Effect Of Autophagy On Oxidative Stress And Senescence In Rat Mesangial Ceils In Vitro

Objective: Glomerular mesangial cell is one kind of kidney residential cells, andtheir changes of senescence play an important role in the process of kidney aging.However, the mechanism in senescence of mesangial cells is unclear. Autophagy is ahighly regulated intracellular process for the degradation of cytoplasmic components,especially protein aggregates and damaged organelles. It is essential for maintaininghealthy cells. Impaired or deficient autophagy is believed to cause or contribute toaging and age-related diseases. To investigate the effects of autophagy on oxidativestress and senescence in cultured primary rat mesangial cells, we suppress or enhanceautophagic function by overexpressing miR-34a expression plasmid and treating thecells in high glucose, high glucose+rapamycin, and high glucose+3-methyladenine(3-MA).Methods: Rat glomerular mesangial cells were isolated and cultured in vitro, andthe cells from passage3and passage25were used in the present study. To examine theeffect of the miR-34a on autophagy related gene (Atg9A) expression, miR-34a andAtg9A shRNA were transfected into the mesangial cells. We also used dual luciferasereporter gene assay system to verify target gene of miR-34a. Rat glomerular mesangialcells were isolated and cultured in normal glucose, high glucose, high glucose+3-methyladenine(3-MA), or high glucose+rapamycin.Cellular senescence was detected by SA-β-gal （senescence-associatedβ-galactosidae） staining and SHFA (senescence-associated heterochromatic foci)formation analysis. MiR-34a levels were quantified by qRT-PCR. Western blotanalysis was performed to assess LC3, p62/SQSTM1and polyubiquitin aggregates.Expressions of8-OHdG in cells were detected by the immunofluorescence staining.We also examined the content of malondialdehyde (MDA) and protein carbonyl. Results: An increased of SA-β-gal staining was observed in old rat glomerularmesangial cells. The protein expression levels of p62/SQSTM1, polyubiquitinaggregates were significantly increased in old rat glomerular mesangial cells. Theprotein expression level of LC3was significant decreased in old rat glomerularmesangial cells. The8-OHdG staining was increased in old rat glomerular mesangialcells. Levels of protein carbonyl and MDA were increased in old rat glomerularmesangial cells.Dual luciferase reporter assay confirmed that Atg9A was the target gene ofmiR-34a. Overexpression of miR-34a in young glomerular mesangial cells decreasedAtg9A and LC3protein levels, increased the protein expression levels of p62/SQSTM1and polyubiquitin aggregates, and lead to increases in8-OHdG,protein carbonyl，MDA,SA-β-gal staining and SAHF formation, compared with the control group.Compared with those in normal cells, the exposed to high glucose for72h and10days showed a down-regulated LC3expression, up-regulated p62/SQSTM1expression,elevated MDA and protein carbonyl levels, and increased SAHF formation andpercentage of SA-β-gal-positive cells. These changes were reversed in cells exposed tohigh glucose and rapamycin for72h and10days, but exacerbated in cells incubatedwith3-MA.Conclusions: The results suggest that autophagic function decreases, andoxidative damage increases during the cellular senescence in rat glomerular mesangialcells；miR-34a suppress autophagy by inhibiting autophagy related protein Atg9Aexpression, resulting in oxidative damage and cell senescence；high glucose cansuppress autophagic function of to mesangial cells result in oxidative damage and cellsenescence. Rapamycin can attenuate autophagy impairment, oxidative damage andsenescence induced by high glucose, whereas3-MA can further aggravate highglucose-induced cell injuries in rat glomerular mesangial cells.