The Molecular Mechanisms Of RGC-32 In Regulating Tubular Epithelial Cell Repair Through G2/M Checkpoint

Background:Acute kidney injury is a common serious disease with high morbidity and mortality in clinic and it is extremely easy to develop to chronic kidney injury.The key of preventing AKI to chronic kidney injury and reducing the mortality is to know the mechanism of renal tubular epithelial cells repair.The common cause of AKI is acute ischemia-reperfusion kidney injury?AIKI?,nephrotoxicity injury and sepsis.The pathological basis of AKI is acute tubular necrosis.It has been reported that G2/M checkpoint has an important role in the renal tubular epithelial cells repair process.G2/M checkpoint determined the outcome of renal tubular repair after injury.As one of important molecular regulating factor on cell cycle G2/M check point,Response Gene to Complement 32?RGC-32?participates in tubular epithial cell repair process.However,the molecular mechanisms underlying its regulation of G2/M check point still remains to be elucidated.Objectives:?1?To study the direct role of RGC-32 in the tubular epithial cell repair process.?2?To explore the possible mechanisms of RGC-32 regulating the G2/M checkpoint inthe tubular epithial cell repair process Methods:?1?Cultivating RGC-32 knockout mice using homologous recombination?2?AIKI and Sham group:RGC-32^-/-and RGC-32^+/+mice were randomly divided into2 groups,including AIKI group?n=48?and Sham group?n=48?.The mice of AIKI group were clip bilateral renal pedicle for 45 min,and the mice of Sham group were not clip bilateral renal pedicle.Mice were sacrificed following reperfusion 6h,24h,48h,72h,1week?w?and 2w.?3?AAN and Sham group:RGC-32^-/-and RGC-32^+/+mice were randomly divided into2 groups,including AAN group?n=48?and Sham group?n=48?.The mice of AAN group were injected of Aristolochic acid.The mice of Sham group were not.Mice were sacrificed following intraperitoneal injection of Aristolochic acid 6h,24h,48h,72h,1week?w?and 2w.?4?Differentially expressed proteins in the kidney of RGC-32 knockout mice were investigated with the isobaric tags for relative and absolute quantification?iTRAQ?technique.Gene ontology analyses?GO?,Kyoto encyclopedia of genes and genomes?KEGG?pathway mapping analysis and functional network analysis were also performed.?5?Renal tubular epithelial cells of rat?NRK-52E?cells were cultured in vitro and we made the NRK-52E cells RGC-32 expressed in either high or low expression by transient transfection.Then we induced RGC-32 normal expressed,RGC-32 high expressed and RGC-32 low expressed cell damage by TNF-??10ng/ml?.The cell cycle distribution was measured by flow cytometry,the mRNA expression of NGAL,FN,DNA-PK,?-SMA were measured by qPCR.Results:?1?The weight of 3w-5w age RGC-32^-/-mice was lower than RGC-32^+/+mice.After6w age,the weight of RGC-32^-/-mice was indiscriminate comparing with the RGC-32^+/+mice.?2?The platelet of peripheral blood was increased and the number of CD8⁺T cells wasdecreased in RGC-32^-/-mice.?3?Comparing with WT AIKI and AAN mice,the duration of the increase of serumcreatinine and KIM-1 expression of RGC-32 knockout mice was longer,theproportion of G2/M phase cells of RGC-32 knockout mice was higher and thedeposition of Collagen fibers in kidney of RGC-32^-/-mice was more.?4?Comparative proteomic analysis revealed 361 differentially expressed proteins inRGC-32^-/-mice?knockout/wild ratio>+/-1.2 and P<0.05?.The differentlyexpressed protein DNA-PKcs participated in the regulating of G2/M phase.?5?High-expressed RGC-32 in NRK-52E can reduce renal DNA-PKcs mRNAexpression decreased degree,inhibite the G2/M phase arrest and reduce theepithelial-mesenchymal transition in the process of renal tubular epithelial celldamage and repair.Low-expressed RGC-32 in NRK-52E can worsen DNA-PKcsmRNA expression decreased degree,promote the G2/M phase arrest and increasethe epithelial-mesenchymal transition in the process of renal tubular epithelial celldamage and repair.Conclusions:?1?For the regulation of G2/M checkpoint,RGC-32 had direct effect on the renal tubular epithelial cell damage and repair.?2?RGC-32 may regulate G2/M checkpoint through DNA-PKcs in the process of renal tubular epithelial cell damage and repair.