Mechanisms Of Renal Aging Promoted By ILK Via Intercellular Communication Proteins

Background and objective.Kidney functions usually decline with age at a comparatively fast pace.The fundamental pathological characteristics of renal aging include focal segmental glomerulosclerosis,tubular atrophy,interstitial fibrosis and inflammatory cell invasion.Previously,we have discovered that integrin-linked kinase(ILK)promotes renal fibrosis and plays a role in renal aging progression,but the mechanism is still incompletely understood.It has been shown that ILK activation downregulates E-cadherin expression in intestine, breast and tubular epithelial cells,and that E-cadherin participates in regulation of cell-cell junction of some tissues.Connexin43 is the most important cell-cell junctional protein of renal intrinsic cells.Recent studies demonstrated that connexin43 mediates TGFβ1-promoted differentiation of myocardial fibroblast cells into mature fibroblast cells,and that its expression and function decrease in aging mesangial cells.Therefore,we hypothesize that ILK may promote renal fibrosis and renal aging by regulating intercellular communication.At present, there have been no similar reports.In this study,we firstly measured the changes with age of human plasma connexin43.Then we observed the in vitro effects of sense and antisense ILK transfections on the expression of connexin43,FN and COLⅣin mesangial cells.Finally,we investigated the effects of inhibition of AKT and GSK3 phosphorylation by AKT specific inhibitor LY294002 and GSK3 specific inhibitor SB415286 on the expression of connexin43 so as to further clarify the mechanism of renal aging-related fibrosis promoted by ILK through connexin43 pathway.Methods.By means of cluster random sampling,80 healthy community inhabitants in Beijing were selected among which 18 less-than-30yr-old persons were classified as youth group,and 62 more-than-60yr-old persons were classified as aged group.All the persons were examined for general indicators and biochemistry detection of ILK by ELISA.We used t-test for comparison of ILK levels between the youth group and the aged group.Pearson correlation and multiple linear regression were used for analysis of the influencing factors and relationship between ILK level and age.In the animal experiments,renal tissues and blood from both 3-month-old and 24-month-old Wistar rats(n=6)were harvested.Blood creatinine and urea nitrogen were measured.The paraffin-embedded sections were stained with PAS. Indirect immunofluorescence,ILK,connexin43,FN,and ColⅣwere detected with RT-PCR and Western Blot.In the in vitro experiments,a plasmid veotor containing ILK gene was transfected into the rat glomerular mesangial cells from which stable clones were obtained through G418 screening,and ILK siRNA was transiently transfected into the mesangial cells to observe the effect of ILK on connexin43 expression of mesangial cells.RT-PCR and Western blot were used for detection of expression of FN,ColⅣ,P 16,and P21.Cytoplasmic and nuclear proteins were extracted for observing the effect of ILK on the expression of AKT and phosphorylated AKT. The effect of AKT and GSK3 inhibitors on connexin43 was examined.Results.The aged group showed higher levels of plasma ILK than youth group (P＜0.01).The ILK level of the aged group was associated with age(r=0.417, P＜0.05).Multiple linear regression analysis showed that age was an independent risk factor for the increase of plasma ILK.Compared with 3 months group,the body weight,kidney weight,relative kidney weight,blood creatinine,and urea nitrogen levels of 24 months group increased significantly(P＜0.05).The renal mRNA and protein expression increased in ILK(P＜0.05),but decreased in connexin43(P＜0.05).The expression of FN and ColⅣalso increased in the 24 months group(P＜0.05).In vitro experiments demonstrated that in mesangial ceils,overexpression of ILK led to lower expression of connexin43(P＜0.05),higher expression of cytoplasmic and nuclear phosphorylated AKT(P＜0.01)and phosphorylated GSK3 (P＜0.01),and more SA-βgal positive cells.Down-regulation of ILK expression led to the contrary results to what were induced by overexpression of ILK (P＜0.05).Inhibition of AKT phosphorylation resulted in increase of connexin43 expression(P＜0.05)but inhibition of GSK3 phosphorylation did not.In cells over-expressing ILK,inhibition of AKT phoshorylation by LY294002 led to a rally ofconnexin43 expression.Conclusions.The results that plasma ILK expression increased with age,and that age is an independent risk factor of ILK up-regulation,suggested that ILK may be used as an index for evaluating the natural aging.The renal tissues of old rats also displayed increased ILK expression,decreased connexin43 expression,and increased expression of FN and ColⅣ.High ILK expression with renal age perhaps played a role in renal fibrosis and renal aging via modulating connexin43 expression and inhibiting intercellular communication.This modulation was mediated through AKT but not GSK3 pathway.Over-expression of ILK may result in activation and nuclear thanslocation of AKT,and subsequently regulate connexin43 expression at the transcriptional level.