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

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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.