Significance of proteolytic regulation of the epithelial sodium channel ENaC by serine proteases in vivo

上皮性钠通道（ENaC）在远端醛固酮敏感小管晚期表达，决定最终尿钠浓度。由于ENaC对钠稳态的重要作用，通道活性受到无数因素的冗余调节。在各种调节机制中，细胞内和细胞外丝氨酸蛋白酶在蛋白水解裂解后激活是ENaC的一个特定特征，并导致α-和γ-亚基上的抑制肽的去除，从而导致通道打开的概率非常高。虽然丝氨酸蛋白酶对ENaC蛋白水解的调控在体外得到了很好的研究，但在体内缺乏这种调控的存在及其相关性的数据。间接地，通过用丝氨酸蛋白酶抑制剂抑酶蛋白治疗小鼠的实验表明，申请者可以切割γ-ENaC，并表现出对enac特异性钠重吸收的抑制。在蛋白尿小鼠中，抑酶蛋白可阻止enact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γ-ENaC在小鼠和人肾组织中完全切割的证据。本应用程序的目的是研究ENaC在体内蛋白水解调节的存在和意义，并确定参与这一过程的蛋白酶。为此，将产生针对γ-ENaC切割位点的特异性抗体，以检测组织样品中的切割。此外，将合成与γ-ENaC裂解区域对应的荧光肽底物，该底物可以与纯化的丝氨酸蛋白酶或尿样品中存在的丝氨酸蛋白酶孵育，以阐明裂解模式。最后，将产生一个含有γ-ENaC的突变基因（RKRK186AAAA）的敲入小鼠。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析，将收集高质量的证据，证明ENaC蛋白水解激活的存在及其意义。这和丝氨酸蛋白酶参与这一调控的鉴定可能为开发一种靶向ENaC体内蛋白水解激活的治疗策略奠定基础。

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.