Role of Ion Channels in Kidney Diseases

描述(由申请人提供)：该项目的长期目标是确定肾脏离子通道的调节，并将我们的发现整合到肾脏疾病中。这一提议将决定肾上皮钠通道(ENaC)如何受ATP结合盒转运体A1(ABCA1)的调节。拟议的体内和体外实验将检验一个中心假设，即ABCA1基因的缺失会增加皮质集合管(CCD)主细胞中的CHO，这种升高的CHO可以稳定顶端微绒毛中的磷脂酰肌醇-4，5-二磷酸(PIP2)，而这种位于微绒毛中的PIP2会增加ENaC的活性，从而增强钠滞留并导致高血压。这一假说是基于之前的研究和两个关键的初步数据，这些数据表明ABCA1 KO小鼠的血压和ENaC活性都会升高，抑制CHO合成会导致PIP2扩散到微绒毛外，降低ENaC活性。这一建议具有临床意义，因为对ABCA1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基础。这个项目具有创新性，因为它 这将为ABCA1在调节ENaC活性和PIP2在两个特殊的顶膜结构域(微绒毛和平面区域)之间的横向移动中的作用提供第一个证据。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各种实验方法将被用来检验三个假说(1)ABCA1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PIP2而增加ENaC活性；以及(3)PIP2在平面上 区域促进含有不活跃的ENaC的胞内凹坑的形成。

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.