SrGAP3 Deficiency Mediates Podocyte Injury By Promoting Cdc42 And Rac1 Activity In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is one of the most serious chronic complications of diabetes.Podocyte injury is the central target of the development of DN.Previous studies have found that Rho GTPase activity balance is crucial to the development of podocyte and glomerular filtration barrier function.Rho GTPase activation plays a key role in cytoskeletal rearrangement and apoptosis of podocytes in DN.Slit-Robo Rho GTPase Activating Protein 3(SrGAP3),a Rho GTPase activating protein,inactivates the downstream Rac1 or Cdc42 by interacting with the Robo family in neurons,as a positive regulator of neurite growth.Previous studies have also shown that SrGAP3 is expressed in renal tissue.Therefore,SrGAP3 may be involved in the progression of DN by regulating Racl or Cdc42 activity.ObjectThis study aimed to verify the expression of SrGAP3 in kidney and its role in podocyte injury of DN,and to further explore the mechanism of SrGAP3 deficiency inducing podocyte injury of DN by the activating of Cdc42 and Racl.MethodsImmunofluorescence was used to detect SrGAP3 expression in podocytes of renal tissue from DN patients and DN animal models.Overexpressed SrGAP3 in renal tissues of DN mice were constructed with adenovirus overexpression of SrGAP3,and the expression efficiency of SrGAP3 was verified by immunofluorescence.The effect of SrGAP3 on the podocyte injury and renal structure of DN mice was observed by western blotting,transmssion electron microscopy and PAS staining.In vitro,with the stimulation of high glucose,cultured podocytes were treated with siRNA silencing and adenovirus overexpression of SrGAP3 to observe podocyte injury,including the expressions of SrGAP3,podocyte marker proteins,cytoskeleton,cell motility and apoptosis.DN mice overexpressing SrGAP3 were established by adenoassociated virus(AAV).The expressions of Cdc42 and Racl were detected by irnmunofluorescence.The expressions and activities(GTP)of Cdc42 and Racl in podocytes with silenced and overexpressed SrGAP3 were determined by western blotting and enzyme activity detection.The western blotting,F-actin staining,scratch assay and flow cytometry were used to detect changes in podocyte marker proteins,cytoskeleton,activity and apoptosis in the rescue experiment.ResultsSrGAP3 was expressed in glomerular podocytes of human and mouse renal tissues,and down-regulated in podocytes of DN patients and DN mice.Overexpression of SrGAP3 in DN mice reduced urinary protein excretion rate,reduced foot process fusion,basement membrane thickening and mesangial matrix proliferation,and also restored the protein expression of podocin,a marker of podocyte in DN mice.Overexpression of SrGAP3 in podocytes cultured by HG can improve the expression of marker proteins nephrin and synaptopodin,inhibit F-actin skeleton rearrangement,reduce motility and apoptosis.The expressions of Cdc42 and Rac1 in podocytes of renal tissues from DN mice were increased and the expressions of GTP-Cdc42 and GTP-Racl were decreased after SrGAP3 was overexpressed in these mice,the increased activity of Cdc42 and Racl in HG cultured podocyte in vitro were inhibited by SrGAP3 overexpression.Furthermore,inhibiting Cdc42 and Rac1 activities could restore the silenced SrGAP3-mediated decreased expression of podocyte marker protein,inhibit F-actin skeleton rearrangement,and reduce motility and apoptosis.ConclusionsSrGAP3 was expressed in podocytes and was low in DN patients.SrGAP3 deficiency mediates DN podocyte injury by promoting Cdc42 and Rac1 activation,which may provide a clinical therapeutic target for protecting podocyte injury of DN.