The Role Of SQSTM1/p62 In The Inflammation Of Renal Tubular Epithelial Cells In Diabetic Kidney Disease By Regulating TLR4 Signaling Pathway

Diabetic kidney disease(DKD)is a microvascular complications of diabetes mellitus(DM).DKD has become a global public health problem and the leading cause of end-stage renal disease(ESRD).WHO data show that there are currently more than 300 million diabetic patients in the worldwide,40%of whom will develop ESRD within the next year[1].The high incidence of DKD has also brought huge consumption of medical resources.Statistics shows that in 1998,16 billion US dollars was spent on DKD treatment in the US,while in 2010 this figure increased to 39 billion US dollars.At present,DKD has become a public health and social problem,seriously threatening people's health.Recent studies have found that Toll-like receptor 4(Toll-like receptor 4,TLR4)mediated secretion of inflammatory factors in renal tubular epithelial cells is an important pathogenic factor for DKD.The multifunctional protein SQSTM1/p62 can directly bind to TRAF6 of TLR4 signaling pathway,thus exerting a regulatory effect on TLR4 signaling pathway.Our study found that the expression of.p62 in renal tubular epithelial cells decreased under high glucose environment,while whether p62 participates in the dysregulation of TLR4 signaling pathway in DKD renal tubular epithelial cells and affects the inflammatory state remains unclear.In the present study,the renal tubular epithelial cell line HK-2 was employedAfter 72 hours of high glucose stimulation in vitro,we found that the levels of inflammatory factors IL-1?,IL-6 and IL-10 in renal tubular epithelial cells were higher than those in the control group,with the expression of E-cadherin decreased,and the expression of Vimentin increased,indicating an enhanced EMT.Meanwile,the expression of p62 both in high glucose stimulated HK-2 cells in vitro and in the sections of kidney of diabetic mice in vivo was dramatically decreased,accompanied by the activation of TLR4 signaling pathway.Further study reavealed that the overexpression of p62 partially attenuated the secretion of inflammatory factors and EMT of renal tubular epithelial cells caused by high glucose.In mechanism,the interaction of p62 and TRAF6 evaluated by Co-IP diminished by high glucose treatment,which was ameliarated by p62 overexpression.In conlusion,this study shows that p62 may become a potential effective intervention target,which can protect DKD by regulating TLR4 signalling pathway.This study is also of great significance for better understanding the intracellular mechanism of activation of TLR4 signaling pathway in DKD tubular epithelial cells.Objectives:1.Whether the abnormality of p62 in renal tubular epithelial cells in diabetic environment is involved in the over-activation of TLR4 signaling pathway.2.Whether up-or down-regulation of p62 expression can affect the progress of DKD by regulating the inflammatory factors secretion through TLR4 signaling pathway in renal tubular epithelial cells.Methods:1.Human renal tubular epithelial cells HK-2 lentivirus packaging plasmid overexpressed p62,then co-transfected HK-2 cells according to the instructions of Lipofectamine 3000,and detected p62 transfection efficiency by real-time quantitative PCR and immunoblotting respectively.2.The expression of E-cadherin,Vimentin,TLR4 and TRAF6 were detected by Western blotting in vitro and IHC in vivo on paraffin embedded sections of kidneys of mice.3.The total RNA was extracted from the cultured cells by TRIZOL method,and the total RNA was retranscribed into CDNA.The expression of IL-1?,IL-6 and IL-10was detected by real-time quantitative RT-PCR,IL-1?,IL-6 and IL-10 levels in the culture supernatant were detected by BD Milliplex kits via Luminex.4.The interaction between p62 and TRAF6 was detected by co-immunoprecipitation(Co-IP)in renal tubular epithelial cells.After overexpression of p62,the HK-2 cells was stimulated by high glucose for 72h,and the supernatant was collected fo IL-1?,IL-6 and IL-10 detection by BD Milliplex kits.Total RNA was extracted from kidney homogenate of STZ-induced diabetic mice and normal controls,and p62 expression was detected by qRT-PCR.The total protein was extracted from kidney homogenate for western blotting and immunohistochemical staining was performed in paraffin sections of kidney.Results:1.The expression of IL-1?,IL-6 and IL-10 in renal tubular epithelial cells was higher than that in control group after high glucose stimulation for 72 hours,while the expression of E-cadherin was lower and Vimentin was higher,with the expression of TLR4 and TRAF6 increased.2.EMT was enhanced with TLR4 signaling pathway activated in renal tubular epithelial cells of diabetic mice.3.The expression of p62 was decreased in diabetic enviroment,and the interaction between p62 and TRAF6 was reduced.4.Overexpression of p62 can inhibit the EMT and the release of inflammatory factors in HK-2 cells induced by high glucose stimulation,mainly through the enhancement of the interaction between p62 and TRAF6.Conclusions:1.p62 level was decreased in renal tubular epithelial cells in high glucose environment.2.p62 is involved in TLR4 overactivation in DKD renal tubular epithelial cells as a downstream mechanism.3.p62 can be a potential effective intervention target,and it has a protective effect on DKD by regulating TLR4 signaling pathway.