Protective Effccts Of Vitamin D On High-Glucose-Induced Lipid Accumulation And Inflammation In Renal Tubular Epithclial Cells

Objective:Occupational and environmental harzards are risk factors of diabetes,and increase the incidence of complications in diabetic patients.Diabetic nephropathy is one of the serious complications.Renal tubular lesions are highly correlated with renal function insufficiency and prognosis.In the initiation of diabetic nephropathy,damages occur in tubular epithelial cells prior to glomerular cells.Lipid accumulation and inflammation are vital events in the progression of diabetic nephropathy.1 a,25-dihydroxyvitamin D(1 a,25(OH)2D3),the active form of vitamin D,is considered to have a protective effect on diabetic nephropathy.However,it remains unclear whether vitamin D can inhibit lipid accumulation and attenuate inflammation,and the potential mechanisms involved in are imcompletely understood.In this study,we evaluated the effects of vitamin D on lipid accumulation and inflammation in high-glucose exposed renal tubular epithelial cells.Methods:The mainly work of this study contains four sections.(1)Effects of high glucose on lipid metabolism in renal tubular epithelial cells.HK-2 cells and NRK-52E cells were treated with 30 mM glucose for 0,3,6,12,24 and 48 h.The lipid accumulation in HK-2 cells and NRK-52E cells were determined by Oil Red O staining,intracellular triglyceride and cholesterol content evaluation.(2)Effects of vitamin D on high-glucose-induced lipid accumulation in renal tubular epithelial cells.The viability of NRK-52E cells were detected by CCK-8 assay after treatment of 1?,25(OH)2D(10 nM and 100 nM)and 250HD(10 nM and 100 nM)in high glucose medium.Oil Red O staining was used to analyze the effects of 1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)on high-glucose-induced lipid accumulation.The effects of la,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)on lipid efflux was analyzed by blood biochemical analyzer.SCAP,SREBP2 and VDR were detected after 1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)treatment in high glucose medium by Western blotting(3)1?,25(OH)2D3 inhibits high-glucose-induced lipid accumulation in renal tubular epithelial cells by regulating SCAP-SREBPs pathway.After treatment of 1?,25(OH)2D3(10 nM and 100 nM)and high glucose,the apoptosis of HK-2 cells and NRK-52E cells were detected by flow cytometry,the viability of HK-2 cells and NRK-52E cells were detected by CCK-8 assay.Oil Red O staining was used to analyze the effects of 1?,25(OH)2D3 on high-glucose-induced lipid accumulation,as well as quantitative analysis of intracellular triglyceride and cholesterol.The effects of 1?,25(OH)2D3(10 nM and 100 nM)and high glucose on lipid oxidation and lipid efflux were analyzed by enzyme labeling and blood biochemical analyzer.SCAP,SREBP-1c,SREBP2,FASN and HMGCR,the lipogenic factors in SCAP-SREBPs pathway,were detected after 1?,25(OH)2D3(10 nM and 100 nM)and high glucose treatment by real-time qPCR and Western blotting,respectively.(4)Effects of 1?,25(OH)2D3 on high-glucose-induced inflammation in renal tubular epithelial cells.Real-time qPCR results showed the effects of high glucose on inflammation and the effects of 1?,25(OH)2D3(10 nM and 100 nM)on high-glucose-induced inflammatory factors,including TGF-?1 and IL-6.Results:1.(1)High glucose(30 mmol/L)enhanced lipid accumulation.(2)Lipid droplets increased in HK-2 cells and NRK-52E cells after being treated with high glucose for 24 h,and continued to increase at 48 h.The quantitative analysis of intracellular lipid droplets confirmed this results.(3)Intracellular cholesterol were significantly increased after being treated with high glucose for 24 h and 48 h.2.(1)After treatment of 1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10nM and 100nM)in high glucose medium for 48 h,the viability of NRK-52E cells did not change significantly.