In vivo contribution of serine proteases to the proteolytic activation of γENaC in aldosterone-infused rats

Abstract

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.

The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

“…The approximately 90-95-and approximately 75-kD bands were compatible with previously published molecular sizes of intact, full-length gENaC protein and the C-terminal fragment of gENaC after proteolytical processing by furin, respectively. 10,12,20,23 The mAb inhibit was directed against an epitope that resides in the inhibitory tract, and putative two-hit cleavage distal to the furin cleavage site would remove the epitope. In case of proteolysis by a distally acting protease only (e.g., prostasin or kallikrein), a third fragment containing the N terminus of gENaC would be observed with mAb inhibit .…”

“…By inference, this result is consistent with but does not show regulation of gENaC level by extracellular volume contraction/aldosterone. 21 In rats, the majority of collecting duct gENaC at the apical plasma membrane is cleaved in low-salt intake conditions and after aldosterone infusion, 20,21,23 and the shift is inhibited by a serine protease/prostasin inhibitor. 23 The mAb inhibit antibody would not detect two-hit cleaved gENaC directly.…”

“…21 In rats, the majority of collecting duct gENaC at the apical plasma membrane is cleaved in low-salt intake conditions and after aldosterone infusion, 20,21,23 and the shift is inhibited by a serine protease/prostasin inhibitor. 23 The mAb inhibit antibody would not detect two-hit cleaved gENaC directly. Cleavage at, for example, the putative prostasin cleavage site would lead to Higher levels of gENaC in kidney tissue from patients treated with diuretics and patients with proteinuria.…”

“…Aldosterone stimulation leads to an increased level of proteolytically processed gENaC, [20][21][22] and in rat, prostasin expression is positively regulated by aldosterone. 23 Prostasin is a glycosylphosphatidylinositolanchored apical serine protease 24 expressed in the collecting duct [25][26][27] that activates gENaC at a site close to but in the C-terminal direction to the furin cleavage with putative release of an 43-residue inhibitory tract. 9,14,16,28 We and other groups have shown that, in pathologic conditions with proteinuria, urine contains aberrantly filtered plasmin that activates ENaC current in vitro-either directly or in low concentrations, through prostasin.…”

Cutting It Out

“…11 The challenge is demonstrating that a subunit has been cleaved twice, shedding an inhibitory track. 10,12 In this issue of JASN, Zachar et al address important questions regarding the proteolytic processing of the g subunit in human kidney. 13 Specifically, they examined whether g subunit processing in human kidney is affected by the administration of an angiotensin-converting enzyme inhibitor, angiotensin-receptor blocker, or a diuretic, and whether proteolytic processing is altered in the setting of nephrotic syndrome.…”