| Objective: Study on the mechanism of podocyte injury in the process of membrane nephropathy, explore the role of endoplasmic reticulum stress(ERS) and oxidative stress in the pathogenesis of membranous nephropathy(MN).Methods : 85 SD male rats were randomly divided into five groups: normal group(no pre-free, not immune, to be the next day intravenous injection 1ml saline(NS) when other experimental groups formal experiment, while NS 1ml/d orally, n = 20); model group(C-BSA to be copied in accordance with the modified rat model of MN Border Act, while NS 1ml/d orally, n = 20); NAC1 intervention group(C-BSA rats copy MN, while N-acetyl gavage cysteine(NAC) 100mg/kg/d, n= 20); NAC2 intervention group(C-BSA rats copy MN, while fed NAC 200mg/kg/d, n = 20); NAC drug group(no pre-free, not immune, to be the next day intravenous injection 1ml NS formal experiment when other experimental group, while the gavage NAC 200mg/kg/d, n= 5). The drug control group were detected by 24 h UTP in the first, second, third, fourth weekend after the official immunity, at the end of the fourth week were killed; five rats were randomly seized to be kildel at the first, second, third, fourth weekend after the official immunization in the rest of the group; when each group seized kill heart blood examination ALB,TG,TC and other biochemical markers; spectrophotometry detection of serum SOD, MDA; rat kidneys were examined with light microscope, immunofluorescence identification model and pathological changes; TUNEL method to detect the glomerular intrinsic apoptosis while apoptotic index(AI);immunohistochemical method to detect the glomerular GRP78, TRAF2, Caspase12, WT-1 protein content at different time points, and calculates the podocyte density according to WT-1 immunohistochemical results;meanwhile Laboratory Parameters row Pearson correlation analysis.Results: ⑴ Biochemical Indexes Each test indicators of normal group were normal;compared with the normal group, 24 h UTP, TG, TC of model group was time-dependent increase, ALB gradually decreases with the progress of the experiment,24 h UTP, ALB at the second, third, fourth weekend were statistically significant(P<0.05),TG, TC in the third and fourth weekend were significantly different(P<0.05);after NAC treatment, NAC1 group, NAC2 group 24 h UTP, TG, TC levels drop,ALB level rise, 24 h UTP compared with the model group were significantly different(P<0.05) in the third and fourth weekend, ALB than the model group were significantly different(P<0.05) in the four weekend, TG of NAC2 group closer to normally in the four weekend, there are significant differences(P<0.05) with the model group;clinical trials of the drug control group compared with the control group had no significant indicators of anomalies. ⑵ Renal morphological changes normal group and drug control kidneys were normal color,no significant changes in kidney tissue morphology, no immunofluorescence deposition;compared with the normal group, model group with the experimental progress, kidney volume increases, the color of pale, pathological lesions gradually increased, fluorescence intensity gradually increased in glomerular capillary loops;in the four weekend, the naked eye to see the kidney volume increased significantly, blunt edge, was "white kidney" like appearance, swollen glomeruli, a small part of the balloon stenosis, basement membrane(GBM) diffuse increase thick, podocytes side "spikes" formation, some spikes on top fusion, was "false track" structure, little visible renal interstitial collagen deposition, immunofluorescence intensity of capillary loops +++- ++++;compared with the model group, NAC1 group, NAC2 group pathological lesions significantly reduced, some GBM thickening, was "satin" kind of degeneration, showing a small amount of "spikes" formation, a little immune complex deposition in the skin, no obvious " false track " change, immunofluorescence intensity between the two groups compared with the model group decreased slightly, but there was no significant difference. ⑶The changes of renal intrinsic apoptosis normal group and the control group of drugs rare apoptosis;apoptosis in model group increased significantly compared with the normal group, which in glomerular were statistically significant compared with the normal group(P<0.05) in the the first, second, third, fourth weekend;compared with the model group, glomerular cell apoptosis of NAC1 group and NAC2 group decrease, which were significantly different(P<0.05) compared with the model group in the four weekend, but still were higher than normal group(P<0.05). ⑷Podocyte