| ObjectivesDiabetes mellitus is one of the main causes of the end stage renal disease. And the morbidity of diabetic nephropathy is increasing faster than ever. With the study of the diabetic nephropathy, the function of podocytes in the pathogenesis of diabetic nephropathy is becoming more importance. And the latest discovery shows that podocyte is uniquely insulin sensitive in the filtration barrier of the kidney. podocytes in the patients of diabetes nephropathy exist insulin resistance, and angiotensin II(AngII) can impair glucose uptake through downregulation of glucose transporter in the adipocytes and smooth muscle cells. At present, there is no evidence to show that whether AngII can induce insulin resistance and dysfunction in podocytes. Our study is to investigate the function of AngII of different concentration in the expression of GLUT1 and the organization of F-actin. meanwhile, we pre-treat cells with losartan before stimulating cells with AngII, in order to study the relationship between dysfunction of podocytes and AngII in the development of diabetes nephropathy and the mechanism of AT1RA in the protection of podocytes. Methordâ‘ mouse podocytes were divided into 5 groups: control, 10-10mol/L angio- tensinII, 10-8mol/L angiotensin II, 10-6mol/L angiotensin II, 10-6mol/L angiotensin II plus 10-6mol/L losartan, expression of GLUT1 mRNA was detected by RT-PCR , expression of GLUT1 protein was measured by Western Blot.â‘¡Then the mouse podocytes were divided into another 5 groups: control,10-6mol/L angiotensinII, 10-6mol/L insulin, 10-6mol/L angiotensinII plus 10-6mol/L insulin, 10-6mol/L losartan plus 10-6mol/L angiotensinII and 10-6mol/L insulin, fluorescence microscope was used to detect the localization of GLUT1 and the organization of F-actin.â‘¢Image-J is used through Semiquantitative analysis to analyze the result of PCR and Western blot , to investigate the AngII'S function in the expression of GLUT1; IPP is used to analyze the presence and the localization of GLUT1.Result1,the change of GLUT1 transcriptionAngII groups significantly increased GLUT-1 transcription by 5.89%, 21.46% and 30.95% respectively, and this effect was concentration dependent(P<0.01). losartan group decreased GLUT-1 transcription by 30.64% compared with AngII(10-6mol/L) (P<0.01).2,the change of GLUT-1 protein expressionAngII groups significantly increased GLUT-1 protein expression by 8.40%, 11.79% and 20.57% respectively, and this effect was concentration dependent(P<0.01). losartan group decreased GLUT-1 transcription by 17.10% compared with AngII(10-6mol/L) (P<0.01).3,the localization of GLUT1Insulin increases the amount of GLUT-1 in the plasma membrane by 16.33%(P<0.01). AngII decreases the amount of GLUT-1 in the plasma membrane. Compared with Insulin, the amount of GLUT-1 in the plasma of the cells pre-treated with AngII before stimulating with insulin significantly decreased(P < 0.01). There is no difference between control group, AngII+Ins group and losartan group. 4,the change of F-actin organizationInsulin rearranged the acin cytoskeleton and actin filaments became thicker after treatment with insulin. Pretreatment of podocytes with AngII can disassemble actin filaments, and the effect of AngII can not be reversed by Insulin but can be partly reversed by pretreatment with losartan.Conclusion1,angiotensinII increases the expression of GLUT1, this effect was concen- tration dependent.2,Losartan can block the effect of angiotensinII on the mouse podocytes.3,angiotensinII prohibits the insulin-induced increasing of GLUT1 in the plasma membrance.4,Insulin rearranged the acin cytoskeleton and actin filaments became thicker after treatment with insulin, angiotensinII prevents the effects of insulin via disarrangement of F-actin. |
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