Expression Of Podocin, Laminin And Fibronectin In Adriamycin-induced Nephropathy Rat Model And Its Significance

Objectives:The model of rat adriamycin-induced nephropathy(AIN) was reported by the foreigndocuments in the early 1980s, which is recognized currently as a better animal model to simulatethe diseases of human kidney. The adriamycin-induced nephropathy processes the characteristicsof causing the chronic progressive renal damage. Adriamycin causes proteinuria throughdamaging the glomerular filtration barrier. Podocin, laminin (LN) and fibronectin (FN) arecomponents of the glomerulus filtration barrier, which play an important role to maintain thenormal functions of glomerular filtration. How do podocin、laminin and fibronectin changeduring the cause and development of kidney disease? It is still unclear that if they have anyinteractions and mechanism with the cause of proteinuia. Therefore, Beginning with the model ofAIN, the experiment through the dynamic observation of the expression and distribution ofpodocin, laminin, fibronectin in kidney tissue and the relationship with 24-hour urine protein,makes a further exploration of their mechanism in the cause and development of urinary proteinand its correlation.Methods:1. Divide 30 male Sprague Dawley (SD) rats into two groups at random: 1) The normal controlgroup with 6 male SDs, 2) the group of AIN with 24 male SDs. The model is built trough thefirst injection of adriamycin (6.5mg/kg) by tail vein to the group two, and then after 7 days, thesecond injection of adriamycin(4mg/kg) to the group two. After building the model, the model ofAIN group is divided into four subgroups randomly——2 weeks, 4 weeks, 6 weeks and 8 weeks,each group with 6 SDs. The normal control group is injected with the same amount of salinesolution as the group two at the same time.2. We will observe the 24-hour urine protein quantitative (24hnp) of the SDs and detect thevariations of plasma albumin (Alb), cholesterol (Chol), creatinine (Scr) and blood urea nitrogen(BUN) by automatic biochemical analyzer. After that we will make a comparison of biochemicalindexes between the two groups.3. Observe the pathological changes in renal tissue of rat through light microscope, and comparethe several subgroups of group two with group one.4. Detect the expressions and locations of podocin, laminin and fibronectin in renal tissue by themeans of immunohistochemistry two-step and frozen immunofluorescence, and then make acomparison of expression of proteins podocin, laminin and fibronectin between the two groups,and its relationship with 24-hour urine.Results: 1. Characteristics of pathological changes of kidney in ratsUnder the light microscope, we detect that the capillary lumen open level of glomeruluswere right in normal control group, and the tubule epithelia cells were arranged regularly, notappear focal segmental glomerular sclerosis. As the increase of the time, the amounts ofglomerular mesangial cells and extracellular matrix multiply gradually in model groups. Thetubule epithelia cells were atrophy, lots of albumen tubule type were appeared.2. Comparison of biochemical indexesModel 2, 4, 6, 8 week group of rats proteinuria continue to rise. Compared with the controlgroup, model 6, 8 week group of rats 24-hour urine protein quantitative and blood biochemicalparameters (plasma albumin, cholesterol, blood creatinine and blood urea nitrogen) differencesare significant (P<0.05). Model group appears proteinuria, low protein blood disease,hyperlipidemia, renal failure, which characteristics are typical of nephrotic syndrome.3. Podocin, laminin, fibronectin protein expression (Immunohistochemistry)Immunohistochemistry result indicate that podocin protein of control group was brownparticles expression of strongly positive, continuous linear distribution only along the glomerularbasement membrane. Renal tissue of negative control without positive expression. Theexpression of podocin protein in model 2, 4, 6, 8 week group were significantly lower than thoseof the control group, the difference was statistically significant (P<0.01). The protein of laminin,fibronectin in control group were brown particles small amount expression in mesangial area,basement membrane. The expression of laminin, fibronectin in model 2, 4, 6, 8 week group weresignificantly higher than those of the control group, the difference was statistically significant(P<0.01).4. The localization of podocin, laminin, fibronectin protein by immunofluorescenceThe result of frozen immunofluorescence indicated that podocin protein of control groupwas mainly located in the glomerular, linear distribution evenly along the glomerular capillaryloop. With the disease progress, the distribution of podocin protein in model 2, 4, 6, 8 weekgroup began to appear discontinuous distribution. Laminin protein of control group were mainlylocated in mesangial matrix and (including glomerular, renal capsule and the basementmembrane of renal tubule); fibronectin was distributed mainly in the mesangial matrix. Withthe time progress, the expression of laminin, fibronectin protein in model 2, 4, 6, 8 week groupwere all enhanced. As the time of model extended, the expression of podocin in each modelgroup was lower than the control group (P<0.01) which expressed a declining trend. Theexpression of laminin,fibronectin of each model group were higher than those of the controlgroup (P<0.01), and with the time progress expressed a increasing trend.5. The relationship among podocin, laminin, fibronectin and 24-hour urine protein 24-hour urine protein quantitative was negatively correlated with podocin proteinexpression level, the difference was statistically significant (P<0.01). 24-hour urine proteinquantitative was positively correlated with laminin, fibronectin expression levels, the differenceswere statistically significant (P<0.01).Conclusion:1. As the model cycle extended, adriamycin nephrotic syndrome model performance for a largenumber of proteinuria, gradually reduced albumin, blood creatinine、blood urea nitrogen andcholesterol rising, lesions of glomerular mesangial cells and matrix on the rise.2. The occurrence and development of proteinuria is closely related to abnormal expressionof podocin,which may be important factors involved in the progression of renal disease.3. The over expression of laminin and fibronectin are likely to involved in increasingnumber of glomerular mesangial cells and excessive accumulation of extracellular matrix, andplays an important role in the process of glomerular sclerosis.