Mesenchymal Stem Cell-derived Exosomes Ameliorate Diabetic Kidney Disease Through NLRP3 Signaling Pathway

Diabetic kidney disease(DKD)is one of the most common complications of diabetes,which is mainly characterized by proteinuria,hypertension and progressive decline of renal function.The pathogenesis of DKD is complex,recent studies have shown that,glucose metabolism disorders,renal hemodynamics changes and the activation of innate immunity are very important in the development of DKD.Nucleotide-binding domain and leucine-rich repeat pyrin 3 domain(NLRP3)as the important part of the innate immune system and can recognize damage-associated molecular patterns(DAMPs),recruit and activate the pro-inflammatory protein Caspase-1 through the apoptosis-associated speck-like protein containing caspase recruitment domain(ASC),which can cleave interleukin 1β(IL-1β)and interleukin 18(IL-18),produce mature cytokines and trigger inflammatory responses.Podocyte is an important component of the glomerular filtration barrier.Podocyte injury and dysfunction is crucial to the development of DKD.Studies have shown that the activation of NLRP3 signaling pathway plays significant roles in podocyte injury in DKD.It is reported that mesenchymal stem cells(MSCs)have therapeutic effects on DKD.However,due to its several disadvantages,such as safety,immune rejection and ethical problems,there are limitations for its application in clinical treatment.In addition to possess the function of MSCs,the adverse reactions of MSCs can be avoided to a certain extent by MSCs derived exosomes(MSCs-Exo),which may have greater clinical application potential than that of MSCs.Therefore,the mechanism of NLRP3 in podocytes injury in DKD and the role of mesenchymal stem cell-derived exosomes(MSCs-Exo)in this process need to be further explored and it is of great significance for the treatment of DKD.Objective1.To clarify the protective effects of MSC-Exo on podocytes under high glucose(HG)and DKD.2.To clarify the effect of MSC-Exo on NLRP3 signaling pathway in podocytes under HG and DKD.3.To explore the mechanism of MSC-Exo on the NLRP3 in podocytes and DKD.Methods1.The effect of MSCs-Exo on podocytes and DKD and NLRP3 pathway1.1 MSCs-Exo extraction,identification and labelingExosomes of MSCs were extracted by ultracentrifugation.Then the exosomes were detected by western blot(WB),nanoparticle tracking analysis(NTA),and transmission electron microscopy(TEM)to detect their marker proteins(CD81,CD63,TSG101),particle size and morphology.Exosomes were labeled with PKH-67 dye to observe the localization of MSCs-Exo in podocytes and DKD mice.1.2 Effect of HG and other injury factors on podocytes and the protective effect of MSCs-Exo on podocytesPodocytes were cultured in vitro with different concentrations of HG,AGEs and tumor necrosis factor alpha(TNF-α)with or without MSCs-Exo co-cultured.The activity and apoptosis of podocytes were detected by CCK8 and flow cytometry.Immunofluorescence(IF)was used to detect the expression and arrangement of F-actin in podocytes.The level of reactive oxygen species(ROS)was determined by the dihydroethidium(DHE)and the mitochondrial membrane potential(MMP)of podocytes was detected by the TMRE.1.3 The effect of MSCs-Exo on the NLRP3 pathway in podocytes under HGPodocytes were cultured under HG with or without Exo.After 24 hours,the expression of inflammatory factors IL-6,IL-18,IL-1β,TNF-α were detected by ELISA;RT-qPCR,WB were used to detect the mRNA and protein expression of NLRP3 and its downstream signaling proteins Caspase-1,ASC,IL-1β.1.4 The effect of MSCs-Exo on renal function and NLRP3 pathway in DKD10-week type Ⅱ male DKD mice(db/db mice)were used as the experimental groups and were randomLy divided into 3 groups:DKD group;MSCs-Exo injection group(DKD+Exo group);insulin treatment group(DKD+Ins group),db/m mice were used as the normal control group(Con group).After 4 weeks of treatment,blood glucose,weight,24-hour urine protein were detected.Hematoxylin-eosin staining(HE),IF,Immunohistochemistry(IHC),Periodic acid schiff(PAS)and Masson staining were used to detect the pathological changes of glomeruli.The ultrastructural changes of the basement membrane of the foot process in the glomerulus were observed by TEM.ELISA,RT-qPCR,WB was used to detect the expression of inflammatory factors,NLRP3 and its downstream signaling proteins.2.The mechanism of MSCs-Exo in podocytes and DKD NLRP3 pathway2.1 Confirm the target miRNA in MSCs-Exo that regulate NLRP3 in podocytes and DKDTo explore the protective mechanism of MSCs-Exo on podocytes and DKD,we analyzed the bioactive substances in MSCs-Exo.Bioinformatics method was used to predict the target genes of the top ten miRNAs in terms of expression,and find out the target miRNAs in MSCs-Exo that may play a role in regulating NLRP3 in podocytes and DKD.Then the double luciferase report experiment was conducted to confirm the regulatory relationship between the target miRNA and the 3’UTR of NLRP3 mRNA.2.2 The effect of miR-22-3p on podocytes viabilityExo-K(Exo with low expression of miR-22-3p)and Exo-M(Exo with high expression of miR-22-3p)were obtained by transfecting MSCs with miR-22-3p inhibitor and mimic respectively.The effect of miR-22-3p on podocyte viability in MSCs-Exo was