Activation of the Epithelial Sodium Channel (ENaC) by Aldosterone in mCCDcl1 Mouse Renal Cortical Collecting Duct Cells is Dependent on SGK1 and Can be Prevented by Inhibiting Endogenous Proteases

The epithelial sodium channel (ENaC) is a heteromeric channel composed of three subunits (α, β, γ) critically important for renal sodium homeostasis and the long term control of arterial blood pressure. In the cortical collecting duct aldosterone is the main hormonal regulator of ENaC activity, and SGK1 (serum and glucocorticoid‐regulated kinase 1) is thought to play a pivotal role in mediating ENaC stimulation by aldosterone. Proteolytic cleavage by the Golgi‐associated convertase furin at three putative furin sites (two in α‐ and one in γENaC) is thought to be important for ENaC maturation in the biosynthetic pathway before the channel reaches the plasma membrane. The final step in proteolytic ENaC activation probably takes place at the plasma membrane where γENaC is cleaved by membrane‐bound proteases and/or extracellular proteases in a region distal to the furin cleavage site. In this study we investigated whether ENaC activation by aldosterone is dependent on SGK1 and requires proteolytic channel activation. A highly differentiated murine principal cell line (mCCD cl1 ) derived from microdissected cortical collecting duct was used, and cells were grown on permeable supports. ENaC‐mediated sodium transport was assessed by recording the amiloride‐sensitive equivalent short circuit current ( I SC‐Ami ) in Ussing chambers for up to 6 h. Exposure of the mCCD cl1 cells to 3 nM aldosterone for 2.5 h stimulated I SC‐Ami about 2.5‐fold. Subsequent application of chymotrypsin (24 or 240 μg/ml) had no additional stimulatory effect. The stimulatory response to aldosterone was completely abolished by the SGK1 inhibitor GSK650394 (10 μM). In untreated control cells apical application of chymotrypsin also showed no stimulatory effect on I SC‐Ami . Therefore, we hypothesized that in mCCD cl1 cells ENaC is fully activated by endogenous proteases in the presence and absence of aldosterone. To inhibit these endogenous proteases, we used an intracellularly acting convertase inhibitor (furin inhibitor‐1) and extracellularly acting serine protease inhibitors (aprotinin or nafamostat). A combination of aprotinin (30 μg/ml)/nafamostat (1 μM) and furin inhibitor‐1 (40 μM) essentially abolished baseline I SC‐Ami . Subsequent apical application of chymotrypsin stimulated I SC‐Ami back to baseline levels. Importantly, pre‐treatment of the cells with furin inhibitor‐1 in combination with aprotinin/nafamostat essentially prevented ENaC stimulation by aldosterone. Subsequent chymotrypsin rapidly and completely rescued the stimulatory effect of aldosterone in mCCD cl1 cells treated with protease inhibitors. We conclude that stimulation of ENaC activity by aldosterone is not only dependent on SGK1 but also requires proteolytic channel activation. It is tempting to speculate that SGK1 may contribute to the regulation of endogenous proteases necessary for channel activation. Endogenous proteases relevant for proteolytic ENaC activation under physiological and pathopyhsiological conditions remain to be elucidated. Support or Funding Information This work was supported by The Alexander von Humboldt Foundation (MKM) and Bayerische Forschungsstiftung (CK).