| Background and objective:Diabetic nephropathy(DN)has become the main cause of ESRD and is one of the most important microvascular complications of diabetes.But its pathogenesis has not been clarified.Our studies and those of other scholars have shown that macrophage-mediated inflammation is closely related to disease progression of DN.However,in addition to producing inflammatory mediators leading to tissue damage,whether macrophages can further aggravate kidney injury through other mechanisms has also attracted great attention in recent years.Exosome is a kind of extracellular vesicle surrounded by phospholipid bilayer,with a diameter of 40-150 nm,widely existing in body fluids,including serum,plasma,saliva,urine and milk.Exosomes can enter recipient cells by carrying proteins,nucleic acids,lipids and other information substances,and affect the function of recipient cells to complete the information exchange between cells.In recent years,more and more studies have been conducted on the influence of exosomes on cell function by carrying mi RNA.mi RNA is a small single-stranded RNA that inhibits the transcription and post-transcriptional regulation of target genes by forming a mi RNA silencing complex(RISC)with target m RNA.A large number of studies have confirmed that exosomes can carry mi RNA into target cells and affect the biological functions of target cells.Our previous study confirmed that macrophages not only promote their own activation through exosomes during DN process,but also macrophage-derived exosomes can promote secretion of extracellular matrix by glomerular mesangial cells.Recent studies have shown that renal tubular injury plays a more prominent role in the pathogenesis of DN,and the mechanism has become one of the hotspots in this field.In the early stage of DN,the hyperglycemic microenvironment will lead to the proliferation and hypertrophy of tubule cells,reduction of organic ion transport and other lesions,and eventually develop into irreversible fibrosis with the progression of the disease.Among them,autophagy homeostasis plays an extremely important role in maintaining renal tubule function.Autophagy is a self-protective mechanism that can maintain intracellular homeostasis by engulfing damaged or aging organelles.Excessive or insufficient autophagy will lead to the destruction of the homeostasis of the intracellular environment,resulting in cellular inflammation and damage.Previous studies have found that the autophagy activity of renal tubular epithelial cells is inhibited in both DKD mice and patients with type 2 diabetic nephropathy,and the deletion of Atg7 can lead to renal autophagy defect in diabetic mice,resulting in more serious renal hypertrophy,renal tubular injury,inflammation,fibrosis and proteinuria.Therefore,homeostasis of autophagy activity is important for the functioning of renal tubular epithelial cells.On this basis,the present study explored the effect and specific mechanism of macrophage-derived exosomes stimulated by high glucose on the autophagy function of renal tubular epithelial cells.Methods:Part I: 1.The effect of macrophages on renal tubular epithelial cells was observed by co-culture RAW264.7 with m TEC and application of exosome releasing inhibitor GW-4869.The concentrations of exosomes derived from macrophages in each group were quantitatively detected by BCA,the expressions of inflammatory factors TNF-?,IL-1? and MCP-1 in supernatant of each group were detected by ELISA,and the expressions of TNF-?,IL-1?,MCP-1,KIM-1,Col-I and FN in each group were detected by RT-PCR.2.Exosomes derived from macrophages stimulated by normal glucose and high glucose were extracted,respectively.Exosomes morphology and concentration were observed by electron microscopy and quantitative BCA detection;exosome markers Alix,CD63 and TSG101 were detected by WB;3.The optimal concentration and time of m TEC stimulated by exosomes were screened by setting different concentrations and time gradients,and the expression changes of KIM-1,Col-I and FN in each group were observed by WB and immunofluorescence.The expressions of inflammatory cytokines TNF-?,IL-1? and MCP-1 in cells and supernatant of each group were detected by ELISA and RT-PCR,the expression of autophagic proteins LC3 and p62 in cells of each group was detected by WB,the expression of p62 in cells of each