| Objective: From the molecular level explore to the pathogenesis of glomerular sclerosis, observed blood biochemistry ,proteinuria and renal Histopathological changes in the performance of Glomerulosclerosis in rats. Application of RT-PCR, western blot and indirect immunofluorescence staining Observate expression and distribution of nephrin,podocin,α-actinin-4 and Ubiquitin in Glomerular sclerosis model.Methods: Setting up the glomerulosclerosis model, forty SD rats, weight 100-130g, were made into glomerulosclerosis model into Control group (Sham-operated group) and model group. Model group( n=30), injection of adriamycin 5mg/kg in tail vein when right kidney removed a week later. Control group (group A, n=10) injection of saline equivalent. At the five weeks will be divided model group into the intervention group and non-intervention group. Intervention group 20, intraperitoneal injection MG-132, Divided into low-concentration group(group B, n=10) and high concentration of group(group C, n=10), continuous injection of 3 days. Non-intervention group(group D, n=10), intraperitoneal injection Saline equivalent of MG-132, continuous injection of 3 days. All rats were divided into the cage rearing, freedom standard feed and water.Blood and urine specimen were collected at the week 6 after setting up the glomerulosclerosis model and the week 8 after setting up the glomerulosclerosis. Blood albumin, cholesterol, urea nitrogen(BUN), creatinine and the excretion amount f 24 hours urine protein were determined. The kidney was resected, part of it was placed into 10% formaldehyde solution, and the renal cortex was separated from other part of kidney and put into -70℃for frozen saving to be tested.Detection of mRNA level of nephrin,podocin,α-actinin-4,ubiquitin were measured by RT-PCR; The protein levels of nephrin,podocin,α-actinin-4,ubiquitin were measured by means of western blot analysis; Observation the distribution change of nephrin,podocin,α-actinin-4,ubiquitin by Indirect immunofluorescence. Hematoxylin-Eosin staining was used to observe the renal pathological change.The datum shown with mean±SD(x—±s),were analyzed by One-way ANOVA,LSD-t, nonparametric test, liner correlation using SPSS 17.0. The difference was considered when P <0.05.Results:1. Changes in urinary proteinB group and C group were compared with A group ,urinary protein showed a visible rise,there were significant differences(P<0.05).Group B and C of the urinary protein group there was no significant difference(P>0.05)The degree of urinary in the 8 weeks protein than the 6 weeks are heavier.With the D group has statistically significant differences, The 8 weeks than increase to the level of the 6 weeks(P<0.05).2. Biochemical changes in bloodThe 6 weeks, B group of CHOL, BUN, Cr increased significantly with the A group's ,there were significant differences(P<0.05).ALB of C Group is Lower than A group,There were significant differences(P<0.05).CHOL, BUN, Cr increased significantly, with the A group's 5.17 times, 2.7 times, 2.48 times.With the A group, ALB of D Group is 0.43 times,There were obvious differences(P<0.05).CHOL, BUN, Cr increased significantly with the A group's, there were obvious differences(P<0.05).Analysis of variance between the four groups, the target in four group were statistically significant differences(P<0.05).ALB of B group, C group, D group significantly decreased, CHOL increased；BUN, Cr than the A group has ignificantly increased, there were obvious differences(P<0.05).The 8 weeks, with the A group, BUN, Cr of B group has statistical significance (P<0.05).With the A group, ALB of C Group is 0.43 times,the difference has statistical significance (P <0.05). CHOL, BUN, Cr increased significantly, with the A group's 5.09 times, 6.10times,3.62times.ALB of group D significantly decreased with B group and C group, CHOL, BUN, Cr increased, the difference has statistical significance, (P <0.05).ALB of D group is 0.32times compared with A group. CHOL, BUN, Cr increased significantly, with the A group's 7.03 times, 6.10times,4.16times. the difference has statistical significance (P <0.05).Analysis of variance between the four groups, the target in four group were statistically significant differences(P<0.05).B, C, D group significantly decreased ALB blood, CHOL increased；BUN, Cr than the A group has significantly increased.3．Pathological changesA group of glomerular renal tubular evenly distributed, no tube, no swelling of glomerular hyperemia.The 6 weeks and 8 weeks were no significant changes.In 6 weeks, in part glomerular of B group and C group hyperemia, swelling, tubular epithelial cells loose change, vacuolation, focal renal tubular expansion, visible protein cast. In D group a small number of rat kidney have segmental glomerular sclerosis, focal hyperplasia, diffuse infiltration of inflammatory cells.The 8 weeks, B group and C group have been seen more podocyte, mild mesangial matrix hyperplasia, the ball is mild wall thickening, mild interstitial inflammatory cell infiltration, focal expansion have a small tube, tube-type protein could be found. Part of D group rats segmental glomerular sclerosis group, adhesion balloon, telangiectasia, moderate interstitial fibrosis.4．