Effect Of Arctiin On Mouse Podocyte Epithelial-mesenchymal Transition Induced By Advanced Oxidation Protein Products

BackgroundKidney fibrosis is an inevitable outcome of all kinds of progerssive chronic kidney disease (CKD). It is closely related with various primary golmerular diseases, diabetes, ureteral obstruction, and kidney damage. It is important to reduce the incidence of end-stage renal disease (ESRD), because kidney fibrosis lesions have been unable to resistance or reverse.Podocyte is the terminal differentiation cell which is covered in glomerular basement membrane.Podocyte injury can led to the damage of filtration membrane charge barrier and aperture barrier, which is an important reason for the formation of proteinuria.The integrity of hiatus between the foot process membrane protein of glomerular filtration mechanical barrier is crucial, and these proteins injury or decreased expression can cause podocyte structure and features dysfunction and proteinuria. Proteinuria is not only the result of glomerular injury, but also the independent risk factor to the progress of kidney injury. Proteinuria gathers in the renal tubular cavity and causes renal tubular epithelial injury, inflammatory material release, inflammation immersing in renal interstitial infiltrates, renal fibrosis, which leads to kidney function decline and end-stage renal failure eventually.Epithelial-mesenchymal transition (EMT) occurs in early stage of renal fibrosis caused by many scholars’attention. In recent years, it was found that glomerular podocyte occured EMT excepting renal tubular epithelial cell and vascular endothelial cell. Podocyte expresses symbolic protein:a-smooth muscle actin and filtration membrane barriers. EMT plays an important role in the damage of glomerular filtration barrier, proteinuria, and glomerular sclerosis, and it is a new prospect in preventing podocyte diseases.Advanced oxidation protein products (AOPP) exists in the plasma of chronic renal failure (CRF) patients as a kind of inflammation and oxidation materials. It causes immune function disorder, atherosclerosis, and long-term complications in CRF patients. It is shown that AOPP is not only the productor but also executor of oxidate stress in diabetes, CKD patients, and fat patients. Increaseing expression of α-SMA in renal tubular epithelial cell caused by AOPP has been proved by Chen Yongmei’s experiment.Endoplasmic reticulum is the main place of storaging protein and Ca2+Endoplasmic reticulum stress (ERS) is a kind of protecting mechanism to cells. Moderate ERS can restore the steady-state of endoplasmic reticulum and inner environment, but strong and long time ERS can lead to cell factors release and cell apoptpsis. It has been proved that ERS plays an important role in atheroscierosis, diabetes, nervous system degenerative disease, cancer, virus infection, aging and so on. In recent years, it was found that ERS had been participated in the pathogenesis of primary glomerular diseases, secondary glomerular diseases, such as diabetes nephrosis, drug/poison correlation renal tubular-interstitial injury, kidney ischemia-reperfusion injury, and chronic kidney diseases, etc. Markan found that the expression of Grp78and CHOP increased significantly in membranous proliferative glomerulonephritis and acuterapidly progressive glomerulonephritis, but the expression of antiapoptotic gene Bcl-2decreased. It has been shown that a variety of harmful materials motivate excessive ERS and trigger cell apoptosis by activationg the apoptotic signal way, Which lead to a grogressive damage of glomerular. Internal and external pathology show that podocyte injury is associated with ERS. What’s more, recent studies found that thyroid epithelial cells and renal tubular epithelial cells ERS can cause EMT directly, so we surmised that AOPP may cause EMT througe ERS and oxidative stress.Traditional Chinese medicine plays an important role in delay the progress of CRF with a long history, which can improve glomerular sclerosis and renal fibrosis. Arctium lappa L and Arctiin reduce the