Creation and analysis of transgenic and knockout animals in hypertension and its related diseases.

1)血管紧张素原基因敲除小鼠的功能分析血管紧张素原缺乏遗传改变小鼠(AGT-KO)表现出预期的表型，如明显的低血压，但意外的表型包括肾脏形态异常，新生儿存活率下降，冷损伤后血脑屏障功能受损。为了验证血管紧张素原基因的破坏是否与观察到的表型有关，我们在小鼠金属硫蛋白-I启动子(MT-AGT)的控制下产生了一系列血管紧张素原基因杂合子的小鼠，并将转基因小鼠与AGT-KO小鼠杂交。得到的小鼠(MT-AGT(+/-)/AGT(-/-)：MT-AGT/KO)产生与野生型小鼠相当的血浆血管紧张素I水平，突变的表型得到挽救。2)血管紧张素原基因敲除小鼠的新功能血管紧张素原基因敲除小鼠(Ag-KO)的形态分析显示，海马区和小脑颗粒层细胞密度降低，提示Ag-KO的神经细胞易发生细胞凋亡。通过降低血清浓度的海马神经元培养进一步研究了这一现象。然而，在血管紧张素II(ALL)和血管紧张素IV(AIV)存在的情况下，在较低浓度的血清中出现数小时内出现凋亡的AG-KO神经元细胞存活的时间要长得多。这种抗凋亡作用不受AII受体拮抗剂的干扰。这些结果表明，肾素-血管紧张素系统可能在中枢神经系统中发挥关键作用，不仅可以通过ALL，也可以通过AIV来阻止神经细胞的凋亡。

The important research results in this project are summarized in the folowing two sections.1) Functional analysis of angiotensinogen-knockout miceGenetically altered mice lacking angiotensinogen (Agt-KO) showed an expected phenotype, such as marked hypotension, but unexpected ones including abnormal kidney morphology, reduced survival rates of newborns, and impaired blood-brain barrier function after cold injury. To examine whether disruption of the angiotensinogen gene is responsible for the observed phenotypes, we generated a line of mice heterozygous for the mouse angiotensinogen gene under the control of a mouse metallothionein-I promoter(MT-Agt) and crossmated transgenic mice with Agt-KO mice. The resulting mic (MT-Agt(+/-)/Agt(-/-) : MT-Agt/KO) produced the plasma level of angiotensin I comparable to that of wild-type mice, and the mutant phenotypes were rescued.2) New function of angiotensinogen knock-out miceThe morphological analysis in a congenic line of angiotensinogen knock-out mice (Ag-KO) revealed the decreased density in granular layer cells of hippocampus and cerebellum, suggesting neuronal cell of Ag-KO susceptible to apoptotic cell death. This phenomenon was further studied by culture of the hippocampal neurons with decreased concentration of serum. Ag-KO neuronal cells, which showed apoptosis by lower concentration of the serum within several hours, however, survived much longer in the presence of angiotensin II (All) and IV(AIV). This anti-apoptotic action was not interfered by AII receptor antagonists. These results suggest that the renin-angiotensin system could play a critical role in central nervous system, preventing neuronal cells from apoptosis not only by All but also AIV.