Identification of proteases relevant for ENaC activation in nephrotic urine

Abstract

In this thesis, the mechanisms of sodium retention in nephrotic syndrome (NS) were investigated, focusing on the proteolytic overactivation of the epithelial sodium channel (ENaC) in the distal tubule of kidney by aberrantly filtered plasma serine proteases due to a damaged glomerular filter system. This is known as overfill theory, complementary to the classical aldosterone dependent ENaC activation as classical underfill theory. Urine samples from healthy and nephrotic humans and mice were analyzed and compared by the protease activity against a universal substrate peptide mixture and peptides representing the distal cleavage region of γ-ENaC, for full activation of the channel. Inhibitors were added to determine protease class specificities, like AEBSF for all serine proteases. The corresponding active proteases were identified by mass spectrometry (MS) and western blot (WB) experiments. Nephrotic urine samples showed a strong, significant increase in aprotinin-sensitive serine protease activity. Proteomic analysis of this activity revealed a strong enrichment of plasma proteases from the coagulation and complement cascades, including plasminogen, plasma kallikrein, Factor XII, FSAP, and Complement Factors B and D. The active state was confirmed by additional MS preparation with AEBSF and aprotinin coupled beads and WB experiments for detection of active cleavage and degradation products. Substrate assays with peptides representing the distal cleavage site of γ-ENaC and coupled to the fluorophors AMCA and AMC identified a dominant cleavage fragment (FTGRKR↓KISGK) that highly matches with the substrate specificities of several candidate proteases, especially Complement Factor D, Complement Factor B, plasma kallikrein, and FSAP, for both human and different mouse models. The results give a very detailed, comprehensive insight into the overfill theory with proteolytic ENaC overactivation. The nephrotic protease activity presumably involved in this process, called proteasuria, arises from redundant and complex cascades of very active coagulation and complement proteases. The further search, analysis and understanding of key components or regulatory cofactors of these protease networks could be a new basis of more effective therapeutic strategies for preventing and treating nephrotic syndrome in patients.