Effect Of Salidroside On Mouse Podocytes Epithelial-mesenchymal Transition Induced By Advanced Oxidation Protein Products

BackgroundChronic kidney disease (CKD) is the vast majority of kidney disease clinical general designation; most of the kidney diseases can belong to the scope of chronic kidney disease in addition to the acute nephritis with acute urinary tract infection (renal acute inflammatory diseases). Chronic kidney disease is a slowly developing relatively benign disease (or common clinical pathological process).All kinds of diseases of the urinary system (include diabetic nephropathy,primary glomerular disease,toxic nephropathy,benign arteriolar nephrosclerosis,ischemic nephropathy,obstructive nephropathy and so on),if it failed to make a diagnosis and give treatment in time, can lead to disease progression, deterioration, or illnesses out and develop into chronic renal insufficiency, renal failure and uremia.Renal fibrosis is the end result and and the inevitable outcome of a variety of chronic kidney disease. In pathology, renal fibrosis is a common pathological basis of chronic kidney disease. It is also an important way to all sorts of chronic kidney disease progress to end-stage renal failure. Renal fibrosis include glomerular fibrosis and renal tubule-interstitial fibrosis.The three are closely related and directly related to degree of decline in renal function. Regardless of glomerulus and renal tubule and renal interstitial, macrophage activation, extracellular matrix (ECM) within the organization and the ECM transformation out of balance in the their process and the mechanism of fibrosis, such as the constant of cellular and molecular events. Extracellular matrix cells mainly include:fibroblasts, tubular epithelial cells, glomerular mesangial cells and local infiltration of mononuclear cells. These cells transform into MFB which express α-SMA, muscle fibroblasts express product mainly isα-SMA. α-SMA is considered as markers of muscle fibroblasts and is specific. It is a sign that glomerular mesangial cells phenotype transformation of disease and damage to activate. Podocytes are highly differentiated and can’t renew. It formed multiple refers to podocyte foot processes. Connection between adjacent podocyte foot process form a layer in the diaphragm, attached to the outer layers of the glomerular basement membrane. Podocytes stable the glomerular structure by weakening outward expansion effect of glomerular basement membrane. Lack of sufficient cell function in proteinuria and glomerular fibrosis formation process play an important role in the development, eventually can cause kidney fibrosis. The essence of the renal fibrosis is that excessive produce extracellular component replace kidney inherent structure. EMT is an important way that produce extracellular matrix synthesis of fibroblasts and muscle fibroblasts. EMT is a process of renal fibrosis in early and reversible changes. If you can reverse EMT foot cells, it will effectively block the development of chronic kidney disease (CKD).The research of podocyte transdifferentiation mechanism, become further proven renal fibrosis mechanism of the important breakthrough point.Advanced oxidation protein products (Advanced oxidation protein products, AOPPs) was the first to notice in hemodialysis patients with cycle the oxidative modification of proteins. In vitro experiments,it suggests that AOPP can be markers of oxidative stress, not only can stimulate monocytes and neutrophils breathing outbreak, also may be inflammation of the intermediary factor. The accumulation of plasma advanced oxidation protein products is prevalent in diverse disorders such as diabetes metabolic syndromes, and chronic kidney disease. Recent studies have shown that chronic accumulation of plasma AOPPs significantly increases urinary protein excretion and accelerates glomerulosclerosis in a remnant kindey model. Cycle of AOPP level associated with kidney disease progress speed and protein oxidation product retention levels associated with the micro inflammation of chronic kidney disease in Patients with chronic kidney disease (such as IgA nephropathy). Recently, AOPPs gradually caused wide public concern in the role of pathogenesis of chronic kidney disease. Some scholars study found AOPPs can induce renal tubular epithelial cells and podocytes EMT happened. This prompt early key link to the pathogenesis of CKD may be the result podocyte AOPPs EMT happened.Salidroside is resales Crassulaceae Rhodiola genus perennial herbs or shrubs wild plants. It named for flowers, roots, stems are red. Rhodiola