Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC γ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR₂) at R³⁶↓S³⁷ and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR₂ at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR₂ at E⁵⁶↓T⁵⁷, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR₂ coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca²⁺, activate ERK1/2, recruit β-arrestins, or induce PAR₂ endocytosis. Cat-S caused PAR₂-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR₂ or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR₂ antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR₂ in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR₂ that causes PAR₂- and TRPV4-dependent inflammation and pain. They expand the role of PAR₂ as a mediator of protease-driven inflammatory pain.

Proteases that cleave protease-activated receptor-2 (PAR2) at R36/S37 reveal a tethered ligand that binds to the cleaved receptor. PAR2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR2 at non-canonical sites can trigger distinct signaling cascades, the functional importance of PAR2 biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR2, causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR2 at A66/S67 and S67/V68. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein coupled receptor kinase-2 (GRK2) or β-arrestin to PAR2, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR2-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR2- and TRPV4-mediated influx of extracellular Ca2+ in mouse nociceptors. Adenylyl cyclase and PKA mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR2- and TRPV4-mediated mechanisms. Thus, elastase biased agonism of PAR2 causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR2 as a mediator of protease-driven inflammation and pain.

The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.

Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.