Kidney-Specific CAP1/Prss8-Deficient Mice Maintain ENaC-Mediated Sodium Balance through an Aldosterone Independent Pathway.

The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.

Deletion of the serine protease CAP2/Tmprss4 leads to dysregulated renal water handling upon dietary potassium depletion.

The kidney needs to adapt daily to variable dietary K+ contents via various mechanisms including diuretic, acid-base and hormonal changes that are still not fully understood. In this study, we demonstrate that following a K+-deficient diet in wildtype mice, the serine protease CAP2/Tmprss4 is upregulated in connecting tubule and cortical collecting duct and also localizes to the medulla and transitional epithelium of the papilla and minor calyx. Male CAP2/Tmprss4 knockout mice display altered water handling and urine osmolality, enhanced vasopressin response leading to upregulated adenylate cyclase 6 expression and cAMP overproduction, and subsequently greater aquaporin 2 (AQP2) and Na+-K+-2Cl- cotransporter 2 (NKCC2) expression following K+-deficient diet. Urinary acidification coincides with significantly increased H+,K+-ATPase type 2 (HKA2) mRNA and protein expression, and decreased calcium and phosphate excretion. This is accompanied by increased glucocorticoid receptor (GR) protein levels and reduced 11ß-hydroxysteroid dehydrogenase 2 activity in knockout mice. Strikingly, genetic nephron-specific deletion of GR leads to the mirrored phenotype of CAP2/Tmprss4 knockouts, including increased water intake and urine output, urinary alkalinisation, downregulation of HKA2, AQP2 and NKCC2. Collectively, our data unveil a novel role of the serine protease CAP2/Tmprss4 and GR on renal water handling upon dietary K+ depletion.

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl- Cotransporter NCC and Transiently Affects Sodium Handling.

Chronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1 Pax8/LC1 ). Upon standard salt diet, Nr3c1 Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1 Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11ßHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1 Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations.

Plasma Potassium Determines NCC Abundance in Adult Kidney-Specific γENaC Knockout.

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 αENaC 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 of αENaC 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 γENaC subunit in the PHA-I phenotype. Nephron-specific γENaC knockout mice also presented with salt-wasting syndrome and severe hyperkalemia. Unlike mice lacking αENaC or ßΕΝaC, 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 γENaC 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 ßENaC 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 γENaC-deficient mice.

Severe hyperkalemia is rescued by low-potassium diet in renal ßENaC-deficient mice.

In adulthood, an induced nephron-specific deficiency of αENaC (Scnn1a) resulted in pseudohypoaldosteronism type 1 (PHA-1) with sodium loss, hyperkalemia, and metabolic acidosis that is rescued through high-sodium/low-potassium (HNa+/LK+) diet. In the present study, we addressed whether renal ßENaC expression is required for sodium and potassium balance or can be compensated by remaining (α and γ) ENaC subunits using adult nephron-specific knockout (Scnn1bPax8/LC1) mice. Upon induction, these mice present a severe PHA-1 phenotype with weight loss, hyperkalemia, and dehydration, but unlike the Scnn1aPax8/LC1 mice without persistent salt wasting. This is followed by a marked downregulation of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and Na+/Cl- co-transporter (NCC) protein expression and activity. Most of the experimental Scnn1bPax8/LC1 mice survived with a HNa+/LK+ diet that partly normalized NCC phosphorylation, but not total NCC expression. Since salt loss was minor, we applied a standard-sodium/LK+ diet that efficiently rescued these mice resulting in normokalemia and normalization of NCC phosphorylation, but not total NCC expression. A further switch to LNa+/standard-K+ diet induced again a severe PHA-1-like phenotype, but with only transient salt wasting indicating that low-K+ intake is critical to decrease hyperkalemia in a NCC-dependent manner. In conclusion, while the ßENaC subunit plays only a minor role in sodium balance, severe hyperkalemia results in downregulation of NCC expression and activity. Our data demonstrate the importance to primarily correct the hyperkalemia with a low-potassium diet that normalizes NCC activity.

Adult nephron-specific MR-deficient mice develop a severe renal 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. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.

Severe Salt-Losing Syndrome and Hyperkalemia Induced by Adult Nephron-Specific Knockout of the Epithelial Sodium Channel α-Subunit.

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 life-threatening 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(-) 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 αENaC-knockout mice (Scnn1a(Pax8/LC1)) 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.

The CAP1/Prss8 catalytic triad is not involved in PAR2 activation and protease nexin-1 (PN-1) inhibition.

Serine proteases, serine protease inhibitors, and protease-activated receptors (PARs) are responsible for several human skin disorders characterized by impaired epidermal permeability barrier function, desquamation, and inflammation. In this study, we addressed the consequences of a catalytically dead serine protease on epidermal homeostasis, the activation of PAR2 and the inhibition by the serine protease inhibitor nexin-1. The catalytically inactive serine protease CAP1/Prss8, when ectopically expressed in the mouse, retained the ability to induce skin disorders as well as its catalytically active counterpart (75%, n=81). Moreover, this phenotype was completely normalized in a PAR2-null background, indicating that the effects mediated by the catalytically inactive CAP1/Prss8 depend on PAR2 (95%, n=131). Finally, nexin-1 displayed analogous inhibitory capacity on both wild-type and inactive mutant CAP1/Prss8 in vitro and in vivo (64% n=151 vs. 89% n=109, respectively), indicating that the catalytic site of CAP1/Prss8 is dispensable for nexin-1 inhibition. Our results demonstrate a novel inhibitory interaction between CAP1/Prss8 and nexin-1, opening the search for specific CAP1/Prss8 antagonists that are independent of its catalytic activity.

Colon-specific deletion of epithelial sodium channel causes sodium loss and aldosterone resistance.

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 αENaC 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 (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil 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 ∆PDamil 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.

Mutations of the serine protease CAP1/Prss8 lead to reduced embryonic viability, skin defects, and decreased ENaC activity.

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.