Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

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.