RENAL KALLIKREIN-BINDING PROTEIN IN HYPERTENSION

本提案的目的是研究肾脏的功能作用， （组织）激肽释放酶结合蛋白在高血压中的作用。 组织激肽释放酶 一组同源丝氨酸蛋白酶，由多基因家族编码， 参与生物活性肽和激素的加工。 激肽释放 及其产物在细胞稳态和血液中发挥重要作用 压力调节 虽然生物化学和调节 正常和疾病状态下的激肽释放酶基因表达已经被 广泛研究，关于调节 激肽释放酶的功能非常有限。 我们最近发现了一本小说 在大鼠和人中形成的不可逆的激肽释放酶结合蛋白 与组织激肽释放酶复合。 大鼠和人的结合蛋白， 与组织激肽释放酶形成不可逆的复合物。 述结合蛋白 在自发性高血压大鼠（SHR）中有缺陷，这表明它 可能是SHR高血压表型的一个促成因素。 本研究的具体目的是：1）纯化和表征 激肽释放酶结合蛋白;开发单克隆和多克隆抗体 用于确定激肽释放酶的分布、数量和定位- 通过结合测定法、放射免疫测定法、蛋白质印迹法， 免疫组织化学和生理学研究; 2）阐明 激肽释放酶结合蛋白在激肽释放酶调节中的作用 生物利用度通过体内清除和催化剂研究; 3）分离 大鼠肝脏激肽释放酶结合蛋白cDNA克隆 文库，并使用这些克隆来鉴定和定位激肽释放酶结合 通过北方、斑点杂交和原位cDNA-mRNA检测蛋白基因表达 杂交; 4）分离和表征编码所述蛋白的基因， 从大鼠基因组文库中筛选激肽释放酶结合蛋白，并鉴定其 潜在的顺式和反式作用元件参与组织特异性 通过缺失分析和核蛋白结合分析基因的表达 5）分析限制性片段长度多态性相关的 与激肽释放酶结合蛋白基因在遗传性高血压动物 模型 从这些研究中获得的信息应该提供一个更好的 了解激肽释放酶-激肽系统在高血压中的作用。

The objective of this proposal is to study the functional role of renal (tissue) kallikrein-binding protein in hypertension. Tissue kallikrein are a group of homologous serine proteinases, encoded by a multigene family and involved in the processing of bioactive peptides and hormones. Kallikrein and its products play important roles in cellular homeostasis and blood pressure regulation. Although the biochemistry and regulation of kallikrein gene expression in normal and disease states have been extensively studied, information regarding factors which modulate kallikrein function is very limited. We have recently identified a novel kallikreinbinding protein in rat and human which forms and irreversible complex with tissue kallikrein. The binding protein in rat and human which form an irreversible complex with tissue kallikrein. The binding protein is defective in spontaneously hypertensive rats (SHR), suggesting that it may be a contributing factor in the hypertensive phenotype of SHR. The specific aims of this study are: 1) to purify and characterize kallikrein-binding protein; develop monoclonal and polyclonal antibodies for determining distribution, quantities and localization of kallikrein- binding protein by binding assays, radioimmunoassays, Western blotting, immunohistochemistry and for physiological studies; 2) to elucidate the role of kallikrein-binding protein in the regulation of kallikrein bioavailability by in vivo clearance and catabolism studies; 3) to isolate cDna clones encoding the kallikrein-binding protein from a rat liver cDNA library and to use these clones to identify and localize kallikreinbinding protein gene expression by Northern, dot blot and in situ cDNA-mRNA hybridization; 4) to isolate and characterize the gene encoding the kallikreinbinding protein from a rat genomic library and to identify the potential cis- and trans-acting elements involved in tissue-specific expression of the gene by deletion analysis and nuclear protein binding assays; 5) to analyze restriction fragment length polymorphisms associated with the kallikrein-binding protein gene in genetically hypertensive animal models. The information gained form these studies should provide a better understanding of the kallikrein-kinin system's role in hypertension.