The circadian clock protein BMAL and post-translational regulation of ENaC in the kidney

摘要 上皮钠通道（ENaC）表达于远端小管和集合管 负责肾脏对钠重吸收的最终调节。的 豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）作为一种重要的 衔接蛋白之间的阴离子磷脂PIP 2和ENaC。ENaC和 MARCKS受蛋白酶组织蛋白酶B的正调控。首先，我们的初步 数据显示肾脏ENaC活性和MARCKS蛋白表达呈阳性， 由昼夜节律蛋白BMAL 1调节。第二，我们的初步数据显示阿尔法-1 BMAL 1敲除小鼠肾脏中的抗胰蛋白酶与 同时处死野生型小鼠。第三，我们的初步数据显示阿尔法-1 抗胰蛋白酶在肾脏中表达，它强烈抑制组织蛋白酶B活性， 有助于血压调节。在这个项目中，我们将测试我们的假设， 肾ENaC和MARCKS之间的联系及其在心尖部的功能 质膜与α-1抗胰蛋白酶表达呈负相关， 昼夜节律依赖的方式。我们将进行实验来研究蛋白质水解 ENaC和MARCKS的顶膜表达，ENaC活性，钠离子浓度， 使用雄性和雌性BMAL 1敲除小鼠的处理和血压，α-1 抗胰蛋白酶敲除小鼠，α-1抗胰蛋白酶过表达小鼠，组织蛋白酶B 敲除小鼠和野生型对照小鼠。成功完成我们的建议 该项目的研究将揭示BMAL 1在细胞凋亡中作用的新机制。 调节肾脏ENaC和MARCKS以及血压控制。我们的长期目标 是为了更好地了解原发性高血压的发病机制， 可能会导致新的药物靶点和治疗方法。

ABSTRACT The epithelial sodium channel (ENaC) expressed in the distal tubule and collecting duct is responsible for the final regulation of sodium reabsorption by the kidneys. The myristoylated alanine-rich C kinase substrate (MARCKS) plays an important role as an adaptor protein between the anionic phospholipid PIP2 and ENaC. Both ENaC and MARCKS are positively regulated by the protease cathepsin B. First, our preliminary data demonstrate renal ENaC activity and MARCKS protein expression are positively regulated by the circadian protein BMAL1. Second, our preliminary data show alpha-1 antitrypsin is increased in the BMAL1 knockout mouse kidney compared to the kidney of wild-type mice sacrificed at the same time. Third, our preliminary data show alpha-1 antitrypsin is expressed in the kidney and it strongly inhibits cathepsin B activity and contributes to blood pressure regulation. In this project we will test our hypothesis that the association between renal ENaC and MARCKS, and their function at the apical plasma membrane negatively correlates with alpha-1 antitrypsin expression in a circadian dependent manner. We will perform experiments to investigate proteolysis and apical membrane expression of ENaC and MARCKS, ENaC activity, sodium handling, and blood pressure using male and female BMAL1 knockout mice, alpha-1 antitrypsin knockout mice, alpha-1 antitrypsin overexpressing mice, cathepsin B knockout mice, and wild-type control mice. The successful completion of our proposed studies for this project will reveal new mechanisms underlying the role of BMAL1 in the regulation of renal ENaC and MARCKS and blood pressure control. Our long term goal is to provide a better understanding for the pathogenesis of essential hypertension that can potentially lead to novel drug targets and therapeutics.