The Role And Mechanism Of MAD2B In Promoting Podocyte Injury In Diabetic Nephropathy

Objective Damage and loss of podocyte is critical event in diabetic nephropathy.Podocytes are important components of the glomerular filtration barrier.As highly differentiated glomerular epithelial cells,podocytes have limited ability to repair or regenerate.Alternations in the normal structure and function of podocytes lead to glomerular filtration dysfunction,proteinuria and glomerulosclerosis,which in turn promotes the progression of diabetic nephropathy(DN).The mitotic arrest deficient like protein(MAD2B)is an important molecule in the regulation of cell cycle.Previously,our group have found that MAD2 B is abundantly expressed in podocytes.It is involved in high glucose(HG)-induced podocyte injury by regulating cell cycle.However,the exact mechanism of MAD2 B implicated in podocyte injury is still lacking.In this study,we constructed diabetic nephropathy model in vivo and in vitro to investigated the role and mechanism of MAD2 B in promoting podocyte injury in diabetic nephropathy.Our study may provide a new target for the treatment of DN.Methods 1.Construction of DN mice model: diabetes was induced by single intraperitoneal injection of high-dose streptozotocin(STZ,150 mg/kg).Blood,urine and kidney specimens of mice were retained after 12 weeks of feeding.2.Generation of podocyte-specific MAD2 B knockout mice: podocyte-specific MAD2 B knockout mice(Nphs2-Cre+/ MAD2Bfl/fl mice)were generated by using Cre-Loxp recombination system.3.Mouse glomeruli were isolated by using inactivated Dynabeads.Western blot was used to detect the levels of MAD2 B,Numb and NICD in glomeruli.4.Serum albumin,blood urea nitrogen,serum creatinine and urine creatinine levels were measured by enzymatic assays using an auto-chemistry analyzer.Urine albumin was detected using an ELISA kit.5.Immunohistochemical staining was performed to observe the distribution and expression of MAD2 B,Numb and WT1 in glomeruli.6.Immunofluorescence staining was applied to reveal the co-localization between MAD2 B,synaptopodin(a podocyte marker protein)and Numb.It is also applied to analyse the expression of nephrin(a podocyte marker protein)in DN mice.7.Periodic acid-Schiff(PAS)staining was employed to evaluate the mesangial expansion in the glomeruli.8.Transmission electron microscopy was performed to determine the GBM thickness,foot process width,and the number of foot processes per micrometer of GBM.9.Yeast two-hybrid analysis was performed to screen for proteins which interact with MAD2 B.Immunoprecipitation(Co-IP)assay was carried out to identify the binding of MAD2 B to Numb.10.In vitro,the expressions of MAD2 B,Numb,NICD and Hes-1 were detected in HG stimulated podocytes.11.In vitro,podocytes were transfected with adenovirus to induce Numb overexpression.Western blot was applied to detect the levels of Numb,LC3Ⅱ and Desmin(a podocyte damage marker)in HG-stimulated podocytes.At the same time,podocytes were transfected with RFP-GFP-LC3 lentivirus,autophagic flux was observed by fluorescence microscopy.12.F-actin staining was chosen to assess podocyte cytoskeleton arrangement.13.In vitro,podocytes were transfected with lentivirus to interfere with MAD2 B expression under HG condition.Western blot was used to analyse the levels of MAD2 B,Numb,NICD,MDM2 and active β-catenin in podocytes.After knocking down MAD2 B,podocytes were transfected with MDM2 overexpression plasmid.Western blot was performed to detect the levels of MDM2 and Numb.Results 1.MAD2 B was upregulated in the kidney from STZ-induced diabetic mice and in podocytes under high glucose condition.Podocyte-specific MAD2 B deletion alleviated proteinuria and renal injury in DN mice.2.MAD2 B negatively regulated Numb.MAD2 B interacted and co-localized with Numb in mice kidney and cultured podocytes.Numb was downregulated in podocytes under high glucose condition and in the kidneys from STZ-induced diabetic mice,while silencing MAD2 B alleviated the decreased expression of Numb in glomeruli.3.Overexpression of Numb alleviated podocyte injury.In cultured podocytes,overexpression of Numb partially reversed the upregulation of Desmin expression,the disarrangement of podocyte cytoskeleton and the reduction of podocyte autophagy level.4.MAD2 B negatively regulated Numb and thus activated Notch 1 signaling pathway.In vitro,silencing MAD2 B reversed the decline of Numb in podocytes exposed to HG,inhibited the activation of Notch 1 signaling pathway and downregulated the expressions of NICD and Hes-1.In vivo,podocyte-specific MAD2 B deletion inhibited the upregulation of NICD in the glomeruli of DN mice.5.Under HG conditions,Wnt/β-catenin signaling pathway was not involved in MAD2 Bpromoted podocyte injury.6.Under HG conditions,MAD2 B promoted the degradation of Numb by regulating MDM2.Conclusions MAD2 B negatively regulated Numb and activated Notch1 signaling pathway,the downstream pathway of Numb,resulting in podocyte injury during DN progression.In addition,MAD2 B promoted the degradation of Numb by regulating MDM2.