Regulation of NKCC2 and NCC by Protein Protein interactions: From salt losing tubulopathies to salt sensitive hypertension

高血压是影响人类最常见的疾病之一。它是心血管疾病发病率和死亡率的主要危险因素。单基因综合征的分析，已允许将高血压与肾钠重吸收升高联系起来，特别是通过集合管中钠通道（ENaC）活性的增加。然而，这种对ENaC及其激活剂醛固酮的关注在某种程度上掩盖了粗升支（TAL）和远曲小管（DCT）对NaCl转运以及血压调节的关键意义。位于顶端的Na-K-2Cl和NaCl共转运蛋白（NKCC 2和NCC）重吸收三分之一的过滤的NaCl，因此分别是TAL和DCT中氯化钠重吸收的起搏器。NKCC 2和NCC的突变导致Bartter综合征（BS）和Gitelman综合征（GS），这是一种遗传性疾病，其特征是由于肾脏NaCl消耗引起的低血压。我们最近发现MAGE-D2突变导致非常严重的产前BS（BS V型），伴有极早产，因此由于NKCC 2和NCC功能受损导致死亡率高（Laghmani et al，NEJM 2016），这清楚地强调了转运蛋白的调节蛋白对其功能的重要性。MAGE-D2在胎儿和成人肾脏的TAL和DCT中表达。有趣的是，BS V型的所有症状在出生后完全消退，这表明不仅调节因素，而且环境（生理性产前缺氧与出生后常氧）决定了NKCC 2和NCC的功能。尽管调控蛋白的重要性显而易见，也许还有细胞环境，但关于它们对NKCC 2和NCC表达的影响知之甚少。因此，我们建议：1）解开蛋白质网络和关键途径参与调控，在体外和体内，NKCC 2和NCC。2)阐明差异调节突变NKCC 2蛋白表达和功能的分子途径。3)研究MAGE-D2突变携带者中BS短暂性的分子机制。4)确定新的调控途径和/或基因负责产前BS。为此，我们提出了一个多学科的项目结合几个实验方法，包括：分子和细胞生物学，遗传学在不明原因的BS患者，在体内和体外基因沉默技术（使用原代细胞培养，稳定的细胞系，以及果蝇和小鼠作为一个整体的动物模型），在体内生理学代谢笼和肾单位段的体外微灌注。更好地了解作用于NKCC 2和NCC表达和活性的调控途径将有助于深入了解盐潴留的病理生理学。我们的努力将最终确定新的药物靶点，以预防和/或治疗与钠平衡相关的肾脏疾病。

Hypertension in one of the most common diseases affecting human population. It is a major risk factor of cardiovascular morbidity and mortality. Analyses of monogenic syndromes, has allowed to link hypertension to elevated renal Na reabsorption, in particular through the increase in the activity of the sodium channel (ENaC) in the collecting duct. However, this focused attention on ENaC and its activator aldosterone has somehow masked the pivotal significance of the thick ascending limb (TAL) and the distal convoluted tubule (DCT) for NaCl transport and hence for blood pressure regulation. The apically located Na-K-2Cl and NaCl co-transporters (NKCC2 and NCC) reabsorb one third of the filtered NaCl and therefore are the pacemakers of sodium chloride reabsorption in TAL and DCT, respectively. Mutations in NKCC2 and NCC cause Bartter's (BS) and Gitelman's syndromes (GS), inherited disorders featuring low blood pressure due to renal NaCl wasting. Our recent finding that mutations in MAGE-D2 cause a very severe form of antenatal BS (BS type V) with extreme prematurity and hence high mortality due to functional impairment of NKCC2 and NCC (Laghmani et al, NEJM 2016) clearly underlines the importance of the regulatory proteins of the transporters for their functioning. MAGE-D2 is expressed in the TAL and DCT in fetal and adult kidney. Intriguingly, all symptoms of BS type V completely resolve postnatally, indicating that not only regulatory factors but also the context (physiological prenatal hypoxia versus postnatal normoxia) determine the functioning of NKCC2 and NCC. Despite the obvious significance of regulatory proteins and perhaps also the cellular environment very little is known about their impact on NKCC2 and NCC expression. Hence, we propose to: 1) Unravel the protein networks and key pathways involved in the regulation, in vitro and in vivo, of NKCC2 and NCC. 2) Elucidate the molecular pathways differentially regulating expression and function of mutated NKCC2 proteins. 3) Investigate the molecular mechanisms responsible for the transient nature of BS in carriers of MAGE-D2 mutations. 4) Identify new regulatory pathways and/or genes responsible for antenatal BS. To this end, we propose a multidisciplinary project combining several experimental approaches including: molecular and cell biology, genetics in patients with unidentified causes of BS, In vivo and in vitro gene silencing techniques (using primary cell culture, stable cell lines, as well as drosophila and mice as a whole animal model), in vivo physiology using metabolic cages and in vitro microperfusion of nephron segments. A better understanding of the regulatory pathways acting on NKCC2 and NCC expression and activity will help to gain insights into the pathophysiology of salt retention. Our efforts will ultimately identify new drugable targets to prevent and/or treat kidney disorders related to sodium balance.