The Ubiquitin Ligase NEDD4L in Blood Pressure Regulation

描述（由申请人提供）：该R21响应PA-03-145“泛素和泛素样修饰调节疾病过程”。基本原理。上皮钠通道（ENaC）通过醛固酮敏感远端肾元（ASDN）的钠重吸收影响血浆容量和血压（BP）。ENaC的PY结构域突变导致高血压疾病Liddle综合征，泛素连接酶NEDD4L通过该结构域介导通道降解。在两个同源基因NEDD4和NEDD4L中，NEDD4L最近被确定为ENaC的特异性调节因子。NEDD4L被认为缺乏C2顶端靶向结构域。我们发现：(1)哺乳动物NEDD4L指导多种转录异构体的形成，这些转录异构体包括或不包括C2结构域；(2)一个先前未被识别的外显子I产生一个C2（+）异构体，该异构体在人类肾脏中最为丰富；(3)共同变异（a为G， 35%）产生的转录本只能编码C2（-）亚型；(4)除了遗传联系外，这种多态性还与治疗高血压患者收缩压的体位变化有关，并且在另一种影响基底外侧Na+，K+ atp酶的变异的背景下，与未治疗高血压患者24小时血压监测和对钠负荷的反应有关。假设。根据这些数据，我们的假设是NEDD4L异构体的相对表达水平，通过影响ASDN的根尖ENaC密度，是钠稳态和血压的重要调节因子。研究目标。我们建议建立实验动物模型，最终通过小鼠同源重组基因靶向研究这两类NEDD4L异构体对完整动物钠平衡和血压的影响。我们的目标是：

DESCRIPTION (provided by applicant): This R21 responds to PA-03-145 "Ubiquitin and Ubiquitin-Iike Modifications Regulating Disease Processes". Rationale. The epithelial sodium channel (ENaC) affects plasma volume and blood pressure (BP) through sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Mutations in PY domains of ENaC, through which the ubiquitin ligase NEDD4L mediates channel degradation, lead to the hypertensive disorder, Liddle syndrome. Of two paralogous genes, NEDD4 and NEDD4L, NEDD4L has been identified recently as the more specific regulator of ENaC. NEDD4L was thought to lack a C2 apical targeting domain. We have found that: (I) mammalian NEDD4L directs the formation of multiple transcriptional isoforms that include or not a C2 domain; (2) a previously unidentified exon I generates a C2(+) isoform that is the most abundant in human kidney; (3) a common variant (A for G, 35%) generates a transcript that can only encode C2(-) isoforms; (4) in addition to genetic linkage, this polymorphism is associated with postural change in systolic BP in treated hypertensives, and with 24-hour BP monitoring and response to sodium loading in untreated hypertensives in the context of another variant affecting basolateral Na+,K+ ATPase. Hypothesis. With these data, our hypothesis is that the relative level of expression of NEDD4L isoforms, by impacting on apical ENaC density in ASDN, is an important regulator of sodium homeostasis and blood pressure. Research Aims. We propose to develop experimental animal models that will afford ultimately to investigate the impact of these two classes of NEDD4L isoforms on sodium balance and blood pressure in intact animals through gene targeting by homologous recombination in the mouse. Our aims are: (1) To generate two lines of animals that constitutively overexpress a C2(+) and a C2(-) isoform of NEDD41. in the ASDN using the Cre/Lox system for tissue specific activation; (2) To generate a line lacking expression of all NEDD4L isoforms in ASDN both as a test of the global significance of NEDD4L in nephron function and as a background to isolate through crossing the functional effect of the two isoforms overexpressed in Aim 1; (3) To perform a preliminary evaluation of the impact of these genetic manipulations on renal fund ion and blood pressure in response to challenges of sodium balance. Perspective. These animal models will afford in-depth evaluation of the contribution of the ubiquitin ligase NEDD4L. in ENaC regulation and analysis of genetic mechanisms of common forms of human essential hypertension.