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

蛋白质相互作用对 NKCC2 和 NCC 的调节：从失盐性肾小管病到盐敏感性高血压

• 批准号: 391098411
• 负责人: Dr. Martin Kömhoff
• 金额: --
• 依托单位: Centre de Recherche des Cordeliers - UMRS 1138
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391098411?language=en
• 关键词: Regulation; NKCC2; NCC; Protein; interactions

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.

高血压是影响人类人口的最常见疾病之一。它是心血管疾病发病率和死亡率的主要危险因素。通过对单基因综合征的分析，可以将高血压与肾脏钠重吸收增加联系起来，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大的升支(TAL)和远端曲管(DCT)对盐的运输从而对血压调节的关键意义。位于顶端的Na-K-2Cl型和氯化钠共转运体(NKCC2和NCC)分别是TAL和DCT中氯化钠重吸收的起搏器。NKCC2和NCC的突变导致巴特氏综合症(BS)和吉特尔曼综合征(GS)，这是一种遗传性疾病，由于肾脏氯化钠消耗而导致低血压。我们最近的发现是，MAGE-D2的突变导致非常严重的产前BS(BS V型)，极早熟，因此NKCC2和NCC的功能损害导致高死亡率(Laghmani等人，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中均有表达。有趣的是，所有的BS V型症状在出生后完全消失，这表明不仅调控因素，而且上下文(生理性产前低氧与出生后正常氧)决定了NKCC2和NCC的功能。尽管调控蛋白具有明显的意义，也许还有细胞环境，但人们对它们对NKCC2和NCC表达的影响知之甚少。因此，我们建议：1)解开NKCC2和NCC在体外和体内调控的蛋白质网络和关键途径。2)阐明NKCC2基因突变蛋白表达和功能差异的分子调控途径。3)探讨MAGE-D2突变携带者发生一过性BS的分子机制。4)确定与产前BS有关的新的调控途径和/或基因。为此，我们提出了一个多学科的项目，结合了几种实验方法，包括：分子和细胞生物学，不明原因BS患者的遗传学，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完整的动物模型)，体内生理学使用代谢笼和体外肾单位节段的微灌流。更好地了解NKCC2和NCC表达和活性的调控途径将有助于深入了解盐滞留的病理生理机制。我们的努力将最终确定新的可用药靶点，以预防和/或治疗与钠平衡有关的肾脏疾病。