Abstract
The epithelial sodium channel (ENaC) is essential for sodium reabsorption and potassium homeostasis in the distal nephron, where its activity is controlled by mineralocorticoid signaling and downstream proteolytic processing of channel subunits. Although cleavage of the γ-ENaC subunit has been implicated in aldosterone-mediated sodium transport, the identity of mineralocorticoid receptor (MR)-regulated proteases responsible for this process remains uncertain. Here, we investigated the role of kallikrein-1 (encoded by Klk1), a serine protease expressed in the connecting tubule and cortical collecting duct (CNT/CCD), as a mediator of ENaC activation. Using CRISPR/Cas9, we generated a conditional Klk1-floxed allele and established mice with CNT/CCD-specific deletion of Klk1 by crossing with Calb1-Cre (CNT-Klk1(-/-)). On a low-sodium, high-potassium diet, CNT-Klk1(-/-)mice exhibited ∼85% less renal kallikrein-1 expression, yet maintained normal serum electrolytes, urinary potassium excretion, and aldosterone responses. Western blot analysis revealed significantly less cleavage of γ-ENaC and α-ENaC in CNT-Klk1(-/-) kidneys, accompanied by more total NCC abundance. Despite impaired ENaC proteolysis, amiloride-sensitive sodium excretion was preserved, indicating intact ENaC function. These findings identify renal kallikrein-1 as a protease that contributes to ENaC subunit processing in vivo. However, the absence of overt sodium or potassium handling defects in CNT-Klk1(-/-) mice suggests that kallikrein-1 deficiency is not sufficient to disrupt overall ENaC function, likely due to compensatory mechanisms from redundant proteolytic or nonproteolytic pathways. Together, our results refine the role of kallikrein-1 as a modulator, rather than a sole determinant, of ENaC activation and highlight the complexity of aldosterone-dependent sodium transport in the distal nephron.NEW & NOTEWORTHY Using a novel connecting tubule/cortical collecting duct specific kallikrein-1 knockout model, we show that γ- and α-ENaC cleavage is impaired by loss of renal kallikrein-1, without major disturbances in sodium or potassium handling. These findings highlight redundancy among ENaC regulatory pathways and suggest that proteolytic cleavage of ENaC, although useful as an indicator of ENaC-mediated transport under physiological conditions, may not, in and of itself, play a major role in ENaC function.