| BACKGROUNDDiabetic nephropathy (DN) is one of the most dangerous microvascular complications of diabetes mellitus (DM), the exact pathogenesis has not been fully clarified. Glomerular visceral epithelial cells (podocyte) injure in diabetic nephropathy plays an important role in the development of proteinuria. Podocyte injury occurs in early stage of DN, induces proteinuria and accelerates the progress of renal disease. Now, the mechanism of podocyte injury in diabetic nephropathy is not very clear. In recent years, we have recognized some protein molecules of the podocytes involved in signal transmission to maintain the normal structure of podocyte. Mutation or deletion of these protein genes can lead to changes in cell structure and number causing proteinuria and glomerular sclerosis.α3β1 integrin is the main adhesion molecules connecting the glomerular basement membranet and podocyte, maintains normal podocyte morphology and GBM permeability,and involves in signal transduction.α3β1 integrin antagonists could decrease podocyte adhesion to extracellular matrix, and cause cell apoptosis. Rats withα3 orβ1 integrin deficiency have glomerular basement membrane rupture stratification,filtration barrier damage and degradation of foot process at birth. In addition,podocyte density was aslo severely reduced,a large number of proteinuria occurred.α3β1 integrin expressed by podocyte in DM patients or diabetic rats are suppressed, and with the the extension of course and elevated blood glucose, this inhibitory effect increased. Studies have shown that mechanical stress, AngⅡ, TGFβ1, ROS can regulateα3β1 integrin expression in podocytes.A large number of basic and clinical evidence have shown that Irbesartan (AT1 receptor antagonist) plays great role in delaying disease progress on diabetic nephropathy and with dose-dependent. Therefore, we propose the following assumptions:Irbesartan may reduce foot cell loss through regulating renal Angll and partialα3β1 integrin expression in kidney tissue, independent of antihypertensive effect, thereby improving the filtration barrier damage and delaying diabetes kidney disease with dose-dependent.This study by in vivo studies, interfering with irbesartan,we'll observe the renal local AngⅡ, renalα3β1 integrin, podocyte cell density in diabetic rats, to clarify whether the protective effect with dose-dependent of irbesartan and relationship among above-mentioned factors, to provide a basis for animal experiments for the Irbesartan dose-dependent mechanism of renal protection and new ideas preventing diabetic nephropathy.Chapter 1 Effect of irbesartan on podocyte in diabetic rats independent of blood-pressure-lowering and the correlation with dosage[Objective]1. To observe the urinary albumin excretion, renal mass index, the volume of renal glomerulus and glomerular podocyte number, the impact of renal pathology in STZ-induced diabetic rats administrated with different dosage of irbesartan and to confirm the renoprotective effect of irbesartan independent of blood-pressure-lowering.2. To approach the correlation between the renal protection effect and the dosage of irbesartan.[Method]1. Sixty-three adult male Wistar rats, weighing initially 240 to 280g were bought from experimental animal center of Southern medical university and were utilized in this study. Rats were maintained at 24±3℃, with relative air humidity at 60±5%,and under 12/12h dany-night cycle. All rats are randomly divided into 3 groups, normal contrl group (Group Ctrl, n=7), diabetic control group (Group DM, n=14) and drug administrated group (n=42).2. After an overnight fast, rats from diabetic control group and drug administrated group were given a single intraperitoneal injection of 55mg/kg body weight of streptozotocin (Sigma, St. Louis, MO, USA) diluted in 0.1M citrate buffer(pH4.5). Rats with tail vein blood glucose concentration in excess of 16.7 mmol/L,72 hours post-induction of diabetes, were included in the study as diabetic rat.3. Diabetes rats from drug administrated group further were randomly divided into 4 groups.4 weeks after induced, diabetic rats were received 25mg kg-1 d-1 irbesartan (Group DM+Irb25, n=9),50mg kg-1 d-1 irbesartan (Group DM+Irb50, n=9),200mg kg-1 d-1 irbesartan (Group DM+Irb200, n=9), and 30mg kg-1 d-1 Hydralazine (Group DM+Hyd, n=9) with orogastric gavage respectively. Rats from group Ctrl and DM were administrated with equal volume water as control.4. During the four weeks after induced,2-4 U insulin (Lantus, Sanofi-Aventis, France) were provided to all diabetic rats thant blood glucose is 30mmol/L or more every two day to maintain body weigh, prevent ketoacidosis, and improve survival. During the study, all rats receiced standard rat chow and free access to tap water.5. Body weight of all animal was measured with electronic balance before and 8 weeks after administration. Tail-cuff pressure (TCP) was measured with PowerLab system (Adinstrument, Australian) before and 8 weeks after administration.6. Eight weeks after administrated, all animals were