Empagliflozin Improves Diabetic Renal Tubular Injury By Alleviating Mitochondrial Fission Via AMPK/SP1/PGAM5 Pathway

Objective:To verify the role of PGAM5 in mitochondrial fission and the development of diabetic nephropathy and to explore the possible mechanism of Empa in improving diabetic nephropathy.Method:It was divided into two parts in our study:in vivo and in vitro test.1.Thirty 8-week-old KK-Ay mice were randomly divided into two groups(1)KK-Ay group:untreated type 2 diabetic KK-Ay mice(n=15);(2)KK-Ay+Empa group:KK-Ay mice were given Empagliflozin(3.8mg/kg/D)(n=15).In addition,healthy C57BL/6J mice of the same age were set as the control group,n=(15),and all the three groups of mice were fed for 8 weeks.2.At the end of the experiment,the body weight,blood glucose,serum insulin,HbAlc and urinary NAG,urinary NGAL and 24-hour urinary microalbumin of mice were measured;the HE staining,cell apoptosis was detected.We also detected the expression of PGAM5,AMPK,SP1 and mitochondrial fission related proteins in diabetic kidney to test the mechanism of Empa's improving effect preliminarily in diabetic tubular injury.3.In vitro,human renal proximal tubular epithelial cells were divided into four groups:normal glucose group(NG),hypertonic control group(MA),high glucose group(HG)and high glucose+Empa group(HG+Empa).Mito-tracker staining was used to observe the morphological changes of mitochondria,JC-1 staining was used to detect the changes of mitochondrial membrane potential.4.Western Blot was used to detect the changes of cell apoptosis,mitochondrial dynamics related proteins,p-AMPK,SP1 and PGAM5,respectively.5.We also introduced the AMPK activator,AMPK inhibitor and Empa to testify the mechanism of Empa's protective effect in renal tubular injury through under high glucose conditions;we also adopted RNA interference technology to detect the effect of SP1 and PGAM5 on mitochondrial dynamics and apoptosis in diabetic renal tubular injury.6.Finally,the interaction between SP1 and PGAM5 promoter was verified by double Luciferase Report test and CHIP test.Result:1.Compared with the normal control group,the KK-Ay mice not only had larger kidney volume,but also had dilated renal tubules and vacuolated degeneration which was detected by HE staining.The markers of renal injury NAG,NGAL and urinary microalbumin increased and the expression of BAX and Cleaved Caspase 3 significantly increased and BCL-2 and Mito-CYT decreased in KK-Ay mice.The administration of Empa could not only alleviated the renal tubule dilation and tubular injury,but also reverse the change on expression of BAX,BCL-2,Cleaved Caspase 3 and Mito-CYT.2.The expression of DRP1S637 decreased and the Mito-DRP1 increased in the kidney of KK-Ay mice,which suggested an increased mitochondrial fission.The change of expression in DRP1S637 and Mito-DRP1 was reversed in Empa treated KK-Ay mice.The expression of p-AMPK decreased and the expression of SP1 and PGAM5 increased in the kidney of KK-Ay mice;the change of expression in p-AMPK,SP1 and PGAM5 were reversed in Empa treated diabetic mice.3.The expression of BAX,Cleaved-Caspase 3 increased and the BCL-2,Mito-CYT decreased in HK-2 cells cultured with high glucose.The expression of BAX,Cleaved-caspase was decreased and the expression of BCL-2 and Mito-CYT was increased after Empa was added,suggesting that cell apoptosis was decreased by the Empa administration.Mito-tracker staining suggested that the mitochondrial fission decreased and the JC-1 staining suggested that mitochondrial membrane potential increased in high glucose cultured and Empa treated HK-2 cells.4.The expression of DRP1S637 decreased and Mito-DRP1 increased,along with the Mito-tracker and JC-1 staining showed that the mitochondrial fission was aggravated in HK-2 cells cultured with high glucose;After Empa was added,the expression of DRP1S637 increased and the expression of Mito-DRP1 decreased,along with the Mito-tracker and JC-1 staining showed that the mitochondrial fission was alleviated.The expression of p-AMPK decreased in HK-2 cells cultured with high glucose,and the expression of SP1 and PGAM5 increased significantly;after Empa administration,the expression of p-AMPK increased and the SP1 and PGAM5 increased significantly.5.Adding AMPK activator AICAR to HK-2 cells cultured in high glucose can increase the expression of DRP1S637.Meanwhile,the p-AMPK expression is increased and the SPland PGAM5 expression is decreased by AICAR administration;when adding AMPK inhibitor Compound C to HK-2 cells cultured in normal glucose,the expression of DRP1S637 decreased.The expression of p-AMPK was decreased and the expression of SP1 and PGAM5 was increased by Compound C;finally,when we added compound C to the high glucose cultured HK-2 cells,the effect of Empa on the expression of DRP1S637,p-AMPK,SP1 and PGAM5 disappeared.Both si-SP1 and si-PGAM5 can increase the expression of DRP1S637 in HK-2 cells cultured with high glucose;si-SP1 can also reduce the expression of PGAM5 in high glucose treated HK-2 cells,but si-PGAM5 has no effect on SP1 expression;in addition,neither si-SP1 or si-PGAM5 have effect on the expression of p-AMPK;6.The double Luciferase Report System suggested that the 593 site of PGAM5 promoter region was the main site to bind with the transcription factor SP1 and play an important role in the course of transcription;Chip assay suggested that transcription factor SP1 could bind to PGAM5 promoter sequence and regulate the transcription of PGAM5.Conclusion:The present study depicted an AMPK regulated and PGAM5 involved mitochondrial fission pathway in the development of diabetic tubular disease.Our finding revealed a new prospective therapeutic target for DKD.