group was detected by immunofluorescence,and the number of autophagosomes in cells of each group was observed by electron microscopy.4.Exosomes were injected into C57 mice through tail vein,and the changes of general indexes and serum creatinine and urea nitrogen were detected.The changes of renal tubule injury in each group were observed by HE and PAS staining,and the changes of renal inflammation were observed by RT-PCR and immunohistochemistry.The expressions of KIM-1,Col-I,FN and p62 were observed by immunohistochemistry,and the expressions of KIM-1,Col-I,FN,LC3 and p62 were observed by WB.Part II: 1.Exosomes derived from macrophages stimulated by normal glucose and high glucose were extracted for mi RNA high-throughput sequencing,and the most differentially expressed mi RNAs were screened for verification,and downstream target genes were searched by bioassay.2.The binding region of mi R-7002-5p and Atg9 b was predicted by Targetscan website,and the expression changes of mi R-7002-5p and Atg9 b in each group were observed by RT-PCR after transfection with mi RNA mimic and inhibitor.The direct binding of mi R-7002-5p to Atg9 b was verified by dual luciferase assay.3.The expression changes of mi RNA in m TEC after HG-exo stimulation were detected by PCR,the expression changes of Atg9 b protein in cells of each group were detected by WB,and the expression changes of mi R-7002-5p in cells of each group and mouse kidney were observed by fluorescence in situ hybridization.4.mi R-7002-5p expression was reduced by mi RNA inhibitor,and the expression changes of inflammatory cytokines TNF-?,IL-1? and Mc P-1 in each group were observed by ELISA and RT-PCR.The expression changes of Kim-1,Col-I,FN,Atg9 b,LC3 and p62 in each group were detected by WB,the expression changes of p62 in each group were detected by confocal laser,and the number changes of autophagosome in each group were observed by electron microscope.5.Each group was injected into C57 mice by tail vein injection,and the changes of general indexes and serum creatinine and urea nitrogen were detected.HE and PAS staining were used to observe the pathological changes of mice kidney,and the expressions of inflammatory factors TNF-?,IL-1? and MCP-1 were detected by RT-PCR and immunohistochemistry.The expressions of KIM-1,Col-I and FN in kidney of each group were detected by immunohistochemistry.Results:Part I: 1.BCA quantitative results showed that GW-4869 could reduce the release of exosomes derived from macrophages stimulated by high glucose;ELISA results showed that exosome inhibitors could reduce the expression of inflammatory factors TNF-?,IL-1? and MCP-1 in the supernatant of m TEC cells under co-culture condition.Rt-pcr results showed that exosome inhibitors could reduce the m RNA expression levels of inflammatory factors TNF-?,IL-1?,MCP-1,KIM-1,Col-I and FN in m TECs under co-culture condition.2.Under electron microscope,the two groups of exosomes showed double membrane-like structures with different shapes and diameters of 100 nm.WB results showed that CD63,TSG101 and Alix expression were positive in the two groups.BCA quantitative results showed that the release of exosomes in HG-exo group was significantly higher than that in NG-exo group.NTA results showed that the particle size distribution peak of exosomes was about 100 nm,and the concentration of HG-exo group was higher than that of NG-exo group.The results of PKH67 showed that exosome particles could be absorbed by m TECs and distributed in cytoplasm.3.WB results showed that the expression of KIM-1,Col-I and FN was the most obvious when HG-exo concentration was 60?g/m L and stimulation time was 36 h.The expression of KIM-1,Col-I and FN was increased in HG-exo group by WB and immunofluorescence.ELISA and RT-PCR results showed that the expression of inflammatory factors was significantly increased in HG-exo group.WB results showed decreased expression of LC3-II and increased accumulation of p62 in HG-exo group.Immunofluorescence results further confirmed increased expression of p62 in HG-exo group,and electron microscopic results showed decreased number of autophagosomes in HG-exo group.4.Then,the effect of HG-exo on kidney of C57 mice was observed.The results showed that there was no significant difference in kidney weight ratio among all groups,but serum creatinine and urea nitrogen