Impact Of GSK-3β On Epithelial-mesenchymal Transition Of Mice Podocyte In Highglucose Condition

BackgroundDiabetic nephropathy (DN) is one of the frequent microvascular complications for patients with diabetes, and is also an important cause for end-stage renal disease (ESRD). Albuminuria is the most common clinical manifestation of DN and closely correlates with severity and progressive of disease,so the major job for diabetic nephropathy treatment is to control proteinuria.Podocyte is a major component of glomerulus filtration barrier, and its structural and functional changes exert critical role in development of DN albuminuria. However, whether podocyte appears epithelial-mesenchymal transition in high glucose condition and what the role of EMT is in proteinuria remains unclear.According to the present studies, there are three signal pathways mediating the epithelial-mesenchymal transition of podocyte, i.e. TGF-β/smad, integrin/ILK and Wnt/β-catenin pathway, as a widely existent serine/threonine AMPK in eukaryot,glycogen synthase kinase3β (GSK-3β) participant in the above pathways and may exert important role in podocyte mesenchymal transdifferentiation.Inorganic lithium ion (lithium chloride used in this paper) is a medicine for mania and depression.Nowadays lithium has become a useful research tool for broad study of biological functions of GSK-3β. With the effect of lithium, phenotypic and functional changes to podocyte are researched after the inhibition of GSK-3β,this may be beneficial for the clinical treatment of diabetic nephropathy.ObjectiveThe experiment is intended to study the action of GSK-3β in podocyte transdifferentiation by observing the changes of phenotype and function of podocytes, activity and expression of GSK-3β in high glucose condition.MethodsConditionally immortalized mouse podocytes cell line in vitro were adopted.1. Impact of high glucose on phenotype and function alteration of podocyte.(1) Impact on phenotype of podocytes under different concentration of glucose: Podocytes were incubated in RPMI-1640medium with normal glucose (5.6mmol/L), normal glucose+44.4mmol/L mannitol (to achieve isoosmolality) or different concentration of high glucose (12.5mmol/L,25mmol/L,50mmol/L).After36h, cells were harvested for or protein. The expressions of nephrin,podocin and α-SMA were detected by both western-blot and indirect immunofluorescence analysis.(2) To observe whether podocytes that has had phenotypic changes in high glucose condition appear dysfunction:For the permeability assay, a modification of a previously published protocol was adopted. Differentiated podocytes (3x105podocytes/well) plated on12-well Transwell plates (3μm pore; Corning, America) were serum-free starved overnight when confluent, and then was given different treatments respectively.24or36hours later, Cells were then washed twice with PBS supplemented with1mM each MgCl2and CaCl2. The upper compartment was refilled with0.25ml RPMI1640alone and the lower compartment with0.5ml RPMI1640supplemented with40mg/ml FBS and incubated for2hours at37℃. Total protein concentration in the upper compartment was determined using a BCA protein assay.2. Detection of GSK-3β kinase activity and the expression of protein(1) Podocytes were cultured as described above, and, after differentiation in6-well plates, the culture medium was replaced with serum-free RPMI-1640with normal (5.6mM d-glucose) or high glucose (12.5mM,25mM or50mM Glu) for36h. Immunoblotting was performed to detect the expression of GSK-3β and the phosphorylation sites of it. Color was developed on the BCIP/NBT color development kit.(2) Podocytes were treated in accordance with instructions of GSK-3β activity assay kit (American Genmed Scientific). Various reagents were added successively. OD value of each group of samples was detected on spectrophotometer and GSK-3β activity was calculated in accordance with the formula given in instructions of assay kit.3. Phenotypic and functional changes of podocytes after GSK-3p was disturbed by siRNA.Based on the GSK-3p full-length gene (Gene Bank NO.NM-019827.6) of mice provided by Gene Bank database, target sequence mRNA designed by Shanghai Genechem Co., Ltd. was (5’-3’), CCACTCAAGAACTGTCAAGTA., while the sequence of negative control GSK-3βsiRNA mRNA(5’-3’) was UUCUCCGAACGUGUCACGUtt. Podocytes were transfected for36h in GSK-3βsiRNA of30nmol concentration with3μl transfection reagent LipofectamineTM2000per well,then we detect the expression of nephrin,podocin, α-SMA,GSK-3βand the albumin influx using Transwell.4. Relationship between GSK-3β activity change and podocyte transdifferentiation in high glucose conditionIn order to confirm whether GSK-3β participates in the transdifferentiation process of podocyte in high glucose condition, we use Licl, a kind of classic GSK-3β activity inhibitor to observe the change of podocyte phenotype and function after GSK-3β activity is inhibited. The experimental groups were divided into normal glucose contrast group (5.6mmol/L Glu), normal glucose+Licl group, high glucose group (25mmol/L Glu) and high glucose+Licl group to test the marker protein of podocyte and the change of monolayer barrier function. Results1.The results of both western-blot and inderect immunofluorescence stain analysis indicate that:(1)after36h treatment with high glucose, the expression of nephrin and podocin was down-regulated in a dose-dependent manner(P<0.05), and the expression of α-SMA and fibronect was up-regulated in a dose-dependent manner(P<0.05)(2) it was found that HG12.5group and HG25group had increased albumin inflow compared with NG group and the difference had statistical significance(P<0.05), and HG50group had increased albumin inflow compared with HG12.5group and the difference had statistical significance (P<0.05).2.(1) The results of western-blot analysis indicate that:compared with control groups, the expressions of GSK-3β and pTry216GSK-3β were incresed, while the expressions of pSer9GSK-3β were decreased in high glucose group. The differences were statistically significant(P<0.05)..(2) GSK-3β kinase activity kit was used to detect GSK-3β activity in groups of podocytes. It was found that if compared with normal glucose concentration group, the high glucose treatment group had increased total GSK-3β expression level.3.Compared with negative control group, podocytes treated with GSK-3βsiRNA expressed more nephrin and podocin while the expression level of α-SMA decreased and monolayer barrier function was improved.4. GSK-3β participates in epithelial-mesenchymal transition of podocyte in high glucose condition, Licl can improve the monolayer barrier function of podocytes in some condition.Conclusion1. High glucose could induce mice podocyte cells to undergo EMT.2. GSK-3β participates in epithelial-mesenchymal transition of podocyte in high glucose condition.3. Licl can reverse the EMT of podocytes and improve the monolayer barrier function of podocytes to some degree.