Title:Proteolytic Activity against the Distal Polybasic Tract of the Gamma
Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine
Volume: 29
Issue: 42
关键词:
上皮钠通道，ENaC，蛋白水解活化，肾病综合征，蛋白尿症，多碱性束。
摘要:
Background: Experimental nephrotic syndrome in mice leads to proteolytic activation of the
epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit
(183RKRK).
Objective: We sought to determine if urine samples from both nephrotic mice and a cohort of patients
with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC.
Methods: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally
coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with
nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin.
The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence
detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer.
In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting
different cleavage sites within the polybasic tract.
Results: No significant proteolytic activity against the substrate was found in the urine of healthy humans
or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different
models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within
the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product
was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR-
AMC.
Conclusion: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity
against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.
