SphK2 Promotes Diabetic Nephropathy Through The Mediation Of Renal Tubular Epithelial Mitochondrial Dysfunction

Aims:Sphingosine kinase 2（Sph K2）,as an important regulator of cell signaling,is highly expressed in the kidney.And studies have shown that Sph K2 plays crucial roles in the development of acute and chronic kidney injury.Diabetic nephropathy（DN）,as a complication of diabetes,is a leading cause of mortality in diabetic patients.The renal is the organ with the most mitochondrial expression after the heart,and the proximal tubule epithelial cells are the mitochondria enrichment sites.Recent studies have found that diabetes impair kidney mitochondrial dynamics by augmenting fission and diminishing fusion,which results in mitochondrial and renal dysfunction.However,the mechanism of mediating mitochondrial dynamics imbalance in renal tubular epithelial cells during DN formation is still unclear.The purpose of this study was to investigate the effect of proximal renal tubular epithelial cells Sph K2 on diabetic renal injury and mitochondrial dysfunction in the process of DN formation and its regulatory mechanism.Method:Construct renal tubular epithelial cell-specific knockout Sph K2 gene(Sph K2^flox/flox-ggt1-cre)diabetic mice model,and by measurement of biochemical metabolic parameters,including fasting blood glucose（FBS）,24-h albuminuria,serum creatinine and urea nitrogen,to assess the effect of renal tubular epithelial cell deficiency Sph K2 on blood glucose regulation and renal function down-regulation in diabetic mice.The pathological changes of the kidneys and the expression of kidney injury-related factors in Sph K2^flox/flox-ggt1-cre diabetic mice were observed by pathological staining（H&E,Masson,PAS）,immunohistochemistry,western blotting（WB）and quantitative real-time PCR（RT-PCR）.In vitro,observation of the effect of silencing rat renal tubular epithelial cells（NRK52E）Sph K2 gene on the high glucose-induced mitochondrial membrane potential changes and mitochondrial ROS release by cell fluorescence.And in high glucose-induced NRK52E cell damage model,cell fluorescence was used to observe the co-localization of Sph K2 and cell mitochondria.The expression of mitochondrial dynamics-related proteins in NRK52E cells with Sph K2 gene silence induced by high glucose and in the renal tissues of Sph K2^flox/flox-ggt1-cre diabetic mice were detected by WB and RT-PCR,as to clarify the regulatory mechanism of Sph K2 on mitochondrial dysfunction in renal tubular epithelial cells induced by diabetes.Results:Renal tubular epithelial cell-specific knockout Sph K2 gene improved renal function down-regulation and renal injury in diabetic mice.Sph K2^flox/flox-ggt1-cre mice remarkably inhibited the 24-h albuminuria,serum creatinine and urea nitrogen in diabetic mice induced by STZ,but had no significant effect on blood glucose regulation in diabetic mice.Meanwhile,Sph K2^flox/flox-ggt1-cre mice significantly improved glomerular hypertrophy,renal tubular damage and extracellular matrix deposition in diabetic mice,and significantly inhibited the expression of renal FN andα-SMA,and promoted E-cadherin production,but had little inhibitory effect on Kim1.High glucose induction aggravated the decrease of mitochondrial membrane potential and the release of mitochondrial ROS in NRK52E cells,and silencing of Sph K2 gene in NRK52E cells significantly improve the above situation.Meanwhile,high glucose induces direct binding of Sph K2to mitochondria in NRK52E cells.Compared with the normal glucose cultured NRK52E cells,high-glucose induction reduced the expression of mitochondrial fusion factors Mfn2 and Opa1,and increased the expression of mitochondrial fission factor Fis1,but the mitochondrial fission regulator Drp1 was not up-regulated.Interfering the expression of Sphk2 in NRK52E cells reversed the expression of Mfn2,Opa1 and Fis1,but did not affect the expression of Drp1.In addition,in vivo studies showed that renal tubular epithelial-deficient Sph K2 up-regulated the expressions of Mfn2 and Opa1in diabetic mice,while down-regulated the expression of Fis1,and had no effect on Drp1,further demonstrating that renal tubular epithelial-deficient Sph K2 plays a protective role in diabetes-induced mitochondrial dysfunction.Conclusion:In the process of DN,proximal tubular epithelial-specific deficiency of Sph K2 may play a renal protective role by inhibiting the upregulation of Fis1 and promoting the synthesis of Mfn2 and Opa1,thereby improving renal injury and fibrosis caused by diabetes-induced mitochondrial dynamic imbalance.