Significance of proteolytic regulation of the epithelial sodium channel ENaC by serine proteases in vivo
体内丝氨酸蛋白酶对上皮钠通道 ENaC 蛋白水解调节的意义
基本信息
- 批准号:360658146
- 负责人:
- 金额:--
- 依托单位:
- 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 2020-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The epithelial sodium channel (ENaC) is expressed in the late distal aldosterone-sensitive tubule and determines the final urinary sodium concentration. Due to ENaC´s essential role for sodium homeostasis channel activity is regulated by a myriad of factors in a redundant way. Within the various regulatory mechanisms activation after proteolytic cleavage by intra- and extracellular serine proteases is a specific feature of ENaC und leads to removal of inhibitory peptides from the α- and γ-subunit, resulting in a very high channel open probability. While proteolytic ENaC regulation by serine proteases is studied well in vitro, there is a paucity on data on the existence and relevance of this regulation in vivo. Indirectly, cleavage of γ-ENaC was suggested by experiments of the applicant with mice that were treated with the serine protease inhibitor aprotinin and that exhibited inhibition of ENaC-specific sodium reabsorption. In proteinuric mice, aprotinin prevented ENaC-mediated sodium retention and edema formation. However, in the literature and also in the applicant´s experience the proof of full cleavage of γ-ENaC in kidney tissue in both mice and humans is lacking. The goal of the present application is to investigate the presence and the significance of proteolytic ENaC regulation in vivo and to identify proteases that are involved in this process. To this end, antibodies specific for γ-ENaC cleavage sites will be generated that allow the detection of cleavage in tissue samples. In addition, a fluorescent peptide substrate corresponding to the region of γ-ENaC cleavage will be synthesized that can be incubated with purified serine proteases or with those present in urinary samples to elucidate the cleavage patterns. Finally, a knock-in mouse will be generated which harbors a mutated gene for γ-ENaC abolishing its cleavage (RKRK186AAAA). By phenotyping this mouse under physiological (low salt diet, diuretics) and pathophysiological conditions (nephrotic syndrome) high quality evidence will be gathered on the presence and significance of proteolytic ENaC activation. This and the identification of serine proteases involved in this regulation may lay the foundation to develop a therapeutic strategy to target proteolytic ENaC activation in vivo.
上皮钠通道(ENaC)在晚期远端醛固酮敏感性小管中表达,并决定最终的尿钠浓度。由于ENaC对钠稳态的重要作用,通道活性以冗余的方式受到无数因素的调节。在各种调节机制中,通过细胞内和细胞外丝氨酸蛋白酶进行蛋白水解切割后的激活是ENaC的一个特定特征,并导致从α-和γ-亚基中去除抑制肽,从而导致非常高的通道开放概率。虽然丝氨酸蛋白酶的蛋白水解ENaC调节在体外研究得很好,但关于这种调节在体内的存在和相关性的数据很少。间接地,申请人对用丝氨酸蛋白酶抑制剂抑肽酶处理的小鼠进行的实验表明γ-ENaC裂解,这些小鼠表现出对ENaC特异性钠重吸收的抑制。在蛋白尿小鼠中,抑肽酶可防止ENaC介导的钠潴留和水肿形成。然而,在文献和申请人的经验中,缺乏小鼠和人类肾脏组织中γ-ENaC完全裂解的证据。本申请的目的是研究体内蛋白水解ENaC调节的存在和意义,并鉴定参与该过程的蛋白酶。为此,将产生对γ-ENaC切割位点具有特异性的抗体,其允许检测组织样品中的切割。此外,将合成对应于γ-ENaC切割区域的荧光肽底物,可以与纯化的丝氨酸蛋白酶或尿液样本中存在的蛋白酶一起孵育,以阐明切割模式。最后,将产生基因敲入小鼠,其具有消除其切割的γ-ENaC的突变基因(RKRK 186 AAAA)。通过在生理(低盐饮食、利尿剂)和病理生理条件(肾病综合征)下对该小鼠进行表型分型,将收集关于蛋白水解ENaC活化的存在和意义的高质量证据。这一点以及参与该调节的丝氨酸蛋白酶的鉴定可能为开发靶向体内蛋白水解ENaC激活的治疗策略奠定基础。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Professor Dr. Ferruh Artunc其他文献
Professor Dr. Ferruh Artunc的其他文献
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{{ truncateString('Professor Dr. Ferruh Artunc', 18)}}的其他基金
Importance of the alternative complement pathway in nephrotic syndrome
补体替代途径在肾病综合征中的重要性
- 批准号:
457011590 - 财政年份:
- 资助金额:
-- - 项目类别:
Research Grants
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