density change normal group at each time point there was no significant difference;compared with the normal group, the first weekend of podocyte density in model group began to decrease, but not statistically significant, decreased significantly(P<0.05) in the the second, third, fourth weekend;after intervention, NAC1 group, NAC2 group podocyte density increased slightly compared with the model group, significant differences(P<0.05) in the four weekend, podocyte density increases with the dose of NAC has some relationship, but the entire course of treatment there were no statistically significant in the same period;compared with the normal group, the four weekend of drug control group had no significant difference between the density of podocytes. ⑸ SOD, MDA changes in serum compared with the normal group, the expression of SOD in the serum of the model group was increased, and the time dependence decreased, which were significantly different(P<0.05) compared with the normal group in the the first, second, third, fourth weekend; the expression of MDA tended to increase in the model group rats, which were significantly different(P<0.05) compared with the normal group in the the first, second, third, fourth weekend;after NAC intervention, the expression of SOD in NAC1 group and NAC2 group increased, but MDA decreased, SOD and MDA than the model group were significantly different(P<0.05) in the third and fourth weekend, in the four weekend of MDA in NAC2 group near normal group. ⑹Kidney tissue immunohistochemical results GRP78 in the normal rats mainly expressed in the cytoplasm of tubular cells, glomerular expression less;compared with the normal group, GRP78 of model group expression in the same position, which in glomerular showed a timedependent increase before three weeks, followed by a downward trend, the GRP78 of glomerular than the normal group were statistically significant(P<0.05) in the the first, second, third, fourth weekend;compared with the model group, GRP78 in glomerular of NAC1 and NAC2 group expression was decreased, there are significant differences(P<0.05) in the second weekend; compared with the normal group, GRP78 in glomerular of NAC1 and NAC2 group were statistically significant in the first, second, third, fourth weekend(P<0.05). TRAF2 expression of normal rats less concentrated expression in glomeruli, proximal and distal tubule little expression, manifolds almost no expression;compared with the normal group, TRAF2 in glomerular of model group during the experiment was time-dependent increase,which than the normal group showed significant differences(P<0.05) in the first, second, third, fourth weekend;caspase12 little expression in normal rats, mainly located in the manifold and the proximal and distal tubule cell cytoplasm, glomerular little expression; compared with the normal group, Caspase12 in glomerular of model group was time-dependent increase compared with the normal group were statistically significant(P<0.05) in the second, third, fourth weekend.By NAC antioxidant treatment, TRAF2 and Caspase12 in glomerular expression of NAC1 and NAC2 group were decreased in the third and fourth weekend,which compared with the model group were significantly different(P<0.05), but still normal group increased(P<0.05).when the four weekend of drug control group,GRP78, TRAF2, Caspase12 express position with the normal group, the semiquantitative in glomerular no significant difference compared with the normal group. ⑺ The index correlation analysis in model group,24 h UTP correlated with glomerular AI was positively which correlated with podocyte density was negatively correlated;SOD and 24 h UTP, glomerular AI were negatively correlated which positively correlated with podocyte density;MDA and 24 h UTP, glomerular AI were positively correlated which negatively correlated with podocyte density;caspase12 and podocyte density was significantly negatively which correlated with the glomerular AI was positively correlated; glomerular AI and podocyte density was negatively correlated.Conclusion: ⑴ The onset of oxidative stress and endoplasmic reticulum stress in rat membranous nephropathy.⑵In the pathogenesis of membranous nephropathy in rats, endoplasmic reticulum stress in early may play a protective role by unfolded protein response, in late may through activation of the TRAF2-Caspase12 pathway induced podocyte injury. ⑶ In the pathogenesis of membranous nephropathy in rats, oxidative stress may be through activation TRAF2-Caspase12 pathway induce of podocyte apoptosis. |
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