detected by CCK8 method.2.3 The effect of miR-22-3p in the NLRP3 pathway of podocytes under HGPodocytes were cultured under HG with or without Exo-K and Exo-M.After 24 hours,ELISA was used to detect the expression of inflammatory factors;RT-qPCR,WB were used to detect the mRNA and protein expression of NLRP3 and its downstream signaling proteins.2.4 The role of miR-22-3p in NLRP3 in DKD mice10-week type Ⅱ male DKD db/db mice were randomLy divided into 4 groups:DKD group;MSCs-Exo injection group(DKD+Exo group);scrambled control Exo injection group(DKD+Exo-Scram group);Exo-K injection group(DKD+Exo-K group).After 4 weeks of treatment,blood glucose,weight,24-hour urine protein and other indicators were detected.HE,IF,IHC,PAS,Masson staining was used to observe the pathological changes of the glomerulus;TEM was used to observe the ultrastructural changes of glomerulus;ELISA,RT-qPCR,WB was used to detect the expression of inflammatory factors,NLRP3 and its downstream signaling proteins.Results1.MSCs-Exo protected podocytes under HG and DKD and inhibit NLRP3 pathway1.1 MSCs-Exo reduced the damage of HG,AGEs and TNF-α to podocytesMSCs-Exo were identified by NTA,TEM and WB analysis.CCK8 detected that HG,AGEs,TNF-α reduced the viability of podocytes in a concentration dependent manner,but MSCs-Exo alleviated the damage to podocytes.Flow cytometry showed that HG,AGEs,and TNF-α significantly increased podocytes apoptosis,while MSCs-Exo alleviated the process.In addition,MSCs-Exo alleviated the pathological changes of F-actin,reduced the ROS and improved the level of MMP in podocytes under HG.1.2 MSCs-Exo inhibited the activation of NLRP3 pathway in podocytes under HGELISA,RT-qPCR and WB analysis showed that HG activated the NLRP3 pathway in podocytes,increased the expression of inflammatory factors IL-6,IL-18,IL-1β,TNF-α,the mRNA and protein level of NLRP3,Caspase-1,ASC,IL-1β.IF staining showed that HG also decreased the expression of Nephrin(podocyte marker protein)and increased the expression of Desmin(podocyte damage protein),while MSCs-Exo inhibited the activation of the NLRP3 pathway and had a protective effect on podocytes under HG.1.3 MSCs-Exo protected the renal function of DKD mice and inhibited the NLRP3 pathwayAfter 4 weeks of MSCs-Exo and insulin injection,it was found that,MSCs-Exo and insulin reduced blood glucose,body weight,24-hour urine protein.ELISA,RT-qPCR and WB showed that the activation of NLRP3 pathway in MSCs-Exo and insulin injection groups was significantly inhibited,the expression of inflammatory factors was reduced,the level of NLRP3 and its downstream signaling proteins Caspase-1,ASC,IL-1β were decreased.TEM showed that the thickness of glomerular basement membrane,the increased foot process width and the number of podocytes were all improved by MSCs-Exo and insulin.2.Mechanism of the inhibition of MSCs-Exo in NLRP3 in podocytes under HG and DKD2.1 MiR-22-3p in MSCs-Exo can target NLRP3MiR-22-3p is the third abundant miRNA in MSCs-Exo,and NLRP3 is predicted to be the target gene of miR-22-3p.The double luciferase report experiment confirmed that miR-22-3p can directly target the 3’-UTR mRNA of NLRP3,so we hypothesized that MSCs-Exo mediated NLRP3 to protect podocytes and DKD through miR-22-3p.2.2 MiR-22-3p strengthened the protection of MSCs-Exo on podocytes viabilityAfter transfection of MSCs with miR-22-3p Inhibitor and Mimic,MSCs with low expression and overexpression of miR-22-3p were obtained(Exo-K/Exo-M).CCK8 detection showed that compared with MSCs-Exo,the protective effect of MSCs-Exo on podocyte under HG was inhibited in Exo-K but enhanced in Exo-M.2.3 MiR-22-3p enhanced the inhibition of MSCs-Exo in the NLRP3 pathway in podocytes under HGELISA,RT-qPCR,WB showed that compared with MSCs-Exo,the lack of miR-22-3p in MSCs-Exo reduced the inhibitory effect of MSCs-Exo on the activation of NLRP3 pathway in podocytes under HG while the overexpression of miR-22-3p strengthened this effect.2.4 Lack of miR-22-3p impaired the inhibition of MSCs-Exo in the NLRP3 in DKD miceCompared to DKD mice with MSCs-Exo injection group,Exo-K inhibited the therapeutic effect of MSCs-Exo on DKD mice,increased the blood glucose in DKD mice.Moreover,Exo-K weakened the inhibition of NLRP3 in renal tissue of DKD mice by MSCs-Exo.The NLRP3 mRNA level and protein level was increased in Exo-K group.Conclusion1.MSCs-Exo can alleviate the damage to podocytes caused by HG,AGEs,and TNF-α,reduce podocyte apoptosis,and have a protective effect on podocytes.2.MSCs-Exo can inhibit the activation of NLRP3 pathway in podocytes caused by HG,reduce the expression of inflammatory factors and alleviate the inflammatory response.In vivo,MSCs-Exo can alleviate renal injury in DKD mice,protect renal function and inhibit the activation of NLRP3 signaling pathway in renal tissue.3.The mechanism of MSCs-Exo inhibits NLRP3 maybe:miR-22-3p in MSCs-Exo can target the NLRP3 gene in podocytes and kidney tissue,directly downregulate the expression of NLRP3,thereby inhibiting the activation of the NLRP3 pathway and reducing the inflammation.4.This study elucidated that,MSCs-Exo can inhibit NLRP3 pathway through the miR-22-3p,restrain the inflammation and injury in podocytes and DKD.Our results may provide a new target for the treatment of DKD.