The Protective Role Of SMN In The Pathogensis Of Acute Kidney Injury

Objective:Acute kidney injury（AKI）is a multifactorial and multiphasic renal disease characterized by a rapid decline of renal function and there are no effective prevention and treatment in clinic practice.Surival motor neuron（SMN）protein is widely expressed in many types of cells and tissues in the body.More recently,non-neuronal roles for SMN have been uncovered,such as m RNA splicing,cell apoptosis and proliferation.SMN is expressed in the kidney,however whether SMN plays a role in AKI remains unknown.Therefore,this study was design to elucidate the role of SMN in the pathogenesis of AKI.Methods:Part one: The role of SMN in the pathogenesis of renal ischemia/reperfusion injuryThe expression of SMN in the kidneys of clinical ischemic acute kidney injury（AKI）and in the mouse model of IRI were determined.We then used SMN heterozygous knockout（SMN+/-）mice and their wild-type（WT）littermates followed with I/Rinduced AKI.Serum creatinine and blood urea nitrogen were collected to evaluate renal function.HE,TUNEL,immunhistochemtry staining and Western blot analysis of the expression of cleaved caspase-3 and cleaved PARP were used to determine kidney morphology and apoptosis.In vitro,mouse renal proximal tubule epithelial cells（m TEC）were transfected with SMN small interfering RNA（si RNA）or SMN-pc DNA3 plasmid,then exposed with cobalt to induced hypoxia.The expression of SMN and cell apoptosis were evaluated.Part two: Autophagy was mediated in the protective role of SMN in the renal ischemia/reperfusion injurySMN heterozygous knockout（SMN+/-）and wild-type mice were intraperitoneally injected with or without 3-methyladenine（3-MA）before IRI.Renal tubular injuries and cell apoptosis were examined by HE and TUNEL.Autophagy were examined by the expressions of Atg5,p62,LC3 and autophagosomes in transmission electron microscope.In vitro,mouse renal proximal tubule epithelial cells were transfected with SMN small interfering RNA（si RNA）or SMN-pc DNA3 plasmid,then exposed with cobalt to induced hypoxia.Autophagy were evaluated by Western blot.Mouse renal proximal tubule epithelial cells were transfected with SMN si RNA pretreated with or withour 3-MA before Co Cl2-induced hypoxia.Cell apoptosis were examined by Western blot and flow cytometry.Part three: The role of SMN in the pathogenesis of cisplatin-induced acute kidney injuryThe expression of SMN in the mouse model of cisplatin-induced AKI post 1 day,3 days and 7 days were determined.SMN heterozygous knockout(SMN^+/-)mice and their wild-type（WT）littermates followed with cisplatin-induced AKI were conducted.Serum creatinine and blood urea nitrogen were collected to evaluate renal function.PAS,TUNEL,immunhistochemtry staining and Western blot analysis of the expression of cleaved caspase-3 and cleaved PARP were used to determine kidney morphology and apoptosis.In vitro,mouse renal proximal tubule epithelial cells（m TEC）treated with cisplatin for different dose or different time,and the SMN protein levels were examined by Western blot anlysis.m TEC were transfected with SMN small interfering RNA（si RNA）or SMN-pc DNA3 plasmid,then exposed with cisplatin to induced nephrotoxicity.Cell apoptosis were evaluated.Results:Part one: The role of SMN in the pathogenesis of renal ischemia/reperfusion injury（1）SMN levels were significantly reduced in the kidney in ischemic AKI and decreased SMN are correlated with the decline of renal function and the degree of kidney injury score.（2）We further found that the decline in renal function,tubular injury and tubular cell apoptosis were far more severe in experimental AKI in SMN^+/-mice,compared to wild-type littermates.（3）In vitro,hypoxia-induced apoptosis was enhanced by si RNA against SMN and attenuated by SMN overexpression.Part two: Autophagy was mediated in the protective role of SMN in the renal ischemia/reperfusion injury（1）Compared to WT mice,I/R increased the expressions of Atg5,p62,LC3 and autophagosomes in transmission electron microscope to a greater extent in SMN+/-mice.（2）In vitro,hypoxia-induced autophagy activation was enhanced by si RNA against SMN and attenuated by SMN overexpression.（3）Inhibition of autophagy using 3-methyladenine（3-MA）pretreatment attenuated IRI in SMN+/-mice.Hypoxia-induced apoptosis in the groups subjected to SMN-si RNA treatment was attenuated by the addition of 3-MA,while hypoxia-induced apoptosis was aggravated in control cells in the presence of 3-MA.Part three: The role of SMN in the pathogenesis of cisplatin-induced acute kidney injury（1）SMN levels were significantly reduced in the kidney in cisplatin nephrotoxicity and decreased SMN are correlated with the decline of renal function.（2）The decline in renal function,tubular injury and tubular cell apoptosis were far more severe in cisplatin-induced AKI in SMN^+/-mice,compared to wild-type littermates.（3）In vitro,cisplatin-induced apoptosis was enhanced by si RNA against SMN and attenuated by SMN plasmid overexpression.Conclusion: SMN has a significant role in the development of AKI and insufficiency of endogenous SMN could increase the susceptibility to or severity of AKI.These findings suggest a protective role of SMN in ischemic AKI,possibly by modulating the autophagy process.