The Role Of MDM2 In Hyperglycemia Induced Podocyte Mitotic Catastrophe

Podocyte injury and loss are essential events of diabetic nephropathy. However, the mechanism of podocyte damage is not completely understood. It is well known that terminally differentiated cells are restricted in'post-mitosis'state and cannot enter the cell cycle to divide, otherwise the outcome is catastrophic. The death caused by abnormal mitosis is defined as mitotic catastrophe. Murine double minute 2(MDM2) is a positive regulator of cell cycle and modulates the process of mitosis through several signaling pathways. It has been proved that MDM2 expresses in renal tubules and podocytes, but little is known about its role in mediating abnormal cell cycle or mitosis in renal disease.In this current study, we take advantage of patient renal biopsies and STZ induced DN mouse model as well as conditional immortalized podocytes to investigate podocyte mitotic catastrophe in diabetic nephropathy and the role of MDM2. By this investigation, we expect to clarify the molecular mechanism of podocyte injury and depletion in the early stage of diabetic nephropathy and shed light on the exploration of its effective therapies.Part ?MDM2 is implicated in hyperglycemia induced podocyte mitotic catastropheObjective:(1) To observe the expression of MDM2 in glomeruli of DN patient and DN mouse model as well as podocyte treated by HG. (2) To investigate whether podocte undergo mitosis in hyperglycemia and the involvement of MDM2.Methods:(1) DN mouse model was built by a single intra-peritoneally STZ injection. (2) Confocal microscopy and double-labeled immunofluorescence staining was performed to detect the expression of MDM2 in glomeruli and colocalization with podocyte marker synaptopodin in DN mice. (3) The ultrastructure of podocytes in DN patients was examined by TEM method. (4) We take advantage of lentivirus transfection to knockdown the expression of MDM2 in podocyte. (5) We investigated the expression of MDM2, Aurora B and p-H3-ser10 in podocyte under HG exposure with or without MDM2 silencing by western blot. (6) Flow cytometry was performed to detect the cell cycle of podocyte under hyperglycemia with or without MDM2 silencing. (7) F-actin staining by Rhodamine-phalloidin and LDH cytoxicity assay kit was used to investigate podocyte injury.Results:(1) Protein expression of MDM2 was increased in glomeruli of DN patient and DN mouse model while immunofluorescence staining showed an increased colocalization in MDM2 and podocyte marker synaptopodin in DN mice. (2) Podocytes in abnormal mitosis were found in DN patients by TEM. Consistently, Ki67, the protein usually expressed by proliferative cells and mitosis protein marker Aurora B and p-H3-ser10 were enhanced in podocyte under HG exposure. In the meanwhile, podocyes were forced into S phase and G2/M phase by HG. (3) The protein expression of MDM2 was increased in cultured human podocytes under HG exposure. Knocking down MDM2 expression in podocytes partly reversed HG induced upregulation of Ki67, Aurora B and p-H3-ser10, and reduced the number of cells entered into G2/M phase under hyperglycemia. (5) Knocking down MDM2 expression in podocytes alleviated HG induced podocyte injury presented by abnormal F-actin arrangement and increased LDH activity in cultured cell medium.Conclusions:(1) Podocyte mitosis catastrophe exists in DN mouse and podocyte under HG exposure accompanied by MDM2 enhancement. (2) Knocking down MDM2 expression inhibits HG induced podocyte mitotic catastrophe and alleviates subsequent podocyte injury.Part ?The mechanism of MDM2 induced podocyte mitotic catastrophe under hyperglycemiaObjective:To clarify the downstream mechanism and signaling pathway involved in MDM2 induced podocyte mitotic catastrophe in hyperglycemia.Methods:(1) We use a small molecule nutlin3a to block MDM2 induced ubiquitin-mediated degradation of p53. Thus we could investigate the role of MDM2-p53 pathway in podocyte catastrophe. (2) Immunofluorescence staining and western blot was done to investigate the expression of Numb and NICD in glomeruli of DN mouse. (3) We detected the protein expression of Numb, NICD and Hes1 in HG treated podocytes with or without MDM2 silening by Westen blot. (4) To knock down NICD expression, we designed siRNA target NICD and explored the expression of mitotic markers under NICD depletion.Results:(1) In vitro, nutlin3a could upregulate the expression of p53 and MDM2, but had little influence on HG induced podocyte injury. (2) Protein expression of Numb was decreased in glomeruli of DN mouse model while NICD level was enhanced in DN mouse renal cortices. (3) Knocking down MDM2 expression could change the expression pattern of Numb, NICD and Hesl. (4) Depletion of NICD could partly reverses HG induced podocyte catastrophe by allivated Aurora B and p-H3-ser10 abundance.Conclusions:(1) MDM2 involved in HG induced podocyte catastrophe in a p53 independent way. (2) Numb-Notchl pathway is the possible downstream of MDM2 in mediating podocyte catastrophe.