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”。理由。上皮钠通道（ENaC）通过醛固酮敏感性远端肾单位（ASDN）的钠重吸收影响血浆容量和血压（BP）。泛素连接酶NEDD 4L通过ENaC的PY结构域的突变介导通道降解，导致高血压疾病Liddle综合征。在两个旁系同源基因NEDD 4和NEDD 4L中，NEDD 4L最近已被鉴定为ENaC的更特异性调节子。NEDD 4L被认为缺乏C2顶端靶向结构域。我们发现：（I）哺乳动物NEDD 4L指导包括或不包括C2结构域的多种转录同种型的形成;（2）先前未鉴定的外显子I产生在人类肾脏中最丰富的C2（+）同种型;（3）常见变体（A为G，35%）产生只能编码C2（-）同种型的转录物;（4）除了遗传连锁外，该多态性还与高血压治疗后收缩压的姿势改变有关，以及在另一种影响基底外侧Na+，K+ ATP酶的变异的背景下，在未经治疗的高血压患者中进行24小时血压监测和对钠负荷的反应。假说.根据这些数据，我们假设NEDD 4L亚型的相对表达水平通过影响ASDN中的顶端ENaC密度，是钠稳态和血压的重要调节剂。研究目标。我们建议开发实验动物模型，最终将提供调查这两类NEDD 4L亚型对钠平衡和血压的影响，在完整的动物通过基因靶向同源重组在小鼠中。我们的目标是： (1)产生组成性过表达NEDD 41的C2（+）和C2（-）同种型的两个动物系。在ASDN中使用Cre/Lox系统进行组织特异性激活; (2)产生在ASDN中缺乏所有NEDD 4L同种型表达的细胞系，既作为NEDD 4L在肾单位功能中的整体意义的测试，又作为通过交叉Aim 1中过表达的两种同种型的功能效应来分离的背景; (3)初步评价这些基因操作对钠平衡挑战时肾功能和血压的影响。 Perspective.这些动物模型将提供泛素连接酶NEDD 4L的贡献的深入评价。在ENaC调控和人类原发性高血压常见形式的遗传机制分析。

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.