Protective Effect Of Numb On Acute Kidney Injury By Inhibiting Mitochondrial Fragmentation

Background Acute kidney injury(AKI)is a common clinical entity that is associated with high mortality and morbidity.AKI is an independent risk factor for chronic kidney disease and end-stage renal disease.Numb is a multifunctional protein involved in diverse cellular processes.Our previous study showed that Numb is expressed in proximal tubules,depletion Numb from proximal tubular cells attenuated interstitial fibrosis.But,the role of Numb in AKI remains unknown.The aim of the present study is to investigate the role and the mechanism of Numb in AKI.Method we constructed AKI model induced by cisplatin and ischemic/reperfusion(IRI)on PT-Numb-WT mice and PT-Numb-KO mice.Renal function was evaluated by the level of serum creatinine(Scr)and BUN.Renal morphology was examined by Hematoxylin and eosin staining.The apoptosis of tubules was tested by TUNEL assay,flow cytometry and western blotting.The mitochondrial morphology was assessed by tissue electronic microscopy(TEM)and immunofluorescence staining.The mitochondrial damage was evaluated by the release of Cytochrome c(Cyt c).Protein level was examed by Western blot.Result In vivo,we found that renal Numb protein level was significantly upregulated in mouse model of AKI induced by Cisplatin and IRI.Numb deletion specifically from proximal tubules exacerbated AKI induced by cisplatin or IRI.As showned by increased Scr,BUN and more severe morphological damage.Ablation Numb promotes renal Cyt c release and consequent apoptosis in mouse model of AKI.TEM showed that there was more mitochondrial fragmentation in PT-Numb-KO mice,but also severe mitochondrial cristae injury and tubule injury.Western blot demonstrated that loss of Numb promoted renal Drp1 expression,a key mitochondrial fission protein.Furthermore,we proved that pretreatment of mdivi-1,a newly identified pharmacological inhibitor of Drp1,could ameliorate mitochondrial fragmentation、release of Cyt c and consequent apoptosis induced by cisplatin in PT-Numb-KO mice.In vitro,flow cytometric analysis demonstrated that Numb deficiency dramatically increased cisplatin-induced apoptosis in NRK-52E cells.Similarly,compared with scramble siRNA transfected cells,mitochondrial fragmentation and Cyt c release were more severe in Numb siRNA transfected NRK-52E cells after cisplatin treatment.Western blot analysis revealed that silencing Numb stabilized Drp1 from degradation.Moreover,pretreatment with mdivi-1,attenuated mitochondrial fission and cisplatin-induced apoptosis in Numb siRNA trasfected NRK-52E cells.Conclusion Our data suggest that Numb plays a protective role in acute kidney injury by suppressing mitochondrial fragmentation and the subsequent tubular cell apoptosis.