Mechanism Of Kidney Senescence Induced By Mammalian Target Of Rapamycin

ObjectiveNumerous mutations increase lifespan in diverse organisms from worms to mammals.Most genes that affect longevity encode components of the target of rapamycin(TOR) pathway.In the current study,we evaluated the expression of mammalian target of rapamycin(mTOR) in senescent renal cells,the diploid human fibroblasts and aging kidneys, respectively.We also tested the hypothesis that mTOR pathway may promote cellular senescence.MethodsWistar rats at the ages of 3,12,24 months were used for this study. The renal tissues were processed for morphometric and senescence analysis.Sections were prepared and stained with periodic acid-Schiff. The sections were examined in a blinded manner,with each section evaluated twice.Renal senescence was detected byβ-galactosidae staining,mTOR mRNA levels were quantified by RT-PCR.Western blot analysis was performed to assess mTOR status.Expression and location of mTOR in kidneys were studied by immunohistochemistry.In vitro experiments,we utilized normal rat glomerular mesangial cell and the diploid human fibroblast line WI-38 cultures.The senescence-like phenotypes,including enlarged and flattened morphology,increased granularity,expression senescence-associated beta-galactosidae (SA-β-gal) and ploidy changes were performed.The expression analyses of mTOR,phosphorylated mTOR,Cyclin D1 and P21^{WAF1/CIP1/SDI1} were evaluated by RT-PCR or Western blot analysis.WI38 cells were incubated with mTOR agonist(L-Leucine) or mTOR antagonist (Rapamycin).Cell cycle progression was determined by flow cytometry using PI staining,and cellular lifespan was evaluated by passage mumber. The close connections between mTOR and P21^{WAF1/CIP1/SDI1} were detected with a confocal laser scanning microscope.ResultsSubstantial tubulointerstitial injury occurred at the age of 12 months, and significant glomerular structure alteration was observed at the age of 24 months.SA-β-gal staining increased in frequency and intensity with age.The mRNA and protein expression of mTOR in rat kidneys showed a significant increase with advancing age.mTOR staining appeared in mesangial matrix and interstitium in the 12-month-old rats.By contrast, 24-month-old animals showed a marked increase in the staining intensity of mTOR in kidneys.Levels of mTOR,p-mTOR,Cyclin D1 and P21^{WAF1/CIP1/SDI1} both rise in mesangial cells and WI-38 cells during progressive passages.However,L-Leucine increased the levels of Cyclin D1 and P21^{WAF1/CIP1/SDI1},and induced senescence-like phenotypes, including cell hypertrophy,the irreversible arrest in the G1 phase of the cell cycle and appearance of SA-β-gal activity in WI-38 cells.In contrast, blocking mTOR pathway using Rapamycin delayed cellular senescence and decreased the levels of Cyclin D1 and P21^{WAF1/CIP1/SDI1}.Conclusions:Our data suggest that mTOR may play an important role in the age-related cellular and organic alterations,mTOR pathway may potentially exert its promote cellular senescence effects through two pathways by inhibiting P21^{WAF1/CIP1/SDI1} to induce G1 cell cycle arrest as well as cell hypertrophy by increasing metabolism and protein synthesis.