RCAN1 Regulates Mitochondrial Dynamics In Mesangial Cells And Participates In Extracellular Matrix Expression

Objective Kidney hypertrophy and accumulation of extracellular matrix proteins are the main manifestations of diabetic nephropathy,and the damage of mesangium is the core of the development of diabetic nephropathy.Previous studies in our group showed that after overexpression of RCAN1.4 in mesangial cells,mitochondrial ROS increased significantly,and promoted the accumulation of extracellular matrix proteins in mesangial cells.We speculate that RCAN 1.4 induces mitochondrial damage and participates in the up-regulation of extracellular matrix proteins in mesangial cells in diabetic nephropathy.Therefore,this study explored the effects of RCAN1.4 on mitochondrial dynamics and function in mesangial cells,and explored whether RCAN1.4 mediates the up-regulation of extracellular matrix proteins by regulating mitochondrial dynamics by using mitochondrial division inhibitor Mdivi1 and an antioxidant targeting mitochondria Mito TEMPO.The aim is to reveal the pathogenesis of diabetic nephropathy and provide a new target for the treatment of diabetic nephropathy.Methods1.Mesangial cells were treated with high glucose to observe whether the up-regulation of RCAN 1.4 protein preceded Drp1 translocation.Mesangial cells were treated with 30 mmol/L HG.The contents of RCAN1.4 and Drp1 proteins in mesangial cells were detected by Western blot.2.The effect of plasmid transfection on mitochondrial kinetics and function was observed after overexpression of RCAN1.4 in mesangial cells.RCAN1.4 was overexpressed in mesangial cells after plasmid transfection.The contents of Drp1,Mfn2 and other proteins in cytoplasm,mitochondria and whole cells were detected by Western blot.Mitotracker fluorescence staining,confocal detection of mitochondrial morphology changes,kit detection of ATP content were performed.The changes of mitochondrial ROS content and membrane potential were detected by flow cytometry.3.After down-regulation of RCAN 1.4 by RNA interference,cells were treated with HG to observe the effects on mitochondrial dynamics and function.After down-regulation of RCAN 1.4 by RNA interference,the cells were treated with HG.The protein levels of RCAN 1.4,FN,Drp1 and Mfn2 were detected by Western blot.Mitotracker fluorescence staining and confocal detection of mitochondrial morphological changes were performed.The changes of ATP content and membrane potential were detected by flow cytometry.4.To observe whether the mitochondrial dynamics changes induced by overexpression of RCAN 1.4 mediate the up-regulation of extracellular matrix proteins and the specific mechanism.After pretreatment of mesangial cells with 50 μM Mdivi1 or 30 μM Mito TEMPO,p LHCX plasmid was transfected to overexpress RCAN 1.4.Western blot was used to detect the expression of extracellular matrix proteins FN.Mitotracker fluorescence staining and confocal detection of mitochondrial morphological changes were performed.The changes of ATP content and membrane potential were detected by flow cytometry.5.The autophagy of mesangial cells was observed after plasmid transfection overexpressed RCAN 1.4 and down-regulated RCAN 1.4 by RNA interference.Western blot was used to observe the effects of plasmid transfection on the expression of LC3,p62,PINK1,Parkin and other autophagy-related proteins after over-expression of RCAN1.4 in mesangial cells and down-regulation of RCAN1.4 by RNA interference.Results1.30 mmol/L HG treatment could induce Drp1 translocation to mitochondria in mesangial cells.Although RCAN1 did not translocate to mitochondria,the increase of RCAN1.4 protein level induced by high glucose was earlier than that of Drp1 to mitochondria,suggesting that the up-regulation of RCAN1.4 is an upstream event of Drp1 translocation,suggesting that RCAN1.4 may regulate the dynamic changes of mitochondria in mesangial cells.2.After plasmid transfection,the expression of mitochondrial fission-related protein Drp1 increased significantly and the translocation of Drp1 to mitochondria increased,while the level of mitochondrial fusion-related protein Mfn2 decreased significantly.The morphology of mitochondria showed that fragmentation and the length of mitochondria decreased,which indicated that mitochondrial division increased and mitochondrial fusion decreased.Intracellular ATP decreased,mitochondrial membrane potential decreased and ROS level increased significantly.These results suggest that RCAN1.4 can induce changes in mitochondrial dynamics and mitochondrial damage in mesangial cells.3.After down-regulation of RCAN 1.4 by RNA interference and co-treatment with HG,it was found that down-regulation of RCAN 1.4 could significantly inhibit the increase of Drp1 protein induced by HG,the decrease of Mfn2 protein induced by HG,and down-regulation of RCAN 1.4 could significantly alleviate the increase of autophagy-related protein induced by HG.Down-regulation of RCAN1.4 could significantly improve ATP content and mitochondrial membrane potential.These results suggest that knocking down RCAN1.4 can inhibit mitochondrial division in mesangial cells and alleviate HG-induced mitochondrial damage.4.When mesangial cells were pretreated with mitochondrial mitochondria fission inhibitor Mdivi1 and antioxidant mito TEMPO,it was found that the up-regulation of extracellular matrix proteins FN induced by RCAN 1.4 overexpression were inhibited5.The expression of mitochondrial autophagy-related proteins LC3,p62,PINK1 and Parkin increased after RCAN 1.4 overexpression.When RCAN 1.4 was down-regulated by RNA interference and treated with HG,it was found that down-regulation of RCAN 1.4 could significantly inhibit the up-regulation of mitochondrial autophagy-related proteins LC3,p62,PINK1 and Parkin induced by HG.Conclusions1.Overexpression of RCAN1.4 in mesangial cells can induce changes in mitochondrial dynamics and increase mitochondrial division,resulting in decreased mitochondrial membrane potential,reduced ATP production.2.Overexpression of RCAN1.4 activates PINK1/Parkin-dependent mitochondrial autophagy pathway,but may result in accumulation of p62 due to blocked autophagic flow.3.RCAN1.4-induced increased mitochondrial fission is involved in the up-regulation of the expression of extracellular matrix protein FN.