| ObjectiveKidney is a major senescent organ. Glomerular filtration rate falls progressively with increasing age,especially after 45 years old. Accompaniment that older people is the high risk group of chronic kidney disease and the morbidity rate increases gradually, it is much more important to investigate mechanisms responsible for the senescent kidney and to protect the function of kiney aging. Study of kidney aging mechanism and prevention methods are hot spots that scholars have paid close attension.The molecular basis of age-related changes in the kidney is unknown. However, organ aging may reflect aspects of cellular senescence. Glomerular mesangial cells (GMCs) are the most important cells of the kidney.Changes in the phenotype and func-tion of GMCs must produce considerable effect. Studies have revealed that angiotensinⅡ(AngⅡ), tert-butly hydroperoxide(tBHP) and high glucose can induce cells aging, especially focused on umbilical vein vascular endothelial cells,lung fibroblast cells, peritoneal mesothelium cells and vascular smooth muscle cells.But there is no report about their relationship with human glomerular mesangial cells(HGMC). The signal transducers and activators of transcription (STATs) are proteins that play a pivotal role in transmitting cytokine signals. It palys role in myocardial cells, macrophage,T cells aging process.To date, nothing is known about the relationship between aging human GMCs and STAT protein. AngⅡ, tBHP and high glucose were used to stimulate HGMC to induce cells aging.Activity of JAK2/STAT pathway and STAT proteins were examined in aging HGMC. Activity of JAK2/STAT pathway and STAT proteins were also examined after losartan,probucol and AG490 used to investigate their function in delaying GMC aging.Methods1. Selection of factors inducing HGMC aging(1)Cells were stimulated with 10-8,10-7,10-6,10-5,10-4molL-1AngⅡfor 24,48,72,96 h; (2) Cells were submitted to 1h stress per day for 5 day under 10,30,50,70, 100μmolL-1 tBHP; (3)Cells were exposed to10,20,30,40,50mmolL-1 glucose for 24,48, 72,96,120h;(4) Cells in control group were cultured in mesangial cell medium without stimulating factor. Then senescence associatedβ-galactosidase(SAP-gal) staining, cell viability and cell cycle analysis were investigated.That 80% cells were SAP-gal positive and 80% cells were in Go-G1 phase was used to assess cells aging. Then the most suitable concentration and times that they can induce cells aging were identified. Morphologic and transmission electron microscopy changes of aging HGMCs were also examined to test whether cells were in senescent stage.2. Examination of activity of JAK2/STAT pathway and STAT proteins during HGMC agingOptimization factors were used to induce HGMC aging. Groups including control group and aging groups. Control group (without stimulating factor). Aging groups: AngⅡ-induced group(10-6molL-1AngⅡfor 72h), tBHP-induced group(30μmolL-1 tBHP 1h stress per day for 5 day), high glucose-induced group (30mmolL-1 glucose for 96h).Expression of STAT1, pSTATl,STAT3 and pSTAT3 was analyzed by Western blot analysis and immunofluorescence staining to investigate changes and function of STAT proteins during HGMC aging.3.Effect of losartan, probucol and AG490 on the activity of JAK2/STAT pathway and STAT proteins during HGMC agingLosartan, probucol, AG490 were added ahead,then HGMCs were stimulated by factors to induce cells aging. Cell viability, SAβ-gal staining,cell cycle analysis, morphologic and transmission electron microscopy,expression of STAT1, pSTATl,STAT3 and pSTAT3 were examined to investigate their function in delaying GMCs senescence. Groups including AngⅡ-related groups and tBHP-related groups (1)AngⅡ-related groups:Control group(without AngⅡ); AngⅡ-induced group(10-6 molL-1AngⅡ),Losartan(10-5molL-1)+AngⅡ-stimulated group, AG490(10μmolL-1)+ AngⅡ-stimulated group. (2)tBHP-related groups:Control group (without tBHP), tBHP-induced group (30μmolL-1tBHP), Probucol(40μmolL-1probucol)+tBHP-stimul-ated, AG490(10μmolL-1)+tBHP-simulated group. Results1.Selection of factors inducing HGMC agingPromoting aging function of AngⅡ:AngⅡstimulated HGMC proliferation during 0-48h,but suppressed cells proliferation after 72h. After 72h of incubation with 10-7,10-6,10-5molL-1AngⅡ, cell survival rate was 94.34±5.16%,81.09±4.52%, 59.13±3.12%;SAP-gal staining was 23.03±0.91%,81.23±6.71%,92.10±6.48%; G0-G1 phase percent was 72.33±3.83%,90.01±5.62%,95.32±6.03%.Promoting aging function of tBHP:After stimulated with 1 stress per day for 5 day