Am J Physiol Renal Physiol 2025 Jan 01;3286:F766-F774. doi: 10.1152/ajprenal.00087.2024.

Furin-mediated modification is required for epithelial sodium channel-activating activity of soluble (pro)renin receptor in cultured collecting duct cells.

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(Pro)renin receptor (PRR) contains an overlapping cleavage site for site-1 protease (S1P) and furin for the 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 epithelial Na+ channel (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 (EM) control, 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. At last, 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 collecting duct cells.NEW & NOTEWORTHY The present study for the first time examined the functional implication of furin-dependent cleavage in the activation of sPRR during ENaC regulation in cultured CD cells. We found that sPRR with the initial S1P-dependent cleavage remained silent and only became active following furin-dependent cleavage in terms of enhancement of ENaC activity and expression of α-ENaC. These results offer novel insight into the sPRR maturation process during ENaC regulation.

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