Ang Ⅱ Influences Apoptosis Of Podocytes In Membranous Nephropathy Of Rats

Objective: we plan to discuss the effects and its mechanism of Ang II and TGF-β1/P38 MAPK pathway to podocyte apoptosis in MN. Methods:120 male SD rats are randomly divided into normal control group(A), model group(group B), modeling + Benazepril intervention group(group C), Benazepril group(group D), each group 30.A group, D group, no pre-free, no modeling, every other day by intravenous injection of 2ml saline(NS) to replace C-BSA, the other two groups according to the improved Border Act [1] copy The rat model of MN; Meanwhile, group C and group D in formal pre-free first week onwards of daily with Benazepril(10mg / kg / d) orally,while group A and group B with an equal amount of NS instead of Benazepril daily gavage. Official immunization 1,2,3,4,5,6 weekend each group were killed five rats, taking 24 h urine to test 24 h urine protein(24h UTP);Detecting Alb, TG, TC, Scr, BUN from the serum;using pathological biopsy which is from rat kidney tissue to indentify the modeled by the way of light microscopy and immunofluorescence,while using immunohistochemical method dynamicly detecting expression content of the glomerular TGF-β1, P38 MAPK, Bax, Bcl-2, WT1 protein;using TUNEL assay to detect various time points glomerular intrinsic apoptosis and apoptotic index(AI) and by Elisa assay detecting kidney tissue homogenates at various time points Ang II content. Results:(1) Renal morphological changes:In group A and D,at each time point kidney color and form are normal,renal tissue showing no morphological changes,immunofluorescence not showing granular Ig G deposition;B, C group, along with the experimental prolonged,kidney volume increases, the color fades to a pale color, renal pathological lesions gradually severe and glomerular capillary loop granular deposition of Ig G fluorescent obviously.To 4 weekend, the kidneys were "white kidney" like appearance---swollen form, color pale;HE staining under light microscope,glomerular swollen obviously, basement membrane(GBM) diffuse thickening, part of the balloon stenosis obvious;by Masson staining can see part of glomerular subepithelial addicted myoglobin complex deposition,mesangial and renal interstitial showing blue positive material deposition,PASM staining show part of the epithelial side of GBM "spikes" formation, and even the formation of "false-track" sign, mesangial and renal interstitial collagen deposition and fibrosis aggravation;immunofluorescence showed glomerular capillary and mesangial Ig G have stronger fluorescence intensity,about +++~++++.To 6 weekend, renal pathology injury in addition to the performance, HE and Masson staining are seen in renal interstitial and mesangial the deposition of collagen fibers become more pronounced. Comparison between the various time points in each group, A, D group had no significant impairment performance of renal pathology;Group B has much more heavier damage of renal pathological than in the other three groups at each observation time point;Group C renal pathological damage heavier than the A, D group, but has been reduced compared with group B;Ig G fluorescence intensity of kidney tissue, comparing group B and group C have no significant difference(P> 0.05).(2)Dynamic changes of renal inherent cell apoptosis:group A and group D are seen only a small amount of tubular cell apoptosis, Glomerular having no obvious cell apoptosis,and the AI expression of glomerular is no significant difference(P> 0.05) at 1 weekend to 6 weekend,while the AI expression of Glomerular between Group D and group A are no significant differences in each weekend(P> 0.05);In Group B and group C,the apoptotic cell can be expressed in glomerular visceral distribution area,mesangial area,renal tubular epithelial cells from the first weekend,and positive cells are brown granular and glomerular expression AI molded extend over time, showed a gradual upward trend,compared with group A, the difference was significant(P <0.01);Compared with group B, the expression of glomerular AI from the first weekend was significantly reduced in group C, the difference was statistically significant(P <0.05).