Furin-Mediated Modification Is Required for Epithelial Sodium Channel-Activating Activity of Soluble (Pro)Renin Receptor in Cultured Collecting Duct Cells

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M-1 cells. M-1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin-site Mut) or without (WT) mutagenesis of the furin cleavage site. As compared with empty vector control (EM), Furin-site Mut showed the attenuation effect on WT-induced α-ENaC expression and amiloride-sensitive short circuit current. In a separate experiment, M-1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin), indicating overexpression of the two types of sPRR induced a significant and comparable increase in the release of sPRR, but only sPRR-furin showed an increase of ENaC activity. Single channel analysis of ENaC activity in Xenopus A6-2F3 cells confirms sPRR-furin activation of ENaC open probability. Lastly, HEK-293 cells were pretreated with furin inhibitor α1-antitrypsin Portland (α1-PDX) followed by transfection with EM, WT PRR. sPRR in the conditioned medium was enriched by using protein centrifugal filter devices and applied to M-1 cells followed by measurement of ENaC activity, demonstrating that pretreatment with α1-PDX attenuated ENaC-acting activity induced by overexpression of WT PRR. In summary, we conclude that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.