The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We hypothesized that proteolytic processing of gammaENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved gammaENaC or the furin-cleaved-to-full-length ratio of gammaENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved gammaENaC, with no significant change in the furin-cleaved-to-full-length gammaENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length gammaENaC by mAbinhibit. Prostasin/kallikrein-cleaved gammaENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within gammaENaC.
Journal of the American Society of Nephrology, 2015, Vol 26, Issue 1, p. 95-106
Adult Aged Aged, 80 and over Angiotensin-Converting Enzyme Inhibitors/chemistry Antibodies/chemistry Antibodies, Monoclonal/chemistry Diuretics/chemistry Epithelial Sodium Channels/*metabolism Exosomes/metabolism Female Furin/chemistry HEK293 Cells Homeostasis Humans Kallikreins/chemistry Kidney/*metabolism Kidney Tubules, Collecting/metabolism Male Microscopy, Fluorescence Middle Aged Protein Structure, Tertiary Renin-Angiotensin System Serine Endopeptidases/chemistry