RCAN1.4 Mediates High Glucose-induced Mitochondrial Fission Through The Phosphorylation Of Drp1 S616/S637

Objective Renal fibrosis is one of the important pathological changes of diabetic kidney disease(DKD),which is mainly manifested in the increase of fibroblasts and the excessive accumulation of extracellular matrix.However,it is unclear how mitochondrial fission affects the accumulation of extracellular matrix.The previous results of our research group found that overexpression of RCAN1.4 in mesangial cells can induce Drp1 translocation and mitochondrial fragmentation,and promote the accumulation of extracellular matrix(ECM)proteins,but the specific mechanism is still unclear.The literature indicates that Drp1 phosphorylation plays an important role in its translocation,and the main sites are Ser616 and Ser637.Therefore,this study explored the role of RCAN1.4 on the mitochondrial fission protein Drp1S616/S637 phosphorylation and whether RCAN1.4 regulates Drp1 phosphorylation by affecting Ca N activity,and explore whether RCAN1.4 regulates the phosphorylation of Drp1 S616/S637 through AMPK and ROCK1 signaling pathways,change the mitochondrial dynamics,thereby affect the expression of extracellular matrix proteins and promote renal fibrosis.Methods1.Treatment of HK-2 and mesangial cells with high glucose to observe the changes of Drp1 S616/S637 phosphorylation level:HK-2 and mesangial cells were treated with 30mmol/L high glucose for 6h,12 h,24h,36 h,and 48 h,respectively.The changes of Drp1 S616/S637 phos-phorylated protein levels were detected by Western blot at various time points.2.Observe the effect of RCAN1.4 overexpression on the phosphorylation of Drp1S616/S637 in HK-2 and mesangial cells:The transfected plasmid over-expressed RCAN1.4 in HK-2 and mesangial cells,respectively.Western blot was used to detect the changes of p Drp1 S616/S637 protein levels at various time points,to observe the effect of RCAN1.4 overexpression on Drp1 S616 / S637 phosphorylation.3.Observe whether Ca N is involved in mitochondrial division:The cells were pre-treated with Cs A or FK506 and treated with HG.Western blot was used to detect the expression levels of RCAN1 and extracellular matrix protein FN.Cells were pretreated with Cs A or FK506,and then overexpressed RCAN1.4.Confocal observation of mitochondrial morphology,the kit to detect changes in ATP content and calcineurin activity,Western blot detection of Drp1,Mfn2 protein expression.After transfection of plasmid RCAN1.4 for 6h,HG was added to co-treat the cells,and the kit was used to detect calcineurin activity.4.Observe whether AMPK and ROCK1 participate in the regulation of Drp1 phosphorylation by RCAN1.4:HG was used to treat HK-2 and mesangial cells respectively,and Western blot was used to detect the expression of p AMPK T172 protein.After transfection of plasmids and overexpression of RCAN1.4 in the cells,the expression levels of p AMPK T172 and p MYPT1 T696 protein were detected by Western blot.The cells were pretreated with AMPK activator AICAR or ROCK1 inhibitor Y27632,and then treated with HG or overexpressed RCAN1.4.The protein expression levels of FN and p Drp1 S616 / S637 were detected by Western blot.Results1.High glucose treatment can induce phosphorylation of Drp1 S616/S637 in both HK-2 and mesangial cells.2.Plasmid transfection for over-expressing RCAN1.4 in HK-2 and mesangial cells results the phosphorylation of Drp1 S616/S637.3.Ca N inhibitors Cs A and FK506 can reduce the up-regulation of HG-induced RCAN1.4 and extracellular matrix protein FN,and can also reduce the mitochondrial damage induced by RCAN1.4 overexpression,but the overexpression of HG and RCAN1.4 on Ca N Activity has different effects.4.In HK-2 and mesangial cells,HG treatment reduced the protein expression of p AMPK T172.After overexpression of RCAN1.4,the protein expression level of p AMPK T172 was down-regulated,and the protein level of p MYPT1 T696 was up-regulated.After AICAR or Y27632 pretreated the cells,and then added HG treatment,it was found that both can reduce the HG-induced FN upregulation.Whether it is HG treatment or overexpression of RCAN1.4,AICAR pretreatment can reduce p Drp1 S616 / S637 protein expression,while Y27632 pretreatment can only reduce p Drp1 S637 protein expressionConclusions1.RCAN1.4 participates in HG-induced phosphorylation of Drp1 S616 and S637 sites in both HK-2 and mesangial cells.Inhibition of Drp1 phosphorylation can reduce mitochondrial fragmentation and upregulation of extracellular matrix protein FN.2.The process of mitochondrial division induced by RCAN1.4 may not depend on Ca N activity.3.Over-expression of RCAN1.4 increases the phosphorylation of Drp1 S616 / S637 by inhibiting AMPK and activating ROCK1 pathway,then promotes the transfer of Drp1 to mitochondria,resulting in mitochondrial division and increased extracellular matrix proteins.