| Background and ObjectivePopulation aging is an important trend of global and Chinese population.It is of great strategic significance to study the mechanism of aging.During aging process,body’s internal environment is disturbed,damaged or misfolded protein resulting in imbalance of proteostasis.The ubiquitin-proteasome system is one of the main ways to maintain proteostasis.Kidney is an important organ responsible for maintaining internal environment stability.Chronic kidney disease(CKD)patients are more prone to early aging,and aging will increase the incidence of CKD.Whether CKD can affect individual aging process by affecting remote proteostasis has not been reported.Cubilin(dCubilin)and Amnionless(dAMN)on drosophila nephrocytes are two important receptors responsible for protein reabsorption,which are highly homologous to mammalians.This study aims to explore the mechanism of premature aging in patients with CKD and potential therapeutic targets for premature aging related diseases in patients with CKD by studying the effect of dCubilin/dAMN-mediated protein reabsorption in drosophila nephtocytes on aging and its regulatory mechanism.MethodsWild type(WT)flies were used as control groupwas used as the control group.dCubilin-RNAi and dAMN-RNAi knockdown drosophila were obtained by RNAi method.Using USA-Gal4 system,dAMN overexpresssion(dAMN OE)drosophila was obtained by hybridization between sp/cyo;U-CG11592/TM3 male drosophila and PH,PMAR;Dot-Gal4 virgin drosophlia.Drosophila of the above four genotypes were tested as follows:1.Coomassie blue staining and red fluorescence quantitative analysis in nephrocytes were used to to determine the changes of protein reabsorption in nephrocytes and total protein in the haemolymph of four genotypes of drosophila with aging.2.Survival analysis was used to determine the influence of different genotypes on the lifespan of drosophila.3.Negative geotaxis test was performed to determine the climbing ability of different genotypes of drosophila.4.The influence of different genotypes on drosophila head aging process was detected by head HE staining,ubiquitinated protein immunofluorescence staining and Western blotting.5.Immunofluorescence staining and Western blotting were used to detect the expression of ubiquitinated proteins in muscle tissues of different genotypes of drosophila.6.The activity of proteasome in whole body,head and muscle of different genotypes of drosophila were detected by fluorescence absorption intensity assay.Results(1)With aging,the total amount of protein in haemolymph in WT group increased;the reabsorption of proteins in the nephrocytes in the dCubilin and dAMN knockdown groups decreased,the total amount of protein in haemolymph increased significantly compared with the control group,while the dAMN-OE group was the opposite.(2)The lifespan of dCubilin and dAMN RNAi knockdown drosophila decreased significantly compared with the control group,and the lifespan of dAMN-OE group significantly increased compared with the control group.(3)The climbing ability of dCubilin and dAMN RNAi knockdown drosophila decreased significantly compared with the control group,and the climbing ability of dAMN-OE drosophila increased significantly compared with the control group.(4)With aging,the number of head vacuoles and the percentage of vacuoles(d>50μm)decreased in the brain of dCubilin and dAMN RNAi knockdown drosophila;while the number of vacuoles and the percentage of vacuoles(d>50μm)increased in the brain of dAMN-OE group;the expression of ubiquitinated protein in the brain of dCubilin and dAMN RNAi knockdown increased,while the expression in dAMN-OE group decreased.(5)With aging,the expression of ubiquitinated protein in muscle tissue of dCubilin knockdown and dAMN knockdown group increased significantly,while the expression of dAMN-OE group decreased significantly.With aging,the proteasome activities of whole body,head and muscle tissue of dCubilin and dAMN RNAi knockdown decreased,while the proteasome activities of whole body,head and muscle tissue of dAMN-OE increased significantly.ConclusiondCubilin/dAMN-mediated protein reabsorption in drosophila nephrocytes can affect proteostasis in haemolymph,head and muscle tissue of drosophila through the ubiquitin-proteasome pathway,and affect the climbing ability and longevity of drosophila,thus affecting individual aging process. |
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