levels of HG-exo group were significantly increased.HE and PAS staining showed that renal tubule injury was increased and tubule injury index was significantly increased in the HG-exo group.Rt-pcr and immunohistochemistry showed that the expression of inflammatory factors TNF-?,IL-1? and MCP-1 in the HG-exo group was increased.WB results showed that the expressions of KIM-1,Col-I,FN and p62 in the kidney of mice in the HG-exo group increased,while the expressions of LC3-II decreased.Immunohistochemical results showed that the expressions of KIM-1,Col-I,FN and p62 in the kidney of mice in the HG-exo group increased.Part II: 1.After high-throughput sequencing of exosomes released by macrophages stimulated by normal glucose and high glucose,a total of 180 mi RNAs with significant differences were found,including 49 up-regulated mi RNAs and 131 down-regulated mi RNAs;After RT-PCR verification of the top 8 up-regulated mi RNAs,mi R-7002-5p was found to be the most differentially expressed.After by KEGG pathway enrichment analysis,Atg9 b may be the target gene of mi R-7002-5p.2.The binding region of mi R-7002-5p and Atg9 b was predicted by Targetscan website;Rt-pcr results showed that after transfection of mi R-7002-5p mimic into m TECs,the level of mi RNA increased significantly,and the expression of mi RNA decreased after transfection with inhibitor.WB and RT-PCR results showed that the expression of Atg9 b in mi R-7002-5p mimic group was significantly decreased compared with NC group,and the expression of Atg9 b was increased after transfection with mi R-7002-5p inhibitor.Dual luciferase reporting results showed that the luminescence ratio of firefly luciferase and sea kidney luciferase in WT+mi R-7002-5p group was significantly decreased.3.After m TECs were stimulated by exosomes,The results of RT-PCR and fluorescence in situ hybridization showed that mi R-7002-5P expression was increased in HG-exo group,mainly distributed in cytoplasm,and mi RNA mainly distributed in renal tubules in HG-exo group.WB results showed that the expression of Atg9 b in HG-exo group was decreased.4.RT-PCR results showed that macrophages and their derived exosomal mi RNA were significantly decreased after transfection with inhibitor;ELISA and RT-PCR results showed that the expressions of TNF-?,IL-1? and MCP-1 in HG-inhib-exo group were lower than those in HG-exo group.WB results showed that the intracellular expressions of KIM-1,Col-I,FN and p62 in HG-inhib-exo group increased compared with HG-exo group,while the expressions of Atg9 b and LC3-II decreased significantly.The results of immunofluorescence showed that the expression of KIM-1,Col-I,FN and p62 increased in HG-inhib-exo group compared with HG-exo group.The results of cell electron microscopy showed that the number of intracellular autophagosomes in HG-inhib-exo group was significantly increased compared with that in HG-exo group.5.The results of animal experiment showed that there was no significant difference in renal weight ratio among all groups,while the serum creatinine and urea nitrogen levels of mice in HG-inhib-exo group were significantly lower than those in HG-exo group.Immunohistochemical and RT-PCR results showed that,Compared with HG-exo group,TNF-?,IL-1? and MCP-1 of mice renal inflammatory factors were significantly decreased in HG-inhib-exo group.HE and PAS results showed that HG-inhib-exo group had less renal injury than HG-exo group.Immunohistochemical results showed that the expressions of KIM-1,Col-I and FN in kidney of mice in HG-inhib-exo group were significantly lower than those in HG-exo group.Conclusion:1.High glucose can induce the release of exosomes derived from macrophages,and after inhibiting the release of exosomes from macrophages,it can effectively reduce the damage and inflammation of renal tubular epithelial cells in the co-culture environment.2.HG-exo can induce renal tubule injury,accumulation of extracellular matrix and increased inflammation in renal tubule epithelial cells and C57 mice,and induce its autophagy activity to decrease.The mechanism is through mi R-7002-5p/Atg9 b pathway.3.mi R-7002-5p inhibitor can reduce the autophagy inhibition of tubule epithelial cells induced by HG-exo,and reduced the injury and inflammation of tubule cells and kidney of C57 mice. |
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