Roles Of AKAP1 In High Glucose-induced Mitochondrial Fission In Podocyte

Background and Objective:Diabetic nephropathy(DN)is one of the most common diabetic microvascular complications.It is also one of the main causes of chronic kidney disease(CKD)and end-stage renal disease(ESRD).Previous studies have confirmed that high glucose stimulation can cause podocyte injury[1];however,the molecular mechanisms of podocyte injury induced by high glucose is not clear.Recent years,the roles of aberrant podocyte mitochondrial structure and dysfunction in the occurrence and progression of DN have been widely concerned.In addition to mitochondrial dysfunction and abnormal energy metabolism,recent studies have shown that aberrant mitochondria fission and morphology caused by mitochondria dynamic changes also play an important role in the occurrence and progression of diabetes[2].It has been found that Dynamin-related protein1(Drp1)plays a crucial role in the process of podocyte mitochondrial fission.Besides,the Drp1 phosphorylation of is demonstrated to involved in mitochondrial dynamics,dysfunction and podocyte injury induced by high glucose.[3,4];however,the upstream molecular mechanism of high glucose-induced phosphorylation of Drp1 is not clear.AKAP1 is the first member of the A kinase anchoring proteins(AKAPs)family.Its amino terminus contains a protein kinase A(Protein A,PKA)binding sequence,which is involved in the second messenger cyclic adenosine monophosphate(cyclic AMP)-mediated intracellular signal transduction by binding to PKA.Studies have shown that AKAP1 expression changes are involved in the regulation of mitochondrial fission in several diseases [5];however,AKAP1 has not been reported in diabetic nephropathy,and also the down stream molecular pathway of AKAP1 is not clear.This study aims to investigate the effects of high glucose stimulation on mitochondrial morphological structure and podocyte apoptosis in cultured human podocytes.Furthermore,we evaluate the role of AKAP1 signaling pathway in this process to further explore the underline mechanism of high glucose-induced mitochondrial damage in podocytes.Methods:Part 1: Renal biopsy specimens from 31 patients with DN diagnosed by clinical renal puncture in the division of Nephrology,Renmin Hospital of Wuhan University were collected.The control group included six patients with renal neoplasm,and normal kidney tissues were obtained from these patients by renal nephrectomy.Digital images of the mitochondria in each group were obtained using transmission electron microscopy(TEM).The aspect ratio values were calculated by computer-assisted morphometric application for mitochondrial morphology quantitative analysis.Part 2: Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours,and then exposed to different glucose concentration in different time periods.The protein expressions of AKAP1 were observed by immunofluorescence.The expressions of AKAP1,Drp1 and p-Drp1(Ser637)were detected by Western blotting.Part 3: Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours.Podocytes were than divided into normal control group(5mmol/L glucose),hypertonic control group(30mmol/L mannitol+5mmol/L glucose),high glucose(35mmol/L glucose)group,and high glucose+AKAP1 si RNA group.The mitochondrial morphology of podocytes in each group was observed by TEM and Mitotracker Red staining;the length,aspect ratio and form factor values were calculated for mitochondrial morphology quantitative analysis.Apoptosis of podocytes in each group was evaluated by flow cytometry.The expression of AKAP1,Drp1,and p-Drp1 was detected by Western blotting.Results Part 1: compared with the normal group,the renal biopsy tissues from DN group showed increased podocytes foot process fusion with increased mitochondrial fission and decreased mitochondria aspect ratio(P<0.05).Part 2: AKAP1 was distributed in the podocyte cytoplasm.Compared with control group,high glucose stimulation increased the expression of AKAP1 in a time-and concentration-dependent manner.(2)High glucose stimulation increased Drp1,p-Drp1(Ser637)expression and p-Drp1(Ser637)/Drp1 in a time-and concentration-dependent manner.Part 3:(1)Compared with normal group,high-glucose induced increased mitochondrial fission with decreased aspect ratio and form factor(all P<0.05).Compared with high glucose group,transfection of AKAP1 si RNA showed decreased mitochondrial fission with increased aspect ratio and form factor(all P<0.05).There was no significant difference between hyperotonic control group and normal group.(2)Compared with normal group,high-glucose induced increased podocytes apoptosis(P<0.05).Compared with high glucose group,transfection of AKAP1 si RNA showed decreased apoptosis(P<0.05).There was no significant difference between the hypertonic control control group and the normal group.(3)Compared with normal group,high-glucose induced upregulated AKAP1,p-Drp1(Ser637),Drp1 and increased ratio of p-Drp1/Drp1(P<0.05).Compared with high glucose group,transfection of AKAP1 si RNA showed downregulated AKAP1,p-Drp1(Ser637)and p-Drp1(Ser637)/Drp1 ratio(P<0.05);however,no significant Drp1 level change was observed.There was no significant difference between hyperotonic control group and normal group.Conclusion:AKAP1 regulates podocytes mitochondrial fission by phosphorylating Dynamin related protein1(Drp1)in high glucose induced podocytes injury.