Roles Of LOX-1 And Oxidation Stress In Ox-LDL-induced Tubular Epithelial-myofibroblast Transdifferentiation

Dyslipidemia is the independent risk factor for the progression of renal damage. It has been confirmed that proteinuria increased and creatinine clearance rate decreased in diet-induced hypercholesteremic rats. Renal pathology disclose obvious tubule-interstitial injuries, which display with tubular epithelial cell hypertrophy, inflammatory cell infiltration, cytokine generation and tubular interstitial fibrosis, but the mechanism is unclear.Many research indicate that tubular epithelial-myofibroblast transdifferentiation (TEMT) is the major mechanism in the development of tubular interstitial fibrosis, However, there are few reports regarding the TEMT induced by ox-LDL.LOX-1 is a recently discovered specific ox-LDL receptor that is found to be expressed in macrophage, vascular smooth muscle cell, monocyte endothelial cell, cartilage cell and many other cells. It has been demonstrated that LOX-1 not only mediated the digestion and degradation of ox-LDL, but also mediate multiple pathological injuries,such as ischemia-reperfusion,injured myocardium, atherosclerosis, rheumatoid arthritis.A few studies on LOX-1 expression in the kidney have suggested that LOX-1 might have an effect on the development of chronic renal disease. However,there is some controversy whether the LOX-1 is expressed in the tubular epithelial cells and mediate ox-LDL into tubular epithelial cells to lead to tubule injury. Statins is a HMG-CoA reductase inhibitors.It protects kidney through decreasing TG and LDL.On the other hand,it has many lipid-lowering-independent effects which mechanism is not clear.To approach the above question, present study was designed as following four parts:Part I was to evaluate the expression of LOX-1 on tubular epithelial cells and the effect of ox-LDL on LOX-1 in vitro; partⅡwas to investigate the effect of oxidized low density lipoprotein receptor-1 (LOX-1) and ROS on TEMT induced by oxidized low-density lipoprotein (ox-LDL);part III was to investigate the renal protective effect of simvastatin on TEMT induced by oxidized low-density lipoprotein; Part IV was to investigate the renal protective effect of simvastatin on tubular epithelial-myofibroblast transdifferentiation (TEMT) in diet-induced hypercholesteremic rats. PartⅠox-LDL induce tubular epithelial cells to express LOX-1Objective:To evaluate in vitro the expression of LOX-1 in tubular epithelial cells and the effect of ox-LDL on LOX-1 expression.Methods:NRK-52E cells were incubated with ox-LDL (0,25,50,and 100ug/ml) for 24 hours or incubated with ox-LDL of 50ug/ml for 0h,6h,12h,24h or pre-treated with the chemical inhibitor of the LOX-1 receptor polyinosinic acid (polyⅠ) and carrageenan or the antioxidant N-acetyl-L-cysteine (NAC), the cells were then exposed to 50ug/ml of ox-LDL.The expression of LOX-Ⅰ, and reactive oxygen species (ROS) were analyzed by real-time PCR, western blotting, immunofluorescence and confocal laser scanning microscopy. Lipid was examined by oil red O.Results:1. LOX-1 was expressed on the cytomembrane of NRK-52E at low level. 2. Ox-LDL stimulated the expression of LOX-1 mRNA and protein in dose-and time-dependend manners.3. Following the increase in the LOX-1 protein level, the lipid intake and ROS generation were increased.4. Pre-treatment with polyⅠ, carrageenan or NAC could inhibit ox-LDL induced LOX-1 expression, lipid intake and ROS generation.Conclusion:Ox-LDL increased the expression of LOX-1 mRNA and protein in a dose-and time-dependent manners. LOX-1 can promote lipid intake and ROS generation.PartⅡ:Effect of LOX-1 and ROS on Oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiationObjective:To explore the effect of LOX-1 and ROS on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.Methods:NRK-52E cells were incubated with ox-LDL (0,25,50, and 100ug/ml) for 24 hours or pre-treated with the chemical inhibitor of the LOX-1 receptor polyinosinic acid (polyⅠ) and carrageenan or the antioxidant N-acetyl-L-cysteine (NAC), the cells were then exposed to 50ug/ml of ox-LDL.The expression of a-SMA, and E-cadherin were analyzed by Western blotting and immunofluorescence. Results:1. Ox-LDL increased the a-SMA protein expression and decreased the E-cadherin protein expression in a dose-dependent manner from Oμg/L to 100μg/L (P<0.05).2. When LOX-1 was blocked by chemical inhibitor of the LOX-1 receptor poly I and carrageenan or ROS was inhibited by NAC,the increased a-SMA and decreased E-cadherin protein were reversed. Combined with part I, a-SMA protein has a positive relationship with LOX-1 and ROS(r=0.97,0.87), E-cadherin protein has a negative relationship with LOX-1 and ROS(r=-0.94,-0.82).Conclusion:LOX-1 and ROS play a vital role in oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.PartⅢ:Effect of simvastatin on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiationObjective:To explore the effect of LOX-1 and ROS on oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation.Methods:NRK-52E cells were divided into three groups:control group(0ug/ml ox-LDL), ox-LDL group(50ug/ml ox-LDL), simvastatin group(50ug/ml ox-LDL+10 umol/L simvastatin). Following incubation for 24h, expression of LOX-I, a-SMA and E-cadherin and production of reactive oxygen species (ROS) were analyzed by Western blotting, immunofluorescence and confocal laser scanning microscopy.Results:1. Compared with control group, ox-LDL stimulated expression of LOX-1 and a-SMA and ROS generation, but inhibited expression of E-cadherin.2. Simvastatin inhibited LOX-1 expression and ROS generation. Simvastatin also reversed the increased a-SMA and the decreased E-cadherin expression (P<0.05)Conclusion:Simvastatin inhibited oxidized low-density lipoprotein induced tubular epithelial-myofibroblast transdifferentiation through down regulating LOX-1 and ROS. PartⅣ:Simvastatin inhibit tubular epithelial-myofibroblast transdifferentiation in hypercholesteremic rats through down regulation of LOX-1 and oxidative stressObjective:To investigate effect of Simvastatin on tubular epithelial-myofibroblast transdifferentiation in hypercholesteremic rats and the possible mechanism.Methods:The SD rats were divided into three groups as follows:normal group,high fat diet group and simvastatin group(rats were fed with high fat diet plus 10 mg·kg-1·d-1 simvastatin). Six rats in each group were sacrificed at 12th week. Levels of total cholesterol (TC) and triglyceride(TG) in serum were measured by enzymatic colormetric methods.The injury of renal tubulointerstitium was observed under microscope with PAS and masson staining and the expression of renal LOX-1,α-SMA and E-cadherin were determined by immunohistochemistry.In addition, the activity of antioxidant including SOD, GSH-PX and level of MDA in the cortex of kidney were measured.Results:1. Compared with normal group, MDA levels in the cortex of kidney was higher and SOD,GSH-Px activities in the cortex of kidney were lower in high fat diet group.2. Compared with normal group, LOX-1 and a-SMA expression were higher and E-cadherin expression were lower in renal tubular epithelial cell.3. Simvastatin could desease MDA generation and expression of LOX-1 and a-SMA, but increased SOD and GSH-Px activities and E-cadherin expression with improvement of tubular epithelial-myofibroblast transdifferentiation.Conclusion:Simvastatin can ameliorate tubular epithelial-myofibroblast transdifferentiation via the inhibition of LOX-1 and oxidative stress in hypercholesteremic rats.