The Senescent Renal Tubular Epithelial Cells Through Paracrine TGF-β1to Promote EMT

Objective: Kidney is one of vital organs which obvious changes ofstructure and function with aging. The histopathological features of agingkidney: renal cortical thinning, glomerulosclerosis, renal interstitial fibrosis,tubular atrophy and arteriosclerosis. However, the changes of renal interstitialis considered to be the main reason leading to the aging renal dysfunction.Although the researches found that aging kidney often was associated withrenal fibrosis, the relationship between aging and fibrosis have been notcompleted understood. Therefore, we intend to explore the relationship ofaging and fibrosis in kidney, and the mechanisms of the aging renal EMT withaging renal model in vivo and in vitro. It is important that anti-aging kidneydamage and prevention of aging-related kidney disease.Methods:3-month-old and24-month-old F344male rats were used toestablish the model of young and old rat, respectively. In vivo, Aging markerof kidney was detected by SA-β-gal（senescence-associated β-galactosidae）staining and collagen fibers of kidney was deteced by Masson staining andimmunofluorescence stained E-caherin and α-SMA, all of them to certificatethe aging, renal interstitial fibrosis and EMT in aging kidney. Human proximaltubuler cells(HPTC) were isolated and cultured in vitro. To examine therelationship between senescence and EMT, we used Western blot analysisexpressive changes of p53and p21which are the makers of senescence andexpressive changes of E-cadherin, vimentin, α-SMA in PTC which werestimulated by high glucose (33mM) for0h,6h,12h,24h,48h,72h to establishthe model of stress induced senescent cell model. We also usedimmunofluorescence to detect changes of E-cadherin and α-SMA in PTCwhich were stimulated by high glucose (33mM) for0h,24h,72h. Then, to confirm that senescent tubular epithelial cells promote EMT through themechanism of SASP, such as paracrine cytokines. We collected conditionalmedium from normal and high glucose stimulated tubular cells in24h tostimulate PTC for48h in order to detect changes of EMT markers. TransfectedsiRNA to inhibit p53induced senescence, then collect the conditional mediumfrom normal(CM), high glucose(SEN-CM),high glucose induced cells whichsip53(SEN-CM(sip53)), high glucose induced cells which sicon(SEN-CM(sicon)) to culture PTC for48h. ELISA examine TGF-β1concentrations in conditional medium from CM, SEN-CM, SEN-CM(sip53),SEN-CM(sicon). Fanally, we use SB525334, a TGF-β1receptor I inhibitor, tostimulate PTC and then detect the expressive changes of SMAD4, pSMAD3and EMT markers in order to prove SEN-CM induced EMT viaTGF-β1/SMAD signalling.Results: Compare to3-month-old, SA-β-gal and Masson positive stainingwere observed in renal tubulars of24-month-old rat, as well as an decreaseexpression of E-cadherin and an increase expression of α-SMA, it is suggestedthat aging, EMT and fibrosis have existed in aging kidney and tubular cellshave underwent EMT. In vitro, compare to0h, SA-β-gal positive staining wasdetected in high glucose induced HPTC after24h, and senescent markers p53,p21express were significantly increased after24h with Western blot. Theseresult showed that senescent phenotypic markedly appeared after high glucosestimulated HPTC for24h（p＜0.05）. However, protein expression levels ofE-cadherin was significantly decreased and vimentin, α-SMA weresignificantly increased in high glucose induced HPTC after48h, moreover,consistent with E-cadherin and α-SMA staining with immunofluorescence. So,it is reveal that EMT occured after high glucose stimulated for48h（p＜0.05）,and these data verified that senescence was earier than EMT in proximaltubular epithelial cells. To explore whether senescence pomote EMT thoughparacrine SASP,we culture HPTC by conditional medium from normal(CM)and high glucose induced HPTC(SEN-CM). E-cadherin was downregulatedand vimentin and α-SMA were upregulated in SEN-CM compared with CM. RNA interference-mediated knockdown of p53abolished p53/p21inducedsenescence, and then collect conditional medium from CM, SEN-CM,SEN-CM(sip53), SEN-CM(sicon) to culture HPTC. Western blot analyzedexpression of protein level found that EMT did not happen in SEN-CM(sip53)by contrast to CM. Finally, we demonstrate that conditioned medium containhigh level of TGF-β1in SEN-CM and TGF-β1/SMAD signalling was activedin HPTC which were culture by SEN-CM to promote EMT.Conclusions: In this study, we confirmed that there were senescentphenotype and EMT in the senescent renal tubular epithelial cells via vivo andvitro experiments. However, the senescent phenotype occurred earlier thanEMT, and inhibition of senescence also inhibited EMT occurred. Finally, wedemonstrated that senescent tubular epithelial cells paracrined TGF-β1topromote EMT through the mechanism of SASP.