cleaved Caspase-1,Mature IL-1?,GSDMD,Cleaved GSDMD,P-Akt,and P-p65,which were upregulated by COM crystals.Besides,S1 P could gradually increase the protein expression of NLRP3,Cleaved Caspase-1,Mature IL-1?,GSDMD,Cleaved GSDMD,and P-Akt.S1 P at the high concentration also increased the protein expression of P-p65.8.In the HK-2 cells,SPHK1 Overexpression induced the up-regulation protein expression of P-Erk1/2,PI3 K,P-Akt and P-p65 protein expression,while JTE-013 reduce the expression of the four proteins.9.In the macrophages,COM crystals and LPS+IFN-? all induced a significant upregulated transcription of TNF-? and IL-1?.10.In the macrophages,COM crystals significantly increased the transcription and protein expression of S1PR2 in a concentration-dependent manner.Compared with M0 macrophages,the transcription of S1PR2 was significantly upregulated in M1,and downregulated in M2,and there was a significant difference between M1 and M2 macrophages.11.In the macrophages,JTE-013 significantly inhibited cell apoptosis,increased the cell viability impaired by COM crystals.It also enhanced the ability of phagocyting COM crystals and HK-2 extracellular vesicles.Besides,COM crystals significantly increased the transcriptional expression of M1 phenotype-related molecules(TNF-?,IL-1? and IL-6),and decreased the expression of M2 phenotype-related molecules(IL-1RN,CD163 and CD206).While JTE-013 could partially reverse the changes in the above-mentioned phenotyperelated molecules.In addition,COM crystals increased the protein expression of NLRP3,Cleaved Caspase-1,Mature IL-1?,GSDMD and Cleaved GSDMD,and those proteins were decreased under treatment with JTE-013.In the macrophages,S1 P increased the protein expression of NLRP3,Cleaved Caspase-1,Mature IL-1?,GSDMD and Cleaved GSDMD.12.In the macrophages,P-p65 was upregulated,and P-Stat6 was decreased in the S1 P group and the COM crystals group.And S1PR2 inhibition reduced the protein expression of P-p65 and increased P-Stat6 protein expression.13.In the mice of COM crystal-induced renal injury,vitexin reduced the renal pathological injury and crystal deposition in the kidney,and decreased the expression of OPN and CD44 protein in the kidney.Vitexin reduced the renal level of oxidative stress MDA,and upregulated the level of SOD,GSH and CAT.Besides,vitexin inhibited renal cell apoptosis,and reduced macrophage infiltration in kidneys,and decreased renal MCP-1expression.Especially,vitexin significantly reduced the protein expression of SPHK1,NLRP3,Cleaved Caspase-1,Mature IL-1? and GSDMD in the kidney.14.In the HK-2 cells,vitexin restored the cell viability impaired by COM crystals,and inhibited the cell apoptosis and LDH release induced by COM crystals.Besides,vitexin increased the protein expression of Pan-ck and reduced the expression of Vimentin and ?-SMA.Vitexin reduced the protein expression of Wnt and ?-Catenin,and increased the expression of phospho-?-Catenin.Furthermore,vitexin reduced the protein expression of NLRP3,Cleaved Caspase-1,Mature IL-1? and Cleaved GSDMD,which were upregulated by COM crystals.15.In the macrophages,vitexin reduced the upregulation of S1PR2 transcription induced by COM crystals,and reduced the transcription of TNF-? and IL-1?,and inhibited LDH release.Besides,vitexin reduced the protein expression of NLRP3,Cleaved Caspase-1,and Mature IL-1? And Cleaved GSDMD,which were upregulated by COM crystals.Conclusion:Via in vivo and in vitro experiments,the study preliminarily confirmed that COM crystals could activate NF-?B p65 through SPHK1/S1P/S1PR2 in renal tubular epithelial cells and macrophages,which lead to the GSDMD pyrolysis,and could cause EMT in tubular epithelial cell and pro-inflammatory M1 polarization of macrophages.In addition,it's found that SPHK1 and S1PR2 were significantly down-regulated when vitexin played the role of treating kidney stones.In general,the SPHK1/S1P/S1PR2 pathway participates in calcium oxalate crystals-induced renal injury via regulating GSDMD-related pyroptosis... |
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