Importance of the alternative complement pathway in nephrotic syndrome

上皮钠通道（epithelial sodium channel, ENaC）表达于远端醛固酮敏感小管晚期，对钠稳态至关重要。在ENaC的多种调控机制中，细胞内和细胞外丝氨酸蛋白酶裂解蛋白后的活化是ENaC的一个特殊特征。肾病综合征导致钠潴留和水肿形成，并由ENaC活性增加介导，这是由于血浆中异常过滤的丝氨酸蛋白酶通过蛋白水解激活ENaC。这一机制的相关性被申请人在体内证明，即非选择性丝氨酸蛋白酶抑制剂抑酶蛋白保护肾病小鼠免受钠潴留和水肿形成。由于这一关键发现，申请人的小组-由DFG资助-专注于从小鼠和人类尿液样本中鉴定可能参与蛋白水解ENaC激活的必需丝氨酸蛋白酶。使用蛋白质组学方法鉴定了几种丝氨酸蛋白酶，并测试了它们在肾病敲除小鼠模型中的病理生理相关性。目前排除尿激酶、纤溶酶、血浆钾化管、凝血因子XII、因子vii活化蛋白酶以及前列腺蛋白酶的参与。此外，肾病尿液样本中含有丝氨酸蛋白酶补体因子D和B，它们是补体替代途径的一部分，也可能发挥重要作用。此外，在人类和小鼠的肾病样本中，有证据表明替代补体途径被激活。根据该途径可能的相关性，该生理底物CFB上CFD的裂解序列与ENaC的γ-亚基的裂解序列相同，这是通过自己的底物方法实验确定的。在本项目中，申请人旨在阐明替代补体途径在肾病综合征钠潴留和水肿形成中的作用。为此，将在肾病尿液样本中建立补体因子和活化的综合分析。体内相关性将通过肾病小鼠模型进行研究，该模型涉及缺失替代补体途径基本成分（CFD， CFB, C3, properdin）的敲除小鼠。这种替代补体途径作为ENaC蛋白水解激活的原因的鉴定可能为开发肾病综合征的治疗策略奠定基础。

The epithelial sodium channel (ENaC) is expressed in the late distal aldosterone-sensitive tubule and is of essential importance for the sodium homeostasis. Among the various regulatory mechanisms of ENaC, activation after proteolytic cleavage by intra- and extracellular serine proteases is a specific feature of ENaC. Nephrotic syndrome leads to sodium retention and edema formation and is mediated by increased ENaC activity due to aberrantly filtered serine proteases from the plasma which activate ENaC by proteolysis. The relevance of this mechanism was demonstrated in vivo by the applicant whereby the unselective serine protease inhibitor aprotinin protected nephrotic mice from sodium retention and edema formation. Since this pivotal finding, the applicant´s group – supported by DFG grants - focuses on the identification of the essential serine protease(s) from urine samples of mice and humans that might be involved in proteolytic ENaC activation. Using a proteomic approach several serine proteases were identified and tested for their pathophysiological relevance in nephrotic knockout mouse models. Currently, involvement of urokinase, plasmin, plasma kallikrein, coagulation factor XII, Factor VII-activating protease as well as prostasin was excluded. In addition, nephrotic urine samples contained the serine proteases complement factor D and B which are part of the alternative complement pathway and could also play a significant role. Moreover, there was evidence of activation of the alternative complement pathway in nephrotic samples from humans and mice. In accordance with a possible relevance of this pathway, the cleavage sequence of CFD at this physiological substrate CFB is identical with that of the γ-subunit of ENaC which was determined by own experiments with a substrate approach. In this project, the applicant aims to elucidate the role of the alternative complement pathway in sodium retention and edema formation in nephrotic syndrome. To this end, a comprehensive analysis of complement factors and activation will be established in nephrotic urine samples. The in vivo relevance will be investigated using nephrotic mouse models involving knockout mice with deletion of the essential components of the alternative complement pathway (CFD, CFB, C3, properdin). This identification of the alternative complement pathway as the cause of proteolytic ENaC activation may lay the foundation to develop a therapeutic strategy in nephrotic syndrome.