sacrificed.24 hour urine were collected and used for biochemical measurements. Pentobarbital sodium was administered antraperitoneally, and kidneys were perfused with ice-cold PBS at a constant flow rate to flush out blood. Kidneys were removed quickly, cut longirudinally, and fixed with 10% buffered formalin phosphate for 24 hours. Remainder in the tube were put in liquid nitrogen for freezing preservation.5. Determination of urinary albumin excretionThe rats were placed in metabolic cages for 24h to determine urinary albumin excretion by using Rat albumin ELISA quantitation kit from Bethyl Laboratories.6. Kidney tissue were perfused with 10% neutral formalin solution and embedded in paraffin. Sections (2μm) were deparaffinized in xylene and rehydrated through graded clcohols. Wax sections were stained with periodic acid-Schiff (PAS) for light microscopic morphologic study.7. Podocyte cell density measurement:10% neutral buffered formalin solution, fixed, conventional dehydration, dipping wax, embedded,5μm paraffin sections, immunohistochemical SP method detected WT1 expression in glomeruli with WT1 rabbit anti-rat polyclonal antibody (Merk Company); Image-Pro Plus 6.0 Semi-quantitative color image analysis system measure cell density.8. Statistical analysisStatistical analyses were conducted with SPSS 13.0. All data are presented as means±SD. Differences between groups were analyzed using One-way analysis of variance (ANOVA) and covariance analysis; multiple comparisons were analyzed by LSD method when P values less than 0.05. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Games-Howell method when P values less than 0.05. Statistical significance was accepted at a value of P<0.05. Analysis of measurement data before and after intervention use Independent-Samples t test. Rat tail blood glucose compared with a number of independent samples of non-parametric (Kruskal-Wallis H) test. Pairs of variables do not meet the normal distribution, correlation analysis using Sperman correlation analysis; P<0.05 indicated that the difference was statistically significant.[Results]1. The characteristics of experimental animalsOne rat from group DM died probably resulting from ketosis, and one from group DM+Irb50 died of intragastric administration.Before administration, Blood glucose was higher in diabetic rats than that in the normal rats. Blood glucose was not significant difference among the five groups except group Ctrl (x2=0.126, P=0.998). Body weight in group DM was significantly lower than that in group Ctrl (P<0.001),but is not statistically different between group DM,DM+Hyd, DM+Irb25, DM+Irb50 and DM+Irb200 (P>0.05).2. Effect of Irbesartan administration on tail-cuff blood pressure (TCP)Before administration, TCP in group DM was significantly higher than group Ctrl (P<0.001).After drug intervention, the blood pressure before drug intervention as covariates, the differencen of blood pressure among groups is significant (P<0.001).3. Effect of Irbesartan administration on urinay albumin excretionin (UAE) in diabetic rats Urinary albumin excretion (UAE) in group DM was significantly increased versus group Ctrl (P<0.001). Irbesartan or hydralazine treatments brought UAE to levels significantly lower than that in group DM (P<0.001). UAE in the three groups administrated with irbesartan was significantly lower than that in group DM+Hyd (P<0.05), and UAE in group DM+Irb50 and DM+Irb200 was significantly lower compare with that in group DM+Irb25 (P<0.01). There was not statistically different between group DM+Irb50 and DM+Irb200 (P>0.05).4. Effect of Irbesartan administration on Renal mass index (RMI) and volume of glomerulus (Vg) in DM ratsRenal mass index (RMI) in group DM was singnificantly increased compare with that in group Ctrl (P<0.001), which demonstrated kidney hypertrophy in diabetic rats.8 weeks after administration, RMI in group DM+Hyd was significantly decreased compare with that in group DM (P<0.01). RMI in the three groups administrated with irbesartan was significantly decreased compare with that in group DM (P<0.001) and DM+Hyd (P<0.05). RMI in group DM+Irb50 and DM+Irb200 was significantly decreased than that that in group DM+Irb25 (P<0.05). There was not statistically different between group DM+Irb50 and DM+Irb200 (P>0.05).Volume of glomerulus (Vg) in group DM was singnificantly increased than that in group Ctrl (P<0.001). That demonstrated glomerular hypertrophy in diabetic rats.8 weeks after administration, Vg in group DM+Hyd was significantly decreased than that in group DM (P<0.001). Vg in the three groups administrated with irbesartan was significantly decreased compare with that in group DM (P<0.001), and DM+Hyd (P<0.05). Vg in group DM+Irb50 and DM+Irb200 was significantly decreased than that in group DM+Irb25 (P<0.001). There was not statistically different between group DM+Irb50 and DM+Irb200 (P>0.05).5. The average density of glomerular podocyte Glomerular podocyte density of rats from diabetic control group is significantly lower than the normal control group (P<0.001). After Hydralazine intervention, glomerular podocyte density was significantly higher than DM group (P<0.001). After the intervention of different doses of irbesartan, diabetic glomerular podocyte density was significantly higher than DM group (P all <0.01), DM+Irb200 was significantly higher than DM+Hyd (P<0.01), DM+Irb25(P<0.01), and DM+Irb50 (P <0.05); DM+Hyd,DM+Irb25,and DM+Irb50 was no significant difference among the three groups (P> 0.05). Podocyte density and urinary albumin excretion rate was significantly negative correlation, R=-0.794, P<0.001.