| Objective:Discussion the produce situation of AOPP and its preliminary study damage on renal tissue in the pathogenesis of C-BSA induced membranous nephropathy in rats.Methods:80 male Spargue-Dawley(SD) rats Were randomized into four groups: normal group(A group),model group(B group),model+Apocynin group(C group),Apocynin group(D group),20 for each group.B and C group duplicate MN model in rats with the improvement Border way. C, D group began to intragastric administration with Apocynin(200 mg/kg.d) at the beginning of the formal immune,Corresponding to stimulate normal group.Stochastic and partial killed the rats at the 1,2,3,4 weekend when start of formal immune,5 each group each time. To collect urine for 24 hours to test the 24 hUTP before the day to be put to death and detection the AOPP、 PCX stander using elisa method in urine;Suction and centrifugal heart blood inspection BUN、Scr、TC、TG and AOPP using elisa method、SOD and MDA stander using spectrophotometry method with supernatant;take part of right kidney cortex about 2×3mm Wrapped with wet gauze with physiological saline,immediately freeze and Sliced after OCT reagent embedded, and make immunofluorescent staining,fluorescence microscopy to observe the immune complex depositionin in dark room, the left kidney cut to two parties fixed with 4% formalin,made paraffin blocks through the step of dehydrated,embedded in paraffin st,Sliced and made HE 、 Masson 、 PASM staining,observed the pathological changes by light microscopy, the remaining kidney tissue frozen at-80 re℃ frigerator detection AOPP and ROS stangders using elisa method and detection WT-1 express in kidney organization use immunohistochemistry method.Make independent samples ANOVA between each group the indicators at the same time,P≤0.05 for the difference was significant. Results:(1)Changes in renal morphological Group A and group D: the color and shape of the kidneys was normal at each time point, there was no significant pathological changes in renal tissue, and no fluorescent IgG deposition; Group B and group C: the color and shape of the kidneys didn’t have obvious abnormalities in the first week, but we could see most kidneys’ size larger and color paler with the extension of modeling time, and changed to " big white kidney", pathological changes were also the some thing, we could gradually see the basement membrane diffuse thickening, some cysts got narrow, the red addicted complex substance deposited, ribbon-like changed, "two-track disease" and "spikes" formation changed, and compared to group B, group C was minor; immunofluorescence staining could be seen in the group B and group C at first week, the fluorescence intensity was +++-++++ and it didn’t significant modeling enhancements with the extension of time, further, the fluorescence intensity between the two groups was no significant difference.(2) Changes in clinical indicators The 24 hUTP, ALB, BUN, Scr, TC, TG of group A and group D were no significant differences at each time point; the 24 hUTP of group B and group C was significantly higher than group A and group D start from the first week, and continue rose to fourth week, but group B lower than group C; the ALB of group B and group C began to decline from the second week, but group B lower than group C; compared to group B and group C,the BUN, Scr, TC, TG level of group A and group D have increased significantly at fourth week, and the visible BUN level of group C was significantly decreased, but the levels of Scr, TC, TG were no significant decline.(3) Dynamic expression of the AOPP, SOD, MDA in serum Indicators had no significant changes with the extension of modeling time in group A and group D, the expression levels between the two groups had no significant difference, too; The mean level of serum AOPP, MDA,SOD in group B were higher than group A and group D at each time point, the levels of AOPP, MDA showed a upward trend, the level of SOD showed a downward trend with the extension of modeling time, the difference of AOPP, SOD between group B and group A,C from the first week had statistically significance(P≤0.05), the difference of MDA from the second week had statistically significance(P≤0.05); The average level of AOPP, SOD and MDA in group C was higher than group A,D, but lower than group B, the difference of AOPP from the second week had statistically significance(P≤0.05); the difference of AOPP between group C and group B from first to fourth week had statistically significant(P≤0.05), and the difference of MDA from the third week had statistically significance(P≤0.05), while the difference of SOD is not obvious at all time points.(4)Dynamic expression of the AOPP, PCX in urine Group A and group D: the level of AOPP,PCX had not notable changes at each time point, and there is no difference between the two groups; Group B and group C: the level of AOPP was higher notably than the group A,D(P≤0.05), showed a upward trend, the mean expression levels of group C was lower than group B at each time point, but the difference is not notable; the expression level of PCX in urine showed a upward trend in the first and second week, and was higher than group A,D, which got lower in the third an fourth week, the level of PCX in group C was lower notably than group B from first week to third week(P≤0.05), but the difference was not obvious in the fourth week.(5)Dynamic changes of AOPP, ROS, WT-1 in renal tissue Group A and group D:AOPP, ROS and WT-1 had no significant changes at all time points, and no significant difference each groups;Group B and group C: the expreesion of AOPP and ROS is on the rise trend, starting from the second weekend is significantly higher than group A and D,AOPP level in Group C significantly less than B besides the second weekend, the rest of the time difference is not obvious,and ROS level of group C was lower than that in group B since the second weekend(P≤0.05),WT-1 semi-quantitative level in group B and C was lower than that in group A and D, and is on the decline trend,but the expression level of group C higher group B.(6)The comparison of the expression level of serum and urine AOPP on model group AOPP expression level in serum and urine is increased time dependence;urine AOPP average level higher than the expression level in the serum,especially obvious difference before 3 weeks(P≤0.05), no obvious difference to 4 weekend.(7)Correlation analysis of each indicator AOPP and MDA in serum were positively correlated, and negatively correlated with SOD;kidney tissues of AOPP was positively related with 24hUTP、 ROS, and negatively correlated with WT-1, ROS was positively related with 24 hUTP, and negatively correlated with WT-1,WT-1 was negatively related with 24hUTP;Urine PCX and AOPP、ROS、WT-1、24hUTP has no obvious relevance. Conclusion:(1) The whole body and kidney have gradually increase oxidative stress reaction in MN rat models,and AOPP was one of oxidative stress indicaters;(2) Oxidative stress is one of the reason of podocyte damag in MN rat renal;(3) AOPP participate to renal injury in MN rats,may be through activated NADPH oxidase induced sertoli cell oxidative stress, leading to sertoli cell damage, produced proteinuria eventually. |
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