Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the aENaC subunit in colonic superficial cells (Scnn1a KO ) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (ΔPD amil ) than control mice fed a high-salt diet. In Scnn1a KO mice, however, this salt restriction-induced increase in ΔPD amil did not occur, and the circadian rhythm of ΔPD amil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a KO mice. However, Scnn1a KO mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8 KO ) were viable, without fetal or perinatal lethality. Compared with controls, Prss8 KO mice fed regular or low-salt diets exhibited significantly reduced ΔPD amil in the afternoon, but the circadian rhythm was maintained. Prss8 KO mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.

CAP1/Prss8 is a membrane-bound serine protease involved in the regulation of several different effectors, such as the epithelial sodium channel ENaC, the protease-activated receptor PAR2, the tight junction proteins, and the profilaggrin polypeptide. Recently, the V170D and the G54-P57 deletion mutations within the CAP1/Prss8 gene, identified in mouse frizzy (fr) and rat hairless (fr(CR)) animals, respectively, have been proposed to be responsible for their skin phenotypes. In the present study, we analyzed those mutations, revealing a change in the protein structure, a modification of the glycosylation state, and an overall reduction in the activation of ENaC of the two mutant proteins. In vivo analyses demonstrated that both fr and fr(CR) mutant animals present analogous reduction of embryonic viability, similar histologic aberrations at the level of the skin, and a significant decrease in the activity of ENaC in the distal colon compared with their control littermates. Hairless rats additionally had dehydration defects in skin and intestine and significant reduction in the body weight. In conclusion, we provided molecular and functional evidence that CAP1/Prss8 mutations are accountable for the defects in fr and fr(CR) animals, and we furthermore demonstrate a decreased function of the CAP1/Prss8 mutant proteins. Therefore, fr and fr(CR) animals are suitable models to investigate the consequences of CAP1/Prss8 action on its target proteins in the whole organism.

Systemic pseudohypoaldosteronism type 1 (PHA-1) is a severe salt-losing syndrome caused by loss-of-function mutations of the amiloride-sensitive epithelial sodium channel (ENaC) and characterized by neonatal lifethreatening hypovolemia and hyperkalemia. The very high plasma aldosterone levels detected under hypovolemic or hyperkalemic challenge can lead to increased or decreased sodium reabsorption, respectively, through the Na + /Cl 2 cotransporter (NCC). However, the role of ENaC deficiency remains incompletely defined, because constitutive inactivation of individual ENaC subunits is neonatally lethal in mice. We generated adult inducible nephron-specific aENaC-knockout mice (Scnn1a) that exhibit hyperkalemia and body weight loss when kept on a regular-salt diet, thus mimicking PHA-1. Compared with control mice fed a regular-salt diet, knockout mice fed a regular-salt diet exhibited downregulated expression and phosphorylation of NCC protein, despite high plasma aldosterone levels. In knockout mice fed a high-sodium and reduced-potassium diet (rescue diet), although plasma aldosterone levels remained significantly increased, NCC expression returned to control levels, and body weight, plasma and urinary electrolyte concentrations, and excretion normalized. Finally, shift to a regular diet after the rescue diet reinstated the symptoms of severe PHA-1 syndrome and significantly reduced NCC phosphorylation. In conclusion, lack of ENaC-mediated sodium transport along the nephron cannot be compensated for by other sodium channels and/or transporters, only by a high-sodium and reduced-potassium diet. We further conclude that hyperkalemia becomes the determining factor in regulating NCC activity, regardless of sodium loss, in the ENaC-mediated salt-losing PHA-1 phenotype.

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis and blood pressure by regulating sodium reabsorption in the Aldosterone Sensitive Distal Nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR Pax8/LC1 ) using a doxycycline inducible system. Under standard diet, MR Pax8/LC1 mice exhibit inability to gain weight and significant weight loss compared to control mice.

The amiloride-sensitive epithelial sodium channel (ENaC) and the thiazide-sensitive sodium chloride cotransporter (NCC) are key regulators of sodium and potassium and colocalize in the late distal convoluted tubule of the kidney. Loss of the ENaC subunit leads to a perinatal lethal phenotype characterized by sodium loss and hyperkalemia resembling the human syndrome pseudohypoaldosteronism type 1 (PHA-I). In adulthood, inducible nephron-specific deletion ofENaC in mice mimics the lethal phenotype observed in neonates, and as in humans, this phenotype is prevented by a high sodium (HNa)/low potassium (LK) rescue diet. Rescue reflects activation of NCC, which is suppressed at baseline by elevated plasma potassium concentration. In this study, we investigated the role of the ENaC subunit in the PHA-I phenotype. Nephron-specificENaC knockout mice also presented with salt-wasting syndrome and severe hyperkalemia. Unlike mice lacking ENaC orΕΝaC, an HNa/LK diet did not normalize plasma potassium (K) concentration or increase NCC activation. However, when K was eliminated from the diet at the time that ENaC was deleted, plasma K concentration and NCC activity remained normal, and progressive weight loss was prevented. Loss of the late distal convoluted tubule, as well as overall reduced ENaC subunit expression, may be responsible for the more severe hyperkalemia. We conclude that plasma K concentration becomes the determining and limiting factor in regulating NCC activity, regardless of Na balance in ENaC-deficient mice.