[Conclusions]1. Irbesartan significantly reduced microalbuminuria in diabetic rats, kidney hypertrophy and glomerular hypertrophy, decreasing podocyte loss to slow down the progress of diabetic kidney disease; in the context of a certain dose range, renal protective effect of Irbesartan is dose-dependent.2.1n Diabetic rats, the loss of podocyte increases the permeability of glomerular filtration, is also one of the reasons of proteinuria.3. Irbesartan also has other improvements of filtration barrier, slows down diabetic nephropathy beyond lowering BP. The specific mechanism has to be further studied.Chapter 2 Effects of irbesartan on the local kidney AngⅡof diabetic rats and glomerular expression ofα3β1 integrin[Objective]To Observe the influence of irbesartan on renal local AngⅡlevel of STZ-induced diabetic rat, expression ofα3β1 integrin and their dose relatonship. Exploring the mechanism of renal protective by irbesartan to provide animal experiments basis for clinical application of ARBs and theoretical basis for developing new drugs.[Method]1. Renal local AngⅡdeterminationFrozen kidney tissue was moved into 1.5ml EP, wash with ice-cold 0.9% saline, and clipped. Adding 1mL 0.1 mol/L acetic acid with a homogenizer to make homogenate.95℃water bath 10min,4℃,15000rmp centrifugalization 20min and to draw the supernatant.2.Renalα3β1 intergrin mRNA expressionReal-time RT PCR was used to detect the expression ofα3β1 intergrin mRNA in the kidney. The levels ofα3β1 intergrin were present as CT values,GAPDH is reference.3.Statistical analysesStatistical analyses were conducted with SPSS 13.0 for windows. All data are presented as means±SD. Differences between groups were analyzed using One-way analysis of variance (ANOVA), and multiple comparisons were analyzed by LSD method when P values less than 0.05. Welch method was used when equal variances is not assumed, and multiple comparisons was analyzed by Games-Howell method when P values less than 0.05. Statistical significance was accepted at a value of P< 0.05. Multivariate correlation analysis, use Partial correlation analysis method.[Results]1. Effect of Irbesartan on renal AngⅡlevel in diabetic ratsRenal AngⅡlevel in DM group was significantly higher than Ctrl goup(P<0.001). After administration with different doses of Irbesartan, diabetic rat renal AngⅡlevels were significantly lower than that in group DM (P< 0.001). AngⅡin group DM+Irb200 was significantly decreased compared with DM+Irb25 and DM+Irb50 (P<0.05). AngⅡin group DM+Hyd was significantly lower than that in group DM (P<0.001).2. Effect of Irbesartan on renal integrinβ1in diabetic ratsExpression of renal a3 integrin mRNA in DM group is significantly lower than Ctrl group, (P<0.001). DM+Hyd was significantly higher than DM group(P<0.001). After the intervention of different doses of irbesartan, expression of renalα3 integrin mRNA in diabetic rats was significantly higher than DM group (P all <0.01), DM+Irb200 was significantly higher than DM+Irb25 (P<0.001)and DM+Irb50 (P <0.01), but is no significant difference compared with DM+Hyd. (P> 0.05).3. Effect of Irbesartan on renalβ1ntegrin mRNA in diabetic ratsExpression of renalβ1 integrin mRNA in DM group is significantly lower than Ctrl group (P<0.001). DM+Hyd was significantly higher than DM group (P<0.001). After the intervention of different doses of irbesartan, DM+Irb200 DM+Irb50 are significantly higher than group DM (P all <0.05); DM+Irb200 is significantly higher than DM+Irb50 (P<0.001) and DM+Irb25 (P<0.001), but no significant difference compared with DM+Hyd (P> 0.05). There was no significant difference between DM+Irb25 and DM+Irb50 (P> 0.05)4. Correlation analysis4.1 Podocyte density and renalα3,β1 integrin mRNA was significantly related, Rα3= 0.883, Rβ1= 0.853, P all <0.001.4.2 Rena locall AngⅡand renalα3,β1 integrin mRNA was significantly related, Rα3=-0.527, P<0.001, Rβ1=-0.421, P<0.001 The rat tail artery blood pressure and renalα3,β1 integrin mRNA is not related, Rα3=-0.183, Rβ1=-0.263, P all> 0.05.[Conclusions] In diabetic rat, renal local RAS system is activated, local AngⅡwere significantly increased in line with past research. Renalα3/β1-integrin mRNA decreased significantly leading to podocyte loss. Byond blood pressure,Irbesartan may down-regulate renal AngⅡlevels, improves renalα3,β1 integrin mRNA expression,increases the glomerular podocyte density to decrease proteinuria with dose-dependent.
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