glucoside is the main active ingredient. Studies have observed rhodiola glucoside of lipid peroxide (LPO), rat liver and superoxide dismutase (SOD) enzyme activity, serum LPO and reduced GSH-Px (gsh-px), the influence of the results show that rhodiola glucoside can significantly improve the activity of rat liver gsh-px and SOD activity, inhibiting the production of rat blood serum and liver LPO, that rhodiola glucoside has strong anti lipid peroxidation. There was also evidence that rhodiola can improve hypoxia induced renal tubular epithelial cells-the muscle fiber cell transdifferentiation, reduce alpha SMA expression, the increase of extracellular matrix, play an inhibition of renal tubular epithelial cells and stromal cells to the transformation of fibroblasts, which can alleviate the renal interstitial injury, prevention and treatment of renal interstitial fibrosis. In this study, the effects of salidroside on mouse podocyte epithelial-mesenchymal transition induced by AOPPS were detected with the techniques of with Western blotting.ObjectiveThe expressions of Grp78,CHOP, a-SMA, nephrin and podocin in Mouse Podocytes stimulated by AOPP were observed. We also observe the protein expression changes of Grp78, CHOP, a-SMA, nephrin and podocin after the intervention of salidroside. We discuss whether salidroside can reverse EMT on mice podocyte by ERS.Methods1. AOPP preparation:using the method of Witko-Sarsat introduced, human serum albumin (HSA) mixed with hypochlorous acid solution with the molar ratio of1/140,30minutes at room temperature (25～30℃). Hypochlorite is removed by PBS for24hours, the preparation of AOPP HSA to join the β-mercaptoethanol termination reaction,30minutes and the centrifugal ultrafiltration flush, adding suitable amount of PBS centrifugal ultrafiltration again. AOPP content is measured by the value of OD (340nm).2. Podocytes culture to extend into37℃RPMI1640medium which do not containγ-interferon,5%CO2used after cell culture incubator to differentiation.3.The expression of a-SMA in Mouse Podocytes stimulated by AOPP is observed in preliminary experiment.200μg/ml and24hours are the best choice.4. The experiment is divided into10groups:1. control group;2. MPC5+BSA group;3. MPC5+AOPP group;4.MPC5+AOPP+salidroside (0.1μmol/L) group;5. MPC5+AOPP+salidroside (1μmol/L) group;6. MPC5+AOPP+salidroside (10μmol/L) group;7. MPC5+AOPP+salidroside (100μmol/L) group;8. MPC5+AOPP+salidroside (200μmol/L) group;9.MPC5+thapsigargin (0.25μmol/L) group;9. MPC5+thapsigargin (0.25μmol/L)+salidroside (200μmol/L) group.4. The expressions of Grp78,CHOP,α-SMA, nephrin and podocin will be detected by Western blotting for24hours.Results1.The expressions of Grp78and CHOP is detected by Western blotting1to3groups, control group and BSA group don’t express Grp78and CHOP protein. Under the effect of AOPP (200μg/ml), the expression of Grp78and CHOP protein is increased (P<0.01). Different concentrations of salidroside (0.1μmol/L、1μmol/L、10μmol/L、100μmol/L、200μmol/L) intervention, Grp78, CHOP protein expression decreased and were dose dependent (P<0.05). The report has been shown that Thapsigargin is the revulsant of ERS and take a role in endoplasmic reticulum specifically. The expressions of Grp78and CHOP stimulated by Thapsigargin has been observed in the experiment (P<0.01). The expressions of Grp78and CHOP are reduced after the intervention of salidroside (P<0.01)2.The expressions of a-SMA,nephrin and podocin is detected by Western blotting1to3groups, compared with control group and BSA group, Under the effect of AOPP (200μg/ml), the expression ofa-SMA protein is increased while nephrin and podocin protein is reduced (P<0.01). Different concentrations of salidroside(0.1μmol/1μmol/L、10μmol/L、100μmol/L、200μmol/L) intervention, the expression ofa-SMA protein is reduced while nephrin and podocin protein is increased (P<0.05). The expressions of α-SMA stimulated by Thapsigargin has been increased in the experiment (P<0.01). The expressions of nephrin and podocin stimulated by Thapsigargin has been reduced in the experiment (P<0.01). The expressions ofa-SMA are reduced and the expressions of nephrin and podocin are increased after the intervention of salidroside (P<0.01) ConclusionThe increasing expressions of Grp78,CHOP,a-SMA and the reducing expressions of nephrin and podocin stimulated by AOPP of mouse podocytes has been observed in the experiment. The expressions of Grp78,CHOP,a-SMA are reduced and the expressions of nephrin and podocin are increased after the intervention of salidroside. It means salidroside may decrease the EMT by ERS.