(3)Renal tissue dynamic expression of TGF-β1,P38 MAPK,Bax,Bcl-2 and WT-1 protein:Group A and Group D:TGF-β1, P38 MAPK, Bax in glomerular visceral distribution area, mesangial, tubular epithelial cells have little expression,Bcl-2 in the glomerular visceral distribution area, mesangial, tubular epithelial cells have more expression,and WT-1 in the cell nuclei of distribution area of the glomerular visceral have more expression,positive cells showing a brown granular,above indicators in 1,2,3,4,5,6 weekend is no significant difference in the expression amount(P> 0.05);in group D,the expression of glomerular TGF-β1, P38 MAPK, Bax, Bcl-2, WT-1 is no significant difference at each weekend,compared with group A(P> 0.05).Group B and Group C:With the modeling time extending, TGF-β1, P38 MAPK, Bax expression level is gradually rising, and mainly expressed in the glomerular visceral distribution area and mesangial cytoplasm, renal tubular epithelial cells only little expression or intensity is weak, Bcl-2 and WT-1 expression showing a gradual decline with the modeling time extending.Compared with group A, group B and group C glomerular TGF-β1, P38 MAPK, Bax expression increased, respectively, from the second and third weekend difference is statistically significant(P <0.05), Bcl-2, WT-1 decreased expression, both from the second weekend difference is statistically significant(P <0.05);Compared with group B, group C glomerular TGF-β1, P38 MAPK, Bax expression decreased, respectively, from the first weekend 4,6,5 difference is statistically significant(P<0.05), Bcl-2, WT-1 expression increase, respectively, from the first weekend 4,5 difference is statistically significant(P <0.05).(4)Kidney tissue homogenates Ang II content dynamic changes:A, D group Ang II expressed in various time points, the expression of each of the weekend was no significant difference(P <0.05);Compared with group A, group B and group C from the second weekend, with the increasing in test time, Ang II content increased significantly, especially in the 6 weekend of the most significant(P <0.05);Compared with group B, group C decreasing trend in the expression of Ang II showed each weekend, beginning from the third weekend difference was significant(P> 0.05).(5)Clinical indicators of change:A group, D group indexes properly.B, Group C TG, TC, Scr, BUN, 24 h UTP levels were higher than in group A in each weekend, Alb lower than in group A, with Alb, 24 h UTP change significantly(P <0.05).Group C in the each weekend compared with group B, Alb elevated levels of statistical significance differences from fifth weekend(P <0.05), 24 h UTP level is decreasing, but there is no significant difference between the two groups(P> 0.05).(6)6 weekend of the correlation analysis of each parameters: In glomeruli, group B and C rat renal tissue Ang II content with glomerular TGF-β1, P38 MAPK, Bax, AI, 24 h UTP is positively correlated and with WT-1, Bcl-2 is negatively correlated; AI with TGF-β1, P38 MAPK, Bax, 24 h UTP is positively correlated and with WT-1, Bcl-2 is negatively correlated; WT-1 with Bcl-2 is positively correlated, and TGF-β1, P38 MAPK, Bax, 24 h UTP is negatively correlated; TGF-β1 with P38 MAPK, Bax, 24 h UTP is positively correlated and with Bcl-2 is negatively correlated; P38 MAPK and Bax, 24 h UTP is positively correlated and with Bcl-2 is negatively correlated; 24 h UTP is positively correlated with Bax, and Bcl-2 is negatively correlated, group A and group D show no significant correlations among each group. Conclusions:①In modeling process the number of podocyte in MN rat gradually reducing;With the increase of modeling time, WT-1 decreased progressively, AI is progressively increased, WT-1 negative correlation with the AI, to prompts that the reason of reducing the number of podocyte may be related to apoptosis;②MN rats in the modeling process, localized kidney Ang II which probably play a role by activating TGF-β1 / P38 MAPK pathway causes podocyte apoptosis process. ③Benazepril by reducing generation of the MN rat kidney tissue Ang II,thereby inhibiting the activation of TGF-β1 / P38 MAPK pathway,reducing podocyte apoptosis to protect the kidney.