| Purpose:The kallikrein-kinin system(KKS) is a complex multi-enzymatic system that has been implicated in the control of renal circulation, glomerular hemodynamics, and tubular function, which include kininogen, kallikrein and kinin. Diabetic nephropathy is the most common chronic complication of DM, and the leading cause of death in DM patients. Diabetic nephropathy is characterized by thickening of the glomerular basement membrance, mesangial cell proliferation, accumulation of mesangial extracellular matrix with obstruction of the glomerular capillary lumen, and loss of glomerular filtration and function. Up to date, we know kallikrein is the main rate-limiting enzyme of KKS, which releases kinin from low moleculer weight kininogen(LMWK). Kinin by binding to its receptors suppress Mesangial cell and matrix proliferation. In the present study: (1) We used the genetic recombination method to produce kallikrein. (2) We determined renal kallikrein activity in the different stages of diabetic nephropathy (3) We injected the produced kallikrein into DN rats and observed the fuction and morphologic change of kidney to find the influence of genetic recombind kallikrein to DN. Method:(1) Delivery kallikrein gene to Hi-5 insects cell by virus, culture the insects cell and detect the human tissue kallikrein expression by ELISA. Use the ion exchange chromatography, gel-filtration chromatography to separate and purificate the human tissue kallikrein. SDS-PAGE was used to detect the purity. (2) Male Sprague-Dawley rats divided into control rats, diabetes rats and kallikrein treated rats. Diabetes was induced by a single intravenous injection of streptozotocin. 8 weeks after the induction of diabetes, urine was obtained every 4 weeks. At 16th week blood tissure kallikrein and NO, cAMP,ET-1 leverl of kidney were examed in the three groups by enzyme linked immunosorbent assay(ELISA), Nitrate reductase method, radio-immunity and immunohistochemical method. The morphologic changes of kidney were also examed by image analysising meter. Results: 1. Hi-5 insect cell has highest expression when inoculate at 1×106cells/ml and infect at 10pfu/ml. After cultured 96 hours, harvest the cells of supernatant. Molecular weight of the human tissue kallikrein is 39604Da. 2. Body weight, tissure kallikrein activity and NO,cAMP level in the kidney in diabetes group were significantly decreased compared with controls(p<0.01). The blood glucose, blood creatinine , blood ureanitrogen and Kw/Bw, ET-1, urine albumin excretory rate were significantly increased compared with controls(p<0.01or p<0.05). 3. kallikrein treated rats had higher BW and TKA activity than controls, and lower NO and cAMP level than controls(P<0.01); But BG, Kw, Kw/Bw and Ualb excretory rate were higher than controls(p<0.01or p<0.05). No significant diference was observed in blood glucose , Kw and BW between DM and DK groups. The plasma kallikrein activity and NO, cAMP level were much higher in DK group than in DM group(p <0.01). The blood glucose, blood creatinine, blood uria nitrogen, Kw, Kw/Bw, Ualb excretory rate were decreased in DK group than in DM group(p <0.01 or p <0.05). 4. (1)In diabetes rats, the TKA activity in 16th week is significantly decreased than in 8th week(p <0.05), and was significant different from the controls(p<0.01). However, the TKA activity was significantly higher in the kallikrein treated rats(p<0.05) , accompanied with attenuated renal injury. (2)In kallikrein treated rats, the albumin excretory in 12th and 16th week were decreased compared with that of the 8th week(p﹤0.05). (3)In 16th week,significant difference was observed in the albumin excretory in DK and DM groups(p <0.05). Compared with controls , the NO and cAMP level in the kidney in diabetes rats was significantly decreased in 16th week. But comparaed with the diabetes rats,the kallikrein treated rats had higher NO and cAMP level in the kidney(p<0.01 or p<0. 05).Conclusion: 1.Using the genetic recombination technology ,we can make the eukaryon in...
|
PDF Full Text Request