Microarray Analysis Of Exosomes Secreted From Hk-2 Cells Exposed To Oxalate And The Mechanism Signals Macrophages To Differentiate Into M1 Macrophages

Objective:Microarray analysis of exosomes secreted from HK-2 cells exposed to oxalate and the mechanism signals macrophages to differentiate into M1 macrophagesMethods:HK-2 cells were treated with oxalate at the concentration of 0mM~10 mM for 48 h,Cell viability was determined with Cell Counting Kit-8.And then,HK-2 cells were exposed to oxalate in various concentration(0,1,2,4 mM)for 48 h,LDH assay was used to measure the toxic effects of HK-2 cells.The HK-2 cell were exposed to 0,2 mmol/L oxalate for 48 h,culture supernatants were collected,and the exosome were isolated and purified from the supernatants by ultracentrifugation,respectively.Transmission electron microscopy(TEM),Western blot analysis and Nanosight technology were used to examine exosome.A multi-step approach combining microarray mi RNA expression profile and bioinformatics analysis was adopted to analyze dysregulated mi RNAs in exosome from 2 mmol/L group,using 0 mmol/L group as controls.We identified significantly differentially expressed Gene Ontology(GO)terms and KEGG pathways terms that were significantly enriched in 2mmol/L oxalate group.In addition,we successfully constructed the mi RNA-gene network.THP-1 cells was incubated with PBS,Exo(-)/(+),LPS+IFN-? and IL-4+IL-13,and then,the cell markers of M1 and M2 expression were detected by RT-PCR,Western Blot and Elisa.Results: Based on the results of CCK-8 Assay and LDH assay,oxalate induced concentration-dependent injury on HK-2 cells,Cell viability of HK-2cells decreased with raised concentrations treatment of oxalate,from 0 mM group to 0.5 mM group,the injury was minor,from 1 mM grou p to 2 mM group,the injury was obvious,but from 4 mM group to 10 mM group,the degeneration and necrosis were very serious.The exosome were identified by TEM,Western blot analysis and Nanosight technology.We identified 92 mi RNAs as significantly differentially expressed,73 miRNAs were up-regulated and 19 were down-regulated in 2mM group.over 144 GO terms and 16 KEGG pathways terms that were significantly enriched.In addition,we constructed an mi RNA-gene network that suggested that miR-1260 b,miR-4447,mi R-3175,mi R-4259-5p,miR-625-5p,mi R-378 g,mi R-604,miR-193a-3p may play important roles in the regulatory network.Furthermore,miR-28-5p may play an importan role in macrophages polarization of the stone formation.Differentiated THP-1 macrophages were incubated with PBS,Exo(-)/(+),LPS+IFN-? and IL-4+IL-13 for 48 h.Morphology analysis showed that M1 macrophages had elongated fibroblastoid morphology,while M2 macrophages were round cells stretching out pseudopodia.LPS+IFN-? and Exo(+)up regulated the mRNA expression of the M1-markers TNF-?,and IL-1? at 48 h in PMA-treated THP-1cells,increased the secretion of IL-6,and upregulated the protein expression of IL-1? at 48 h.However,Exo(-)did not affect the M1-markers as mentionedabove.IL-4+IL-13 upregulated the mRNA expression of the M2-markers CD206 and IL-1ra at 48 h,increased the secretion of IL-10,and up regulated the protein expression of CD206.Whereas,Exo(-)/(+)had no effect to induce M2-polarized macrophages.These results indicate that Exo(+)promoted M1-polarization of macrophage.Conclusions:Oxalate have toxic effect on HK-2 cells in a concentration dependent.Ultracentrifugation can be used to isolate and purify exosomes efficiently.MiRNA expression in exosome from 2 mmol/L group,and suggests that miRNAs may be important in the regulation of calcium oxalate.The exosomes in oxalate-treated HK-2 cells can program THP-1 cells to polarize into M1 macrophage.