The Role Of Fyn In High Glucose-induced Podocyte Injury

Objective:Diabetic nephropathy (DN) is one of the microvascular complications of diabetes mellitus (DM), and it is also an important cause of end-stage renal disease (ESRD). Progressive development of proteinuria is a major clinical manifestation of diabetic nephropathy. The glomerular visceral epithelial cell, also known as podocyte, residents on the glomerular basement membrane (GBM), is an important part of the kidney filtration barrier. In addition to the GBM and mesangial matrix proliferation, the number of podocyte decreased associated with proteinuria during diabetic nephropathy. Evidence has illustrated that the damage of podocytes is considered to be an early pathological change of diabetic nephropathy. Fyn is one of the Src family tyrosine kinases (SFKs) members, which can be activated by a variety of stimulis, mediating downstream signaling pathways in cell growth, adhesion, motility and so on. Previous studies have demonstrated that Fyn interacts with CD2AP and synaptopodin in podocytes, and Fyn can directly bind to and phosphorylated nephrin in regulating podocyte morphology and function. Whether Fyn is involved in diabetic nephropathy and podocyte injury is yet to be elucidated. This study aims to reveal the role of Fyn in high glucose-induced podocyte injury.Methods:1. Podocyte culture and treatment. Conditionally immortalized mouse podocyte cell line is a kind gift from Professor Peter Mundel (Harvard Medical School), In brief, podocytes were cultured on type I collagen in RPMI1640supplemented with10%fetal bovine serum,100U/ml penicillin, and100mg/ml streptomycin under permissive conditions33℃plus lOU/ml mouse recombinant γ-interferon for5to7days. Podocytes differentiation were induced by maintaining podocytes at37℃without γ-interferon under nonpermissive conditions for about10to14days. And to determine the effects of high glucose on podocytes, the cells were randomly applied to5different treatments:normal glucose treatment, high glucose treatment, hyperosmolar treatment, Fyn siRNA transfection, and Control siRNA transfection.2. Determination of specific podocyte markers. Real-time PCR and western blot were conducted to determine nephrin, synaptopodin, desmin and vimentin in podocytes under different conditions.3. Assessment of Fyn expression and activity and its potential role involved in high glucose-induced podocyte injury. A podocyte specific Fyn siRNA were constructed to knockdown Fyn gene expression. Podocytes transfected with Control siRNA were considered to be controls. Immunoprecipitation and western blot were used to assesse the expression and activity of Fyn (phosphor-Tyr418Fyn).Results:1. High glucose incubation impaired podocytes in vitro. It was found that30mM glucose incubation of podocytes for48hours resulted in remarkable reduction in nephrin and synaptopodin expression at both mRNA and protein levels (P<0.05), whereas the expression of desmin and vimentin were notably up-regulated (P<0.05), compared to podocytes incubated with normal glucose medium.2. Fyn was activated in podocytes incubated with high glucose. Incubation of podocytes with30mM glucose for48hours gave rise to a significant increase of Fyn Tyr418phosphorylation, which is the active form of Fyn (P<0.05), with limited alterations on total Fyn expression (P>0.05), compared to podocytes incubated with normal glucose medium.3. Knockdown of Fyn with specific Fyn siRNA ameliorated high glucose-induced podocyte injury. Compared to podocytes incubated with normal glucose medium, in podocytes pretransfected with Fyn siRNA for24hours and then incubated with30mM glucose for48hours, the expression and activation of Fyn were both significantly abrogated (P<0.05). Contemporarily, the alteration of nephrin, synaptopodin, desmin and vimentin were partially reversed (P<0.05).Conclusion:Fyn involves in mediating the high glucose-induced podocyte injury in vitro, knockdown of Fyn can partially reverse this damage.