The Role Of VDR On Epithelial-mesenchymal Transition Induced By High Glucose Of Mice Podocyte

Background and ObjectiveDiabetic nephropathy(diabetic nephropathy, DN) is one of the most common chronic diabetic complications and microvascular disease. It has become a major cause of end-stage renal disease(end-stage renal disease, ESRD) whose incidence is also increasing year by year. Podocytes, glomerular visceral epithelial cells,is an important part of glomerular filtration barrier which cover glomerular basement membrane. Changes in glomerular pathology of diabetic nephropathy involve basement membrane thickening and mesangial matrix proliferation. However, the change in the number of podocytes and related functions also plays a very important role in the development of diabetic nephropathy. So proteinuria in DN is inseparable with the development of podocyte transdifferentiation(EMT) and impaired barrier function.Vitamin D receptor(VDR) is a member of nuclear receptor superfamily which widely distributed in various tissues and cells, for example, liver cells, pancreatic islet cells, renal tubular epithelial cells and podocytes and so on. Vitamin D(VD)-VDR signaling system can regulate gene expression, calcium and phosphorus metabolism,cell migration and differentiation, immune response depending on the regulation of expression and activity of VDR. Studies have shown that in the patients with chronickidney disease, end stage renal disease and chronic diabetic micro-inflammatory state VDR reduced expression, but research are less about relationship between VDR and diabetic nephropathy podocyte transdifferentiation.Wnt signaling is an important pathway of mediating podocytes occuring EMT.Our early research results have also confirmed that Wnt signaling pathway molecules- glycogen synthase kinase 3β(GSK-3β) and β-catenin expression were increased in podocytes under high glucose,causing the mesenchymal cell marker protein expression increasing and promoting podocytes EMT process so that barrier function is impaired. But the mechanism is unclear how the three interact in high glucose environment.Paricalcitol(Paricalcitol) is a VDR agonist, having many aspects of protective effect on the kidneys. In this study we use VDR agonist, paricalcitol, researching the role of VDR to the expression and regulation of Wnt signaling molecules, and the protective effect of VDR to the podocyte phenotype and functional impairment caused by high glucose.MethodsWe make the conditionally immortalized mouse podocytes(MPC-5) as research objects.1.The relationship between vitamin D receptor expression levels and podocyte phenotype by using podocyte cultured in the normal glucose concentrations.The effect of VDR expression levels to podocyte phenotype: The podocytes cultured under conditions of normal glucose concentrations were divided into three groups:(1)Normal glucose concentrations group: including 5.6 mmol/L glucose;(2) scramble siRNA group: including 5.6 mmol/L glucose + 100pmol/L scramble siRNA;(3)VDRsiRNA group: including 5.6 mmol/L glucose + 100pmol/L VDRsiRNA. After36 hours,we use western blot and qRT-PCR technology to detect the expression of GSK-3β, β-catenin, podocyte marker protein podocin,nephrin and mesenchymal cell marker protein α-SMA,MMP9 in the gene and protein levels.2.Testing that VDR agonist-paricalcitol can exert an alleviated effect on EMTand barrier dysfunction induceded by high glucose.1)The role of paricalcitol on EMT induceded by high glucose:the cultured podocytes were divided into normal glucose concentration group,normal glucose concentration + DMSO group, normal glucose concentration + paricalcitol(0.1μM)group, high glucose concentration group, high glucose concentration + DMSO group and high glucose + paricalcitol(0.1μM) group. We detect the protein expression and activation of VDR,podocyte marker protein podocin,nephrin and mesenchymal cell marker protein α-SMA,MMP9.2) The role of paricalcitol on barrier dysfunction induceded by high glucose:Differentiated podocytes(5×103podocytes/well) plated on Transwell plates were serum-free starved overnight when confluent, and then was given different treatments respectively. 36 hours later, Cells were then washed twice with PBS. The upper compartment was with 0.3ml RPMI 1640 supplemented with 40 mg/ml FBS and the lower compartment was refilled with 1ml RPMI 1640 alone,then incubated for 1hours at 37℃.Total protein concentration in the lower compartment was determined using a BCA protein assay.3.The role of VDR on the expression and regulation of Wnt signaling pathway.(1) The cultured podocytes were divided into normal glucose concentration group, normal glucose concentration + DMSO group, normal glucose concentration +paricalcitol(0.1μM) group, high glucose concentration group,high glucose concentration + DMSO group and high glucose + paricalcitol(0.1μM) group. We detect the protein expression and activation of GSK-3β and β-catenin.(2) Testing that VDR involves in the podocyte transdifferentiation via GSK-3βor not.The podocytes cultured in normal glucose conditions: 1) Normal control group:medium containing 5.6mM glucose; 2) GSK-3β siRNA transfection group; 3)Scramble siRNA transfection group; 4) paricalcitol group; 5) paricalcitol + GSK-3βgroup; 6) paricalcitol + Scramble siRNA transfection group. After 36 hours, we use western blot and qRT-PCR technology to detect the expression of GSK-3β, β-catenin,podocyte marker protein podocin,nephrin and mesenchymal cell marker proteinα-SMA、MMP9 in the gene and protein levels.RESULTS1.Compared to normal glucose group,we use siRNA to interfere the expression of VDR, the protein and gene expression of VDR, podocin, nephrin reduced(P<0.05),and the expression of GSK-3β, β-catenin, α-SMA,MMP9 increased(P<0.05).2.(1) Under high glucose conditions, the protein and gene expression of VDR,podocin, nephrin reduced(P<0.05),and the expression of GSK-3β, β-catenin, α-SMA,MMP9 increased(P<0.05),after adding paricalcitol,the expression of VDR, podocin,nephrin increased(P<0.05), and the expression of GSK-3β, β-catenin, α-SMA,MMP9 decreased(P<0.05);(2)The result of albumin flow indicates that in high glucose, the albumin flow of podocytes increased(P<0.05), after adding paricalcitol,the albumin flow of podocytes decreased(P <0.05).3. We use siRNA to interfere the expression of GSK-3β in podocytes cultured in normal glucose, and then add paricalcitol, the gene and protein expression of podocyte marker protein nephrin, podocin increased(P<0.05), the gene and protein expression of mesenchymal labeled protein α-SMA, MMP9, GSK-3β and β-catenin decreased(P<0.05).CONCLUSIONS1. The lack of VDR will cause podocytes transdifferentiation.2. Paricalcitol can alleviate podocyte injury caused by high glucose.3. VDR plays a role on expression and regulation of Wnt signaling pathway molecule GSK-3β, β-catenin.