The Role Of Autophagic Flux Mediated By TLR4 In Soluble Uric Acid Induced Inflammatory Response In Renal Tubular Epithelial Cells

Objective To investigate the relationship between Toll like receptor 4(TLR4)mediates soluble uric acid(UA)induced flammatory response in HK-2 cells through Autophagic flux disorder.Methods HK-2 cells were stimulated with soluble uric acid solution for 24 h,the expression of MCP-1m RNA was determined by RT-q PCR;the protein expression of MCP-1 was determined by western blotting.Autophagosomes and autophagolysosomes were observed by transmission electron microscopy(TEM).After HK-2 cells were co-stimulated for 12 h with soluble uric acid solution or(and)chloroquine(CQ),western blotting was used to detect the expression of LC3-II and P62;Autophagosomes and autophagolysosomes were observed by transmission electron microscopy(TEM).Next,HK-2 cells were co-stimulated with soluble uric acid solution and TLR4 inhibitor(TAK242)for4h,the m RNA expression of TLR4 was detected by RT-q PCR;HK-2 cells were co-stimulated with soluble uric acid solution and TLR4 inhibitor(TAK242)for24h,western blotting was used to detect the protein expression of TLR4,LC3-II and P62;after using m RFP-GFP-LC3 to transfect HK-2 cells,the autophagic flow was observed by confocal microscope.The protein expression of MCP-1was detected by western blotting after HK-2 cells were stimulated by soluble uric acid solution,TAK242 and CQ for 24 h.Results HK-2 cells were stimulated with soluble uric acid for 24 h,the m RNA and protein expression of MCP-1 is up-regulated in the soluble uric acid group(P<0.05);the number of autophagy increased as observed by TEM,Enzymes are rare,it indicates that the autophagy flow is impaired in renal tubular epithelial cells.soluble uric acid could induce the protein expression of LC3-II and p62 in HK-2 cells to increase,and the expression of LC3-II and p62 in HK-2 cells to further increase after the treatment of soluble uric acid and(or)CQ(P<0.05).RT-q PCR results showed that TLR4 expression levels of soluble uric acid group were higher than those of normal control group(P<0.05),while TAK242 inhibited TLR4 expression levels of soluble uric acid induced HK-2cells(P<0.05);western blotting results showed that TLR4,LC3-II and p62 expression levels of soluble uric acid group were higher than those of normal control group(P<0.05),while TAK242 inhibited TLR4,LC3-II and p62 expression levels of soluble uric acid induced HK-2 cells(P<0.05);The results of confocal microscope showed that the levels of autophagosomes were significantly higher in soluble uric acid group compared with control group,and the levels of autophagolysosomes were lower;while compared with the TAK242 +UA group,the level of autophagosomes decreased and autolysosomes increased in the TAK242 + UA group,indicating that the fusion of autophagosomes and lysosomes increased,and the process of forming autophagolysosomes was smoother.After HK-2 cells were co-stimulated for 24 h with soluble uric acid plus TAK242 and(or)CQ,TAK242 inhibited the expression of MCP-1 stimulated by soluble uric acid in HK-2(P<0.05);the expression level of MCP-1 in UA + CQ group was significantly higher than that in soluble uric acid group(P<0.05);while that in UA +TAK242 + CQ group was higher than that in UA +TAK242 and(or)UA + CQ groups(P<0.05).ConclusionSoluble uric acid can induce the inflammatory response in renal tubular epithelial cells;Soluble uric acid can induce the autophagy flow is impaired in renal tubular epithelial cells;Soluble uric acid can mediate renal tubular epithelial cells autophagic disorder through TLR4;TLR4 mediates soluble uric acid(UA)induced flammatory response in HK-2 cells through Autophagic flux disorder.