(2)The intracellular droplets in NRK-52E cells increased significantly after treatment of high glucose.1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)repressed high-glucose-induced lipid accumulation in NRK-52E cells.(3)1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)had no influence on lipid efflux of NRK-52E cells.The content of extracellular triglyceride and cholesterol did not change significantly.(4)The proteins related to cholesterol synthesis in NRK-52E cells increased significantly after 48 h treatment of high glucose.1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM)inhibited the expression of proteins related to cholesterol synthesis,which were induced by high glucose.The expression level of VDR in NRK-52E cells increased after treatment of 1?,25(OH)2D3(10 nM and 100 nM)and 250HD(10 nM and 100 nM).3.To investigate the potential protective effects of la,25(OH)2D3 on high-glucose-induced lipid accumulation,we incubated cells in normal or high glucose medium containing la,25(OH)2D3(10 nM and 100 nM)(1)The apoptosis of HK-2 cells and NRK-52E cells did not change significantly after 24 h treatment of 1?,25(OH)2D3(10 nM and 100 nM),high glucose and combined intervention.After 48 h treatment,the apoptosis of NRK-52E cells did not change significantly.The apoptosis of HK-2 cells of high glucose group increased significantly compared with normal control group,and 1?,25(OH)2D3(10 nM and 100 nM)relieved the apoptosis induced by high glucose.The effect of 1?,25(OH)2D3 has a dose-dependent manner.(2)After 24 and 48 h treatment of 1?,25(OH)2D3(10 nM and 100 nM),high glucose and combined intervention,the viability of HK-2 cells did not change significantly.However,the viability of NRK-52E cells decreased in 1?,25(OH)2D3(10 nM and 100 nM)and high glucose groups,compared with normal control group.1?,25(OH)2D3(10 nM and 100 nM)increased the viability which reduced by high glucose.(3)The intracellular droplets in HK-2 cells and NRK-52E cells increased significantly after treatment of la,25(OH)2D3(10 nM and 100 nM)and high glucose,as well as triglyceride and cholesterol.1?,25(OH)2D3(10 nM and 100 nM)reduced high-glucose-induced lipid accumulation in HK-2 cells and NRK-52E cells,and the effect of 1?,25(OH)2D3 has a dose-dependent manner.(4)1?,25(OH)2D3(10 nM and 100 nM),high glucose and combined intervention had no influence on lipid oxidation of HK-2 cells and NRK-52E cells.(5)la,25(OH)2D3(10 nM and 100 nM),high glucose and combined intervention had no influence on lipid efflux of HK-2 cells and NRK-52E cells.The content of extracellular triglyceride and cholesterol did not change significantly.(6)To further validate the mechanisms involved in lipid accumulation induced by high glucose,we explored the effects of high glucose and 1?,25(OH)2D3 on the expression of lipogenic factors.SCAP-SREBPs pathway,the master in regulating lipid metabolism,is activated in HK-2 cells and NRK-52E cells after 24 h and 48 h treatment of 1?,25(OH)2D3(10 nM and 100 nM)and high glucose,respectively.However,1?,25(OH)2D3(10 nM and 100 nM)inhibited the expressions of mRNA and protein related to triglyceride and cholesterol synthesis,which were induced by high glucose.4.To gain insight into the effects of 1?,25(OH)2D3 on high-glucose-induced inflammation,we evaluated the effects of 1?,25(OH)2D3(10 nM and 100 nM)on the mRNA expression of inflammatory factors.In HK-2 cells,the mRNA expression of TGF-?1 and IL-6 were increased in high glucose medium for 24 h and 48 h.,and 1?,25(OH)2D3(10 nM and 100 nM)inhibited the expressions of TGF-?1 and IL-6 induced by high glucose.Conclusions:These findings demonstrate that high glucose induces lipid accumulation and the increase of inflammatory factors in renal tubular epithelial cells.High-glucose-induced lipid accumulation is inhibited by 250HD and 1?,25(OH)2D3,and 1?,25(OH)2D3 regulates lipid metabolism by SCAP-SREBPs pathway.la,25(OH)2D3 markedly reduces high-glucose-induced inflammation.It is a new opportunity for pharmacological control of vitamin D in diabetic patients exposed to environmental toxicants.