Expressions Of Bax/Bcl-2 And WT-1 In Renal Tissues Of Membranous Nephropathy In Rats And Effects Of Tetramethylpyrazine

Bjective:To observe apoptosis and expression of Bcl-2, Bax and WT-1 in renal tissues of membranous nephropathy (MN) induced by cationic bovine serum albumin (C-BSA) in rats which were influenced by tetramethylpyrazine (TMP). To find the possible therapeutic mechanisms of which TMP treat MN. Methods:72 male Spargue-Dawley (SD) rats were randomized into four groups:normal group (N group), model group (M group), TMP group (T group),24 for each group. N group, do not preimmuniz and do not make model. At the same time of a formal model in M, T groups, the normal saline replaced C-BSA through the vena caudalis injection(2ml/qod), and the same amount of normal saline taked the place of the TMP through abdominal cavity injection every day.the remaining two groups were duplicated MN model in rats with the improved Border way. Acrossing the formal immune, intraperitoneal injected TMP at a dose of 100 mg·kg-1·day-1 in T group and the same amount of normal saline taked the place of the TMP through abdominal cavity injection in M group.The renal tissue of 6 rats in each group was collected on the 1th,2th,3th and 4th weekends of formal immune. Dynamic detect the urine protein in 24 hours (24hUTP) and concentration of urine creatinine after fasting. To draw the blood from rats and detected clinical biochemical indicators, to compare respectively the condition of creatinine (Scr), blood urea nitrogen (BUN), creatinine clearance(Ccr) per minute, plasma albumin (Alb), triglyceride (TG) and total holesterol(TC). Nephridial tissue was used to judge model’s condition by using light microscope, electron microscope, immunofluorescence technology. The TdT-mediated dUTP nick end labeling (TUNEL) technique was used dynamically observe the expression of apoptosis in each group and the immunohistochemical technique was used dynamically observe the expression of Bcl-2, Bax, WT-1 in each group. We made the correlation analysis of apoptosis, Bcl-2, Bax, WT-1,24hUTP of glomerular and tubular in each group. Results:(1) kidney morphology:light microscope:renal color, morphology and nephridial tissue pathological lesion were normal, and no IgG fluorescence deposit in N group. In M、T groups the kidney morphology normal, the nephridial tissue pathological lesion had mild changes on 1th weekend, glomerular capillary loop and mesangial area had a small amount of IgG fluorescence deposit. With the experiment proceeding, kidney bulk accreted, color pale, the nephridial tissue pathological lesion gradually aggravate, glomerular capillary wall and mesangial area IgG gradually increased. On 4th weekend kidney enlarged to varying degrees, pale color, glomcrulus intumesced obviously; basement membrane thickening diffusely, parts of capsular space angusty, collagen fibers deposited obviously, and interstitium widened even more significantly, fibrosis level heavy. Electron microscopy showed:bulk of electrondense object deposited under glomerular epithelium. Foot process coalesce, the part flatten or vanished. Immunofluorescence showed:glomerular capillary wall IgG fluorescence deposited powerful, the intensity between +++ to ++++. The pathological lesion of M group was more heave than the other groups. The pathological lesion of T group was lighter than M group. Fluorescence intensity had no significant difference in M group and T group. (2) The dynamic change of apoptosis in renal cell:in N group tubular only few apoptotic cells accidentally fund. With the experiment proceeding, the expression of apoptotic gradually rised in M and T groups, the expression of apoptotic were mainly in the renal capsule visceral distribution, glomerular mesangial area, tubular epithelial nucleus and showed brown granular. The expression of apoptotic in M, T group was more than N group, the comparison between M and T group, the expression of T group notably decreased, the difference was significant (P<0.05).(3) The dynamic condition of Bcl-2,Bax,WT-1 in glomerular and tubular:in N group there were many expressions of Bcl-2 in the renal capsule visceral distribution, glomerular mesangial area, tubular epithelial cytoplasm.The expression of Bax was little in the glomerular mesangial area and tubular epithelial cytoplasm.WT-1 was only expressed in the renal capsule visceral distribution, showing brown granular. With the experiment proceeding, the expression of Bcl-2,WT-1 gradually reduced in M and T groups, The expression in M, T group was less than N group, the comparison between M and T group, the expression of T group notably increased, the difference was significant (P <0.05). With the experiment proceeding, the expression of Bax was opposite. (4) Change of clinic indexs:the indexs in N group were nomal. The level of Scr, BUN, TG, TC,24h UTP were step up, the level of Ccr, Alb were degraded in M group, especially for Scr, BUN, Alb,24h UTP (P<0.05). T group compared with M group, the level of Scr, BUN, TG, TC,24h UTP were decreased (P＜0.05), the level of Ccr, Alb were increased (P＜0.05), especially for Scr, BUN, Alb on the 4th weekend (P<0.05).(5) Dependablity of each index on the 4th weekend:In glomerular, AI was positive correlation with Bax, Bax/Bcl-2,24hUTP and negative correlation with Bcl-2,WT-1. WT-1 was positive correlation with Bcl-2, and negative correlation with Bax, Bax/Bcl-2,24hUTP.24hUTP was positive correlation with Bax, Bax/Bcl-2 and negative correlation with Bcl-2 in M and T group. In Tubular, AI was positive correlation with Bax, Bax/Bcl-2,24hUTP and negative correlation with Bcl-2.24hUTP was positive correlation with Bax, Bax/Bcl-2 and negative correlation with Bcl-2 in M and T group. The indexs was not obvious dependability in N group. Conclusion:There’s little cell apoptosis existed in normal renal tissue.Cell apoptosis rised in MN model of rats renal tissue and the cell death included podocyte, intercapillary cells and tubular epithelial cell. Bcl-2,Bax and Bax/Bcl-2 expressed abnormally in MN model of rats renal tissue. WT-1 reduced in MN model of rats renal tissue. This indicated that Podocyte reduced in MN model of rats renal tissue. The reduction of podocyte manybe due to apoptosis which was effected by Bcl-2 and Bax. TMP reduced apoptosis in renal tissue (especially reduced podocyte apoptosis) to protect renal.