RT-PCR detection of renal cortex the mRNA level of nephrin, podocin,α-actinin-4 and ubiquitin,nephrin mRNA:in the 6 weeks and 8 weeks, the level of nephrin mRNA of B group, C group ,D Group slightly decreased respectively, the difference has statistical significance(P<0.05).Analysis of variance between the four groups, The 8 weeks the level of nephrin mRNA in each group have declined than the 6 weeks, the difference as statistical significance(P<0.05).podocin mRNA:in the 6 weeks, the level of podocin mRNA of B group, C group slightly increased, respectively, an increase of 1.30 times, 1.32 times with A group, the difference has statistical significance(P<0.05).in the 8 weeks, D Group podocin mRNA level is 0.57 times with the A group, the difference has statistical significance(P<0.05).The 8 weeks the level of nephrin mRNA in each group haven't declined than the 6 weeks, the difference has no statistical significance(P<0.05).α-actinin-4 mRNA:in the 6 weeks and 8 weeks, the level ofα-actinin-4 mRNA of B group, C group and D group no significant increase or decrease,there was no statistically significant differences(P<0.05).ubiquitin mRNA:in the 6 weeks,the level of ubiqutin mRNA of B group, C group and D group increased, respectively, an increase of 1.93 times, 2.21times, 2.42 times with A group, the difference has statistical significance(P<0.05).in the 8 weeks,the level of ubiqutin mRNA of C group and D group increased, an increase of 1.99 times, 2.03times with A group, the difference has statistical significance(P<0.05).The 8 weeks the level of nephrin mRNA in each group have increase than the 6 weeks, the difference has statistical significance(P<0.05).5．Western Blot detected the protein expression of nephrin,podocin,α-actinin-4 and ubiqutinThe Molecular Weight of nephrin, podocin,α-actinin-4,ubiqutin are180KDa,45K- Da, 100 KDa, 60 KDa . In 100KDa, 40KDa, 100 KDa, 40 KDa and appeared clear strip.Nephrin:The expression of the model group gradually weakened, which the exp- ression of the weakest in group D, In the six weeks and eight weeks the expression w- ere 0.28 and 0.37 times with the A group. Group B in the six weeks and in eight week is 0.74 times and 0.62 times with the A group, Group C in the six weeks and in eight weeks is 0.76 times and 0.58 times with the A group, there were significant difference (P<0.05). Nephrin expression of C group in the 8 weeks reduced than the first six we- eks significantly , there were significant differences(P<0.05). Podocin:B group, C group and D group gradually weakened expression, the exp- ression of D group the weakest. In the 6 weeks and 8 weeks the expression were 0.53 times and 0.44 times; B group and C group expression in much reduced compared wi- th A group,There were significant differences(P<0.05).Α-actinin-4:, the expression of model group there was no significant difference compared with the A group, in the six weeks and in eight weeks(P<0.05).Ubiqutin:B group, C group and D group gradually enhanced the expression, the expression of D group in the 6 weeks and 8 weeks the expression were 1.70 and 2.36 times with the A group. Group B in the six weeks is 1.19 times with the A group, the difference has statistical significance(P<0.05).ubiqutin expression of B group and C group have no significantly in in the 6 weeks and 8 weeks.6．Indirect fluorescence immunostainingA group was found by staining nephrin and podocin immunofluorescence staining showed a row along the glomerular capillary loop-like distribution. The first six weeks nephrin and podocin line along the glomerular capillary loop-like distribution, and gradually to the kind of discontinuous distribution of coarse-grained model,expression gradually weakened. The eight weeks a further weakening of the expression, impaired glomerular increased.The As the condition deteriorated, a-actinin-4 gradually become coarse granular, expression gradually weakened.Under normal circumstances, Ubiquitin distribution in the glomerular and tubular, Mainly located in tubular-Mesenchymal, was fine granular distribution. As the condition deteriorated, expression gradually increase, enhanced staining gradually expanded, throughout the glomerular and tubular, some regions showed linear patchy and continuous enhancement, and the eight weeks of staining than the 6 weeks slightly enhanced.7．The correlation analysis between urinary protein and the expression of ubiquitin, nephrin, podocin,α-actinin-4The expression of nephrin and protein in urine was negatively correlated (r=-0.653,P<0.05);the expression of podocin and protein in urine was negatively orrelated (r=-0.519,P<0.05);the expression of a-actinin-4 and protein in urine was no correlation ((r=-0.294,P>0.05); the expression of ubiquitin and in urine was positively correlated (r=0.837,P<0.005).conclusion:1.Establishment the rat model of adriamycin-induced nephropathy,observed that the nephrin, podocin,α-actinin-4, ubiquitin both in the glomerular expression,Ubiquitin in the renal tubule - interstitial also expression. Ubiquitin expression in the development of disease enhanced, nephrin, podocin expression relative decline,α-actinin-4 expression had no significant change.2.MG-132 is a proteasome inhibitor, when blocked the ubiquitin-proteasome pathway,ubiquitin expression decreased and podocin expressed, nephrotic syndrme symptom improvement.Prompting ubiquitin-proteasome pathway involved in the process of nephrotic syndrome. |
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