under10,30,50μmolL-1tBHP, cell survival rate was 96.03±5.38%,80.12±4.05 %,62.16±3.01%;SAβ-gal staining was 25.22±1.23%,84.52±5.86%,90.30±6.25%; G0-G1 phase percent was72.33±3.83%,90.01±5.62%,95.32±6.03%.Promoting aging function glucose:High glucose stimulated HGMC proliferation during 0-72h,but suppressed cells proliferation after 96h. After 96h of incubation with 10, 30,50mmolL"'glucose, cell survival rate was 99.15±5.48%,82.84±3.45%,53.18±3.08 %; SAβ-gal staining was 22.48±1.03%,82.23±6.05%,90.13±6.13%; G0-G1 phase percent was 68.82±4.13%,89.06±5.46%,91.91±5.22%.After 72h of incubation with 10-6molL-1 AngⅡ,5 successive stresses of 30μmolL-1 tBHP,96h with 30mmolL-1glucose, cellular proliferation was permanently inhibited in the aging groups, with an OD value significantly lower than that of the control group;SAβ-gal staining was significantly increased;The cell cycle was arrested at the Go-G1 phase. Cellular proliferation was obviously inhibited after stimulated with 10-5molL-1Angll,50μmolL-1tBHP,50mmolL-1glucose.Althoug 80 percent cells were arrest at G0-G1 phase and SAP-gal staining positive, death and debris cells increased. Aging cells induced by 10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1glucose were characterized by enlarged cell morphology, polymorphic nuclei, chromatin condensation at the nuclear margin, invagination of the nuclear membrane, the disappearance of mitochondria, dictyosome, tigroid body and pulp-lysosome identified.The results indicated that HGMCs have achieved aging standard.10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1 glucose were used to induce HGMC aging in our studies. 2. Changes of JAK2/STAT pathway and STAT proteins during HGMC agingAfter stimulated by the aging-induced factors, aging indexes were examined. HGMCs were tested to be aging cells. Western blot analysis indicated that expressions of STAT1, pSTAT1, STAT3 and pSTAT3 were increased in aging cells compared to the control cells, especially of pSTAT1 and pSTAT3. Using immunofluorescence detection, we found that STAT1, pSTATl, STAT3 and pSTAT3 were expressed in cytoplasm and cytoblast in normal HGMCs, and their expressions were increased in aging cells, in particular in the cytoblast.It is most obvious in AngⅡ-induced group. Expressions of STAT1, pSTATl, STAT3 and pSTAT3 were slightly decresed after stimulated with 10-5 molL-1 AngⅡ,50μmolL-1tBHP, 50mmolL-1 gluccose,but there is no statistics difference. However their expressions were slightly increased after stimulated with 10-7molL-1 AngⅡ,10μmolL-1tBHP,10mmolL-1 glucose,and there is also no statistics difference.. The results indicated that activation of JAK2/STAT pathway involved in HGMC aging through increasing phosphorylation of STAT1,STAT3 induced by 10-6molL-1 AngⅡ, 30μmolL-1tBHP,30mmol-1 glucose.3. Effect of the three intervention factors on JAK2/STAT pathway and STAT proteins during HGMC agingAfter using 10-5molL-1losartan,10μmolL-1AG490,living cells increased with cell survival percent raised from81.12±3.25% to 90.43±4.12% and 87.82±4.63%; SAβ-gal positive rate falled from 83.10±6.71% to 42.13±3.61% and 46.28+3.73%; proportion of G0-G1 phase reduced from 91.36±6.45% to 71.56±4.28% and 70.97±4.03%; expressions of STAT1,pSTAT1,STAT3 and pSTAT3 decreased.These results indicated that after using losartan and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in AngⅡ-induced aging HGMC.After using 40μmolL-1 probucol,10μmolL-'AG490, living cells increased with cell survival percent raised from 80.12±4.05% to 92.68±4.13% and 89.49±4.06%; SAβ-gal positive rate falled from 81.03±5.86% to 45.32±4.13% and 43.16±3.28%; proportion of G0-G1 phase reduced from 90.76±6.34% to 72.96±4.32% and 72.73±4.17%;expressions of STAT1, pSTAT1, STAT3 and pSTAT3 decreased.These results indicated that after using probucol and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in tBHP-induced aging HGMC.Conclusion1.Ang II,tBHP,high glucose induced HGMC aging in vitro. Conditions of incubation with 10-6molL-1AngⅡfor 72h,5 successive stresses of 1 h per day with 30μmolL-1tBHP,30mmolL-1 glucose for 96h were the most suitable factors to induce HGMC aging.2. Phosphorylation of STAT1,STAT3 were increased in aging HMGC induced by Ang II,tBHP,high glucose,and activation of JAK2/STAT involved in HGMC aging. 3. Antagonizing AT1 acceptor, antioxidation and blocking JAK2/STAT pathway can decrease expressions of STAT1,pSTAT1,STAT3 and pSTAT3 and improve aging HGMCs patho-state to delay HGMC aging. |
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