Role of Ion Channels in Kidney Diseases

描述（由申请人提供）：本项目的长期目标是确定肾脏中离子通道的调节，并将我们的发现整合到肾脏疾病中。本研究旨在确定ATP结合盒转运蛋白A1（ABCA 1）是如何调控肾上皮钠通道（ENaC）的。所提出的体内和体外实验将测试一个中心假设，即ABCA 1的缺失升高皮质集合管（CCD）主细胞中的Cho，这种升高的Cho稳定顶端微绒毛中的磷脂酰肌醇-4，5-二磷酸（PIP 2），并且这种位于微绒毛的PIP 2增加ENaC活性以增强钠潴留并引起高血压。该假设基于先前的研究和两个关键的初步数据，表明ABCA 1 KO小鼠的血压和ENaC活性均升高，并且Cho合成的抑制导致PIP 2扩散出微绒毛并降低ENaC活性。这一建议具有临床意义，因为ABCA 1控制的膜和细胞内Cho稳态的研究可能为使用Cho生物合成抑制剂（如他汀类药物）治疗高血压提供理论依据。该项目具有创新性，因为它 将提供ABCA 1在调节ENaC活性和PIP 2在两个专门的顶端膜结构域（微绒毛和平面区域）之间的横向运动中的作用的第一个证据。包括扫描离子电导显微镜和高分辨率扫描膜片钳技术在内的多种实验方法将用于验证三种假设：（1）ABCA 1缺失增加ENaC活性和Na+吸收，从而导致高血压;（2）Cho通过稳定微绒毛中的PIP 2增加ENaC活性;（3）平面中的PIP 2增加ENaC活性。 区域促进含有非活性ENaC的内吞凹的形成。

DESCRIPTION (provided by applicant): The long term goals of this project are to determine the regulation of ion channels in the kidney and to integrate our findings to kidney diseases. This proposal will determine how the renal epithelial sodium channel (ENaC) is regulated by ATP-binding cassette transporter A1 (ABCA1). The proposed in vivo and in vitro experiments will test a central hypothesis that deletion of ABCA1 elevates Cho in cortical collecting duct (CCD) principal cells, this elevated Cho stabilizes phosphatidylinositol-4,5-bisphosphate (PIP2) in apical microvilli, and this microvilli- located PIP2 increases ENaC activity to enhance sodium retention and cause hypertension. The hypothesis is based on previous studies and two key preliminary data showing that both blood pressure and ENaC activity are elevated in ABCA1 KO mice and that inhibition of Cho synthesis causes PIP2 diffusion out of microvilli and reduces ENaC activity. The proposal is clinically significant, because investigation of ABCA1-controlled membrane and intracellular Cho homeostasis may provide a rationale for using Cho biosynthesis inhibitors such as statins to treat hypertension. The project is innovative because it will provide the first evidence for the role of ABCA1 in regulating ENaC activity and PIP2 lateral movement between two specialized apical membrane domains (microvilli and planar regions). A variety of experimental approaches including scanning ion conductance microscopy and the high resolution scanning patch-clamp techniques will be used to test three hypotheses (1) that deletion of ABCA1 increases ENaC activity and Na+ absorption thereby causing hypertension; (2) that Cho increases ENaC activity by stabilizing PIP2 in microvilli; and (3) that PIP2 in planar regions promotes formation of endocytic pits containing inactive ENaC.