Carrisoza-Gaytan, R. | bioRxiv Science

bioRxiv2024

Proteolytic Cleavage of the ENaC gamma Subunit: Impact Upon Na+ and K+ Handling

The ENaC gamma subunit is essential for homeostasis of Na+, K+, and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (Q4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.

physiology↗

bioRxiv2023

Mineralocorticoid receptor-independent activation of ENaC in bile duct ligated mice

Sodium and fluid retention in liver disease is classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). Aldosterone dives Na+ retention by activating the mineralocorticoid receptor and promoting the maturation and apical surface expression of the epithelial Na+ channel (ENaC), found in the aldosterone-sensitive distal nephron. However, evidence of fluid retention without RAAS activation suggests the involvement of additional mechanisms. Liver disease can greatly increase plasma and urinary bile acid concentrations and have been shown to activate ENaC in vitro. We hypothesize that elevated bile acids in liver disease activate ENaC and drive fluid retention independent of RAAS. We therefore increased circulating bile acids in mice through bile duct ligation (BDL) and measured effects on urine and body composition, while using spironolactone to antagonize the mineralocorticoid receptor. We found BDL lowered blood [K+] and hematocrit, and increased benzamil-sensitive natriuresis compared to sham, consistent with ENaC activation. BDL mice also gained significantly more body water. Blocking ENaC reversed fluid gains in BDL mice but had no effect in shams. In isolated collecting ducts from rabbits, taurocholic acid stimulated net Na+ absorption but had no effect on K+ secretion or flow-dependent ion fluxes. Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease which may provide additional therapeutic options for liver disease patients. SignificanceAdvanced liver disease is often complicated by renal sodium retention, leading to fluid retention, poor outcomes, and increased mortality. This is currently thought to be driven by increased levels of the hormone aldosterone, although numerous published reports demonstrate normal aldosterone levels in many liver patients with volume overload. Management of these patients relies on diuretics and Na+ restriction, or more invasive procedures with increased risks for diuretic-resistant patients. Here, we report a novel mechanism for fluid retention in liver disease, which provides a basis to develop new strategies to treat fluid retention in these patients.

physiology↗