The Protective Role Of Nrf2 To Renal Fibrosis Induced By OxLDL In Diabetes

Objective Hyperglycemia and hyperlipidemia persist in diabetes.Oxidized low density lipoprotein(Oxidized low density lipoprotein,OxLDL),being one of major hyperlipidemia associated damaging factor,have inseparable relationship with oxidative stress injury and renal tubular interstitial fibrosis.Past studies have confirmed that the NF-E2 related factor(Nuclear factor erythroid2-related factor2,Nrf2)plays an important role in regulating oxidative stress,inflammation,metabolic syndrome and increasing of insulin resistance.Our research try to observe the interstitial damage and the expression of CD36,receptor of OxLDL,in kidney tissue samples of diabetic rats with hyperlipidemia;By cytological experiments,to confirm the regulating function of OxLDL to its receptor CD36,to observe if OxLDL can activate Nrf2 related anti-oxidative stress system.To explore the machanism of Nrf2 regulating membrane receptor CD36 and LOX-1 in process of OxLDL induced renal tubular damage;To explore the influence of Nrf2 inhibiter on CD36.E-cadherin and NF-κB and demonstrate the potential protective mechanism of Nrf2 in OxLDL related renal tubular damage,and may provide new therapeutic targets for clinical treatment.Methods1.Chose healthy male SD rats,Diabetic group(DM group)was fed with high sugar and fat feeding and low dose STZ intraperitoneal injection to induce hyperglycemia and hyperlipidemia,continuely fed the rats for 8 weeks;control group(C group)was fed commonly and injected via intraperitoneal with equivalent dose and time of 0.1%citric acid buffer and then observed the pathological change in kidney samples,calculated the tubular injury index,detected the expression of CD36 in kidney specimen by immunohistochemical method.2.Rat renal tubular epithelial cells NRK-52E cultured with DMEM containing 5%FBS,50 mg/L OxLDL was made by cell culture medium mixture and incubated with the NRK-52E cells.Stimulated NRK-52E cells for 0h,5h,10h,24h,48h respectively,then collected groups of cells and test CD36 protein or Nrf2 total protein level.Stimulated NRK-52E cells for 2,3 days With 0 mg/L,100 mg/L,150 mg/L of OxLDLs,collected each groups of cells later and test CD36,NFκB expression.Stimulated NRK-52E cells of over-expressed by KeapI and transfected control cells with 0 mg/L,150 mg/L of OxLDLs for 2,3 days,collected groups of cells and test expression of CD36.Stimulated NRK-52E cells for 2 days with 150 mg/L OxLDLs and starving treated other group for 2 days,extracted both groups of nucleoprotein and cytoplasmic protein at the same time,then detected the Nrf2 protein level.All above processing cell protein and nucleoprotein cytoplasmic protein were test by western blotting method;Detected CD36,LOX-1 mRNA by RT-PCR;293T cells were transfected and corresponding cells were collected for CD36 and LOX-1 protein expression and mRNA detection.Results1.Animal experiment:Comparing to C group,DM group show increased glomerular volume,expansion of medullary loops,mesangial proliferation and sclerosis and thickening of basement membrane and foamy change of endothelial cells,hyalinosis of efferent and afferent arteriol,interstitial and tubular injury;half quantitative analysis results of tubulointerstitium:renal tubular damage index STI of DM group was 5.54±1.5,C group was 0.65 ±0.15,difference was significant statistically,P<0.05;expression of CD36 in tubulointerstitium of DM group increased comparing to C group.2.Cell experiment:CD36 expression and LOX1 mRNA increased in NRK-52E cells after incubated with different concentration and time of OxLDL;Total Nrf2 protein level did not chang significantly at 5h,10h,but decreased at 24h,48h;Nuleus Nrf2 protein level relatively inereased,meanwhile,cytoplasmic Nrf2 protein level did not change significantly;CD36 mRNA,CD36 protein expression decreased and LOX-1 mRNA increased in NRK-52E cells treated by KeapI over expression processing and OxLDL;E-cadherin expression decreased in NRK-52E after KeapI over-expression processing;NFκB expression increased in NRK-52E cells treated by OxLDL,and decreased after KeapI over-expression processing.Conclusion1.LAnimal experiment:CD36 may be one of corresponding adjustment mechanisms of hyperlipidemia associated damage in diabetes with hyperlipidaemia state;2.Cell experiment:OxLDL can be accepted by CD36 and LOX-1 in NRK-52E cells and upregulated their expression;Nrf2 could be activated by OxLDL,but Nrf2 can resist oxidative stress only after it transfer to the nucleus from cytoplasm.Nrf2 play an important role through upregulating CD36 and downregulating LOX1.Nrf2 also play a regulatory role in renal tubule fibrosis process.OxLDL promote the inflammatory response in renal tubule cells,Nrf2 may play a certain role regulating this responding.