Impact Of High Glucose On Phenotype Alteration And GSK3βActivity Of Mice Podocyte Cell Line

BackgroundDiabetic nephropathy (DN) is one of the most important causes leading to end-stage renal disease. As a serious complication, DN threatens the life and health of patients with diabetes mellitus. Proteinuria is the most common clinical manifestation of DN, and it plays an important role in both severity and progress of the disease. Recently, changes of glomerular filtration barrier in structure and function, especially podocyte injury, become research focus of many scholars, because of their effects on pathogenesis and progression of proteinuria.Podocytes are specialized, terminally differentiated visceral epithelial cells that reside on the glomerular basement membrane (GBM) outside the glomerular capillaries. They play an important role in maintaining the integrity of glomerular filtration barrier. Podocytes may undergo epithelial-to-mesenchymal transition (EMT) after injury. They lose the specialized epithelial features and become dysfunction, thereby playing a role in the genesis of proteinuria. Several intracellular signal transduction pathways such as TGF-β/smad, integrin-linked kinase (ILK) and Wnt/β-catenin signaling are essential in controlling the process of EMT. As participant of these pathways, glycogen synthase kinase 3β(GSK3β) may play an important role in the process of podocyte EMT. ObjictiveTo investigate the possible mechanism of glomerular injury and proteinuria in diabetes mellitus by determining whether epithelial-mesenchymal transition (EMT) is caused by high glucose in mice podocyte. The activity of GSK3βunder high glucose condition is also studied in order to find new target and theory for the treatment of DN.MethodsUsing mice glomerular podocyte cell line as an in vitro system.1. Impact of high glucose on phenotype alteration of podocyte:(1) Impact on phenotype of podocyte under different concentration of glucose: Podocytes were incubated in RPMI 1640 medium with normal glucose (5.6 mmol/L), normal glucose+44.4mmol/L mannitol (to achieve isoosmolality) or different concentration of high glucose (12.5mmol/L,25 mmol/L,50 mmol/L).After 36h, cells were harvested for either RNA or protein. The expressions of nephrin and a-SMA were detected by both Western Blot and RT-PCR analysis.(2) Impact on phenotype of podocyte after different time treatment of high glucose: Podocytes were incubated in RPMI 1640 medium with high glucose concentration (25mmol/L) for different time (Oh,12h,24h,36h, and 48h). Then cells were harvested for either RNA or protein. The expressions of nephrin and a-SMA were detected by both Western Blot and RT-PCR analysis.2. Impact of high glucose on GSK3βactivity of podocyte and the relationship between GSK3βactivity and EMT:Grouping according to the following method:①Normal control group:Podocytes were incubated in RPMI 1640 medium with normal glucose concentration (5.6 mmol/L).②High glucose group:Podocytes were incubated in RPMI 1640 medium with high glucose concentration (25mmol/L).③Hypertonic control group:Podocytes were incubated in RPMI 1640 medium with normal glucose (5.6 mmol/L)+19.4mmol/L mannitol (to achieve isoosmolality).④High glucose and lithium chloride group: Podocytes were incubated in RPMI 1640 medium with high glucose concentration (25 mmol/L)+ 20mmol/L lithium chloride (lithium chloride could inhibit the activity of GSK3β). After 36h, cells were harvested for protein. The expressions of nephrin, a-SMA, total GSK3p,pSer9-GSK3(3 were detected by Western Blot analysis.Results1. The results of both Western blot and RT-PCR analysis indicate that:(1)After 36h treatment with high glucose, the expression of nephrin was down-regulated in a dose-dependent manner (P< 0.05), and the expression of a-SMA was up-regulated in a dose-dependent manner (P<0.05).(2) After high glucose (25mmol/L) treatment for different time, the expression of nephrin was down-regulated in a time-dependent manner (P< 0.05), and the expression of a-SMA was up-regulated in a time-dependent manner (P< 0.05)2. The results of Western blot analysis indicate that:Compared with control groups, the expressions of nephrin and pSer9-GSK3βwere decreased, while the expression of a-SMA was increased in high glucose group. The differences were statistically significant (P<0.05). Compared with high glucose group, the expression of pSer9-GSK3βwas increased in high glucose + lithium chloride group. However, the depression of nephrin and the increase of a-SMA were more significantly than high glucose group. The differences were statistically significant (P<0.05). Besides, the expression of total GSK3βhad no significant difference among groups (P>0.05)Conclusion1. High glucose could induce mice podocyte cells to undergo EMT. The expression of nephrin and a-SMA in both protein and mRNA level changed significantly in both time and dose dependent manners during the high-glucose-induced-EMT process.2. Inhibiting the activity of GSK3βwith lithium chloride, podocytes could undergo EMT more significantly, that indicated the activity of GSK3βmight be essential to maintain the normal structure and phenotype of podocytes.3. The activity of GSK3P in podocytes was increased by high glucose treatment. It might be a type of reactivity regulation of podocytes under high glucose stimulation, in order to ease EMT and maintain the normal structure and phenotype of cells.