urine protect and protect the kidney. They had been concerned in preventing chronic kidney disease widely, but the mechanism is unclear. Arctiin is a lignanoids compounds separated from Arctium lappa L Arctigenin forms Arctiin by COMT in the bowel bacteria and liver. The expression of TGF-β1、MCP-1、TNF-α, PKC activity, ECM synthesis and degradation are influenced by Arctium lappa L. It can decrease urinary protein and protect the kidney by increasing the expression of nephrin and podocin. It had been recognized that Arctium lappa L. can prevent primary kidney disease. Arctiin can reduce diabetic rats’urine protein, relieve renal hypertrophy, and prevent diabetes glomerular sclerosis, etc. In addition, Arctiin play a role in chronic nephritis, nephritic syndrome and chronic renal failure prevention by inhibiting TNF-α、NO release largely.The Mouse Podocytes were stimulated by AOPP, and the expressions of Grp78、 CHOP and a-smooth muscle actin (a-SMA) were detected with Western blotting. Arctiin whether can decrease the EMT by ERS in Mouse Podocytes? It will provide new experimental basis for research of renal fibrosis. PurposeThe expressions of Grp78、CHOP and a-SMA in Mouse Podocytes stimulated by AOPP were observed. Arctiin whether can decrease the EMT by ERS in Mouse Podocytes? It will provide new experimental basis for research of renal fibrosisMethods1. AOPP preparation:the proportion of human serum albumin (HSA) and hypochlorite solution is1:140,25to30℃,30minute. Hypochlorite is removed by PBS for24hours. AOPP-HSA is stopped by p-mercaptoethanol and centrifugal ultrafiltration30minute. PBS and centrifugal ultrafiltration are repeatedly. AOPP is measured by the value of OD (340nm).2. The expression of a-SMA in Mouse Podocytes stimulated by AOPP is observed in preliminary experiment.200μg/ml and24hours are the best choice.3. The experiment is divided into9groups:1. control group;2. MPC5+BSA group;3. MPC5+AOPP group;4.MPC5+AOPP+Arctiin (50μmol/L) group;5. MPC5+AOPP+Arctiin (100μmol/L) group;6. MPC5+AOPP+Arctiin (200μmol/L) group;7. MPC5+AOPP+Arctiin (400μmol/L) group;8. MPC5+thapsigargin (0.25μmol/L) group;9. MPC5+thapsigargin (0.25μmol/L)+Arctiin (400μmol/L) group.4. The expressions of Grp78、CHOP and a-SMAwill be detected by Western blotting for24hours.Results1. The expressions of Grp78、CHOP and a-SMA is detected by Western blotting1to3groups’ Western blotting has been observed. The expressions of Grp78、 CHOP and a-SMA are increased in MPC5cells after24hours compared with control group and MPC5+BSA group (P<0.05). It means AOPP can lead to ERS and EMT. The report has been shown that Thapsigargin is the revulsant of ERS and take a role in endoplasmic reticulum specifically. The expressions of Grp78、CHOP and a-SMA stimulated by Thapsigargin has been observed in the experiment.2. The expressions of Grp78、CHOP and a-SMA intervened by Arctiin is detected with Western blottingThe Mouse Podocytes is stimulated by AOPP (200μg/ml) for24hours, and the expressions of Grp78、CHOP and a-SMA is detected by Western blotting, but the Arctiin can reverse it. With the increasing of Arctiin concentration, Grp78、CHOP and a-SMA is decling, and they are inhibited by Arctiin on the dose in a certain range. The expressions of Grp78、CHOP and a-SMA in400μmol/L group of Arctiin is different from AOPPgroup and50μmol/L group. It has statistics significane. ERS and the expressions of a-SMA stimulated by Thapsigargin can be reversed by Arctiin in the experiment.ConclusionThe increasing expressions of Grp78、CHOP and a-SMA stimulated by AOPP of Mouse Podocytes has been observed in the experiment, but the Arctiin can reverse it. With the increasing of Arctiin concentration, Grp78、CHOP and a-SMA is decling, and they are inhibited by Arctiin on the dose in a certain range. AOPP can lead to the mouse podocytes’ ERS and therefor causes EMT. Besides, the expressions of Grp78、 CHOP and a-SMA stimulated by AOPP and Thapsigargin can be decreased by Arctiin. It means Arctiin may decrease the EMT by ERS, and it provides new experimental basis for research of renal fibrosis.