The Ubiquitin Ligase NEDD4L in Blood Pressure Regulation

描述(申请人提供)：此R21响应PA-03-145“泛素和类似泛素的修饰调节疾病过程”。基本原理。上皮性钠通道(ENaC)通过在对醛固酮敏感的远端肾单位(ASDN)重吸收钠来影响血浆容量和血压(BP)。ENaC的PY结构域突变，泛素连接酶NEDD4L通过其介导通道降解，导致高血压疾病，利德尔综合征。在两个相似的基因NEDD4和NEDD4L中，NEDD4L最近被确定为ENaC更特异的调节基因。NEDD4L被认为缺少C2顶靶向结构域。我们发现：(I)哺乳动物NEDD4L指导包括或不包括C2结构域的多种转录异构体的形成；(2)先前未知的外显子I产生在人类肾脏中最丰富的C2(+)异构体；(3)一种常见的变体(A代表G，35%)产生只能编码C2(-)异构体的转录本；(4)除了遗传连锁外，该多态性还与治疗高血压患者的收缩压姿势变化有关，并且在另一种影响基础侧Na+，K+ATPase的变体的背景下，对未治疗的高血压患者进行24小时血压监测和对钠负荷的反应。假设。根据这些数据，我们的假设是，NEDD4L亚型的相对表达水平通过影响ASDN的心尖ENaC密度，是钠稳态和血压的重要调节因素。研究目的。我们建议建立实验动物模型，最终通过在小鼠体内同源重组的基因打靶来研究这两类NEDD4L亚型对完整动物钠平衡和血压的影响。我们的目标是： (1)产生两个动物品系，它们结构性地过度表达NEDD41的C2(+)和C2(-)亚型。在ASDN中，使用Cre/Lox系统进行组织特异性激活； (2)建立一个ASDN中所有NEDD4L亚型均不表达的品系，作为NEDD4L在肾单位功能中的整体意义的检验，并作为背景，通过交叉目的1中过表达的两种异构体的功能效应来分离； (3)初步评估这些基因操作对肾功能和血压的影响，以应对钠平衡的挑战。 透视。这些动物模型将为泛素连接酶NEDD4L的贡献提供深入的评估。在ENaC中调节和分析常见形式的人类高血压的遗传机制。

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.