| BackgroundAbnormal morphology and function of podocytes is one of the important causes of proteinuria and glomerular fibrosis in diabetic nephropathy(DN).Epithelial-mesenchymal transition(EMT)is a process that occurs in the early stage of renal fibrosis and is reversible to some extent.About half of the fibroblasts and myofibroblasts associated with DN kidney fibrosis are derived from transdifferentiation of epithelial cells.Previous studies have shown that EMT is a change in cell phenotype mediated by multiple molecules,and zinc finger transcription factor(Snail)is one of the important upstream molecules involved in this process,Bai et al.found that MicroRNA-130 b improved tubulointerstitial fibrosis by inhibiting EMT induced by Snail in DN.Recently,some scholars have found that high glucose environment in DN can also induce EMT in podocytes,causing glomerular basement membrane damage and even fibrosis,but its molecular mechanism is not clear.Nucleotide binding oligomerization domain-containing protein 2(NOD2)is a pattern recognition receptor,which is mainly expressed in various epithelial cells and inflammatory cells,and participates in the body's innate immune response in inflammatory response.Studies have shown that NOD2 promotes kidney damage by aggravating podocyte insulin resistance in DN.Previous studies by our group have shown that NOD2 can participate in the glomerular endothelial cell EMT process by activating the MEK/ERK signaling pathway,which suggests that it may play a key role in the progression of diabetic nephropathy.But so far,its role in podocyte phenotypic transformation has not been clear.Objective1.To investigate the effect of high glucose on the expression of NOD2,Snail and EMT-related proteins(?-SMA,Desmin,E-cadherin,Nephrin)in human glomerular podocytes.2.To investigate the regulation of NOD2 on the expression of Snail and subsequent EMT-related proteins.3.To determine whether Snail mediates the regulation of EMT-related protein expression.Methods The condition of immortalized mouse HPC was studied.1.HPC was cultured in vitro and divided into four groups: normal control group(glucose: 5.6 mmol/L),20 mmol/L high glucose group,40 mmol/L high glucose group,osmotic pressure control group(5.6 mmol/L glucose + 34.4 mmol/L Mannitol),tested in different medium for 24 h.Cellular immunofluorescence was used to detect the expression of EMT-related protein ? smooth muscle actin(?-SMA)and nephrin.Real-time quantitative PCR and Western blotting were used to detect the expression of NOD2,Snail and podocyte EMT-related proteins ?-SMA,desmin,epithelial cadherin and nephrin.2.The shRNA targeting NOD2 was constructed,and shRNA and empty vector were transfected into HPC respectively,and then stimulated by high glucose.The mice were divided into four groups: normal control group,high glucose group,high glucose+shRNA-NOD2 interference group,high glucose+ Empty vector set.The test is carried out after a certain period of cultivation.Western Blot was used to detect the expression levels of Snail,?-SMA,Desmin,E-cadherin and Nephrin.3.The NOD2-specific activator MDP was used as a stimulator,and the constructed shRNA-Snail plasmid and empty vector were transfected into HPC,respectively,into four groups: normal control group,MDP group,shRNA-Snail interference group,empty vector.group.The test is carried out after a certain period of cultivation.Western Blot was used to detect the expression levels of NOD2 and Snail proteins.4.HPC was cultured in vitro,and shRNA was added to slience the expression of Snail before MDP stimulation.HPC were divided into four groups: normal control group,MDP group,MDP+shRNA-Snail interference group,and MDP+ empty vector group.After a certain period of culture,Western Blot detected the expression levels of ?-SMA,Desmin,E-cadherin and nephrin protein.Results1.High glucose affects the morphology and phenotype of glomerular podocytes:The result of cellular immunofluorescence showed that with the increase of glucose concentration,the foot processes of the high glucose group gradually merged and fell off,and the cell gap increased,and the cell density became smaller.Compared with the normal group,the expression of nephrin was gradually decreased,and the expression of ?-SMA was gradually increased,while the osmotic pressure control group had no significant change.2.High glucose increases NOD2 and Snail expression and promotes podocyte transdifferentiation.:The results of real-time PCR and Western blot showed that the mRNA and protein expressions of NOD2 and Snail in podocytes of high glucose group were significantly increased compared with the control group;the mRNA and protein expression of epithelial phenotype-related proteins E-cadherin and Nephrin were down-regulated;The mRNA and protein expressions of interstitial phenotype-related proteins Desmin and ?-SMA were up-regulated(all P<0.05).The above changes are concentration dependent.There was no significant difference between the osmotic pressure control group and the normal control group(both P>0.05).3.Silencing the NOD2 gene inhibits Snail and podocyte transdifferentiation:After shRNA silencing NOD2 gene,Western blot results showed that the protein expression of E-cadherin and Nephrin was significantly restored in the high glucose+shRNA interference group,and the protein expressions of Snail,Desmin and ?-SMA were significantly decreased compared with the high glucose group(all P>0.05).There was no significant difference in the expression of the above proteins between the high glucose + empty carrier group and the high glucose group(all P>0.05).4.NOD2 regulates the expression of Snail:To directly verify the upstream and downstream regulatory relationships between NOD2 and Snail,the NOD2-specific activator MDP was used as a stimulator.Western blot results showed that the expression of Snail was significantly increased after activation of NOD2 compared with the normal control group(P<0.05).Under normal culture conditions,gene silencing of Snail had no significant effect on NOD2 protein expression,and there was no significant difference in empty vector histone expression(P>0.05).It is suggested that NOD2 is an upstream molecule that regulates Snail expression.5.Silencing the Snail gene attenuates NOD2-mediated podocyte transdifferentiation:After interfering with Snail expression,the podocytes were stimulated with MDP for 2 h.Western blot results showed that compared with MDP-stimulated group,the expression of E-cadherin and Nephrin was significantly increased after interfering with Snail expression;the expression of Desmin and ?-SMA was significantly decreased(all P<0.05);There was no significant difference in the expression of histones in empty vector(P>0.05).It is suggested that Snail is involved in the NOD2-induced podocyte EMT process.Conclusion1.High glucose can induce the expression of NOD2 in podocytes,and promote the transdifferentiation of podocyte epithelial mesenchyme by up-regulating Snail expression.2.Gene intervention with NOD2/Snail/EMT pathway as a target can alleviate podocyte injury induced by high glucose,which may provide new ideas for the treatment of diabetic nephropathy. |
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