Dissection Of Glomerular Transcriptional Profile Derived From Patients With Diabetic Nephropathy: SrGAP2a Protects Podocvte Structure And Function

Part One:Transciptomic analysis of glomerular gene expression in type 2 diabetic nephropathyType 2 Diabetic Nephropathy(DN)is the leading cause of end stage of renal diseases in the worldwild.Though the molecular mechanisms of the DN were widely investigated,the exact molecular mechanisms of the DN were still unclear.We applied the Affymetrix(?)microarray platform(Human Transcriptome Array 2.0,HTA 2.0)tested the gene expression of the microdissected glomeruli from DN patients.There were total 61 biopsy proven DN patients were enrolled and 20 healthy as control.By using the weighted gene co-expression network analysis(WGCNA),we found that the molecular mechanisms of the DN concisely by a number of functional modules of co-expressed genes.This result indicates a sequential order of transcriptional changes in pathways of cell skeleton,immune response,metabolisms,acting in the pathogenesis and progression of the DN.Comparisons analysis of the different stages of the DN,we found that stage-specific pathways are notably changes in the different stages of DN patients.Furthermore,we identify key members(or hub genes)of different stages of DN.These genes represent novel candidates that are likely to be the key in the development and progression of DN.the results provide a valuable resource to dissect gene regulatory mechanisms underlying progression of DN.Part Two:Dissection of glomerular transcriptional profile derived from patients with diabetic nephropathy:srGAP2a protects podocyte structure and functionPodocytes are specialized epithelial cells that play a pivotal role in maintaining glomerular filtration barrier function through their interdigitated foot processes.However,the mechanisms that govern the podocyte cytoskeletal rearrangement and disruption in renal dysfunction such as diabetic nephropathy still remain unclear.Analyzing transcriptional profile of renal biopsy from diabetic nephropathy patients and control donors identified Slit-Robo GrTP activating protein 2a(srGAP2a)as one of the main 'hub'genes that is strongly associated with proteinuria and estimated glomerular filtration rate in type 2 DN patients.Based on this finding,we provide the first evidence that srGAP2a is a key regulator of the unique cytoarchitecture of podocytes and is essential for glomerular filtration.Double immunofluorescnce staining and western blot analysis revealed that human and mouse srGAP2a was primarily localized at podocytes,and primarily co-localized with synaptopodin.Podocyte srGAP2a was downregulated in DN patients and db/db mice at both mRNA and protein level.Reduction of srGAP2a was also confirmed in cultured podocytes treated with high concentration of glucose or TGF?.Functional and mechanistic studies indicate that srGAP2a suppresses the motility of podocytes through inactivating RhoA/Cdc42.The protective role of srGAP2a in podocyte function is also confirmed in zebrafish,in which knockdown of srGAP2a,a srGAP2 ortholog in zebrafish,recapitulated podocyte foot process effacement.Finally,increasing podocyte srGAP2a level in db/db mice via administration of adenovirus expressing srGAP2 significantly mitigated podocyte injury and proteinuria.Our results demonstrate that srGAP2a protects podocytes via suppressing podocyte migration.