Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4

Abstract

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Fig 1. Distribution of wildtype CAP2/Tmprss4 mRNA transcript expression.

CAP2/Tmprss4 mRNA expression profile in WT mice (n = 4) in various organs as indicated; individual values were normalized to β-actin.

Fig 2. Inactivation of the CAP2/Tmprss4 gene locus.

(A) Scheme of the wild-type allele, the targeting vector, and the targeted CAP2/Tmprss4 ^loxneo allele following homologous recombination, and the CAP2/Tmprss4 ^lox and the CAP2/Tmprss4 ^Δ allele following breeding with Flp- and Cre-deleter mice, respectively. Relevant restriction enzymes for cloning and diagnosis of targeted ES cell clones are shown. Exons 8 and 9 and the neomycin cassette (flanked by frt sites) are flanked by loxP sites. 5’ and 3’ probes as well as PCR primers used for ES cell screening and mouse genotyping are indicated. (B) Southern blot analyses of targeted ES cell clones using the external 5’probe (upper left panel) following digestion with SpeI and NheI, the neo probe (upper right panel) following EcoRI digestion, and the external 3’probe following digestion with BamH1; note that clone #2 and #3 harbour additional recombination and integration events as evidenced by Southern blot analyses using the 5’ and neo probe, respectively. (C) Southern blot analysis of CAP2/Tmprss4 ^loxneo/+, CAP2/Tmprss4 ^lox/lox and/or CAP2/Tmprss4 ^lox/+ and CAP2/Tmprss ^Δ/Δ mice using the 5’ probe following SpeI/NheI digestion. (D) PCR-based genotyping of mice harbouring the wild type (+, 250bp, lane 1 and 3), lox alleles (lox, 350bp, lane 2) and knockout alleles (Δ, 500bp, lane 3 and 4).

Fig 3. Phenotype of CAP2/Tmprss4-deficient mice.

(A) Representative pictures of 3 months old (male) CAP2/Tmprss4 wildtype (WT) and CAP2/Tmprss4 knockout (KO) littermates. (B) Mean body weight (g) of 3-month-old male and female wildtype (WT, n = 6), heterozygous mutant (HET, n = 11 and n = 9, respectively), and knockout (KO, n = 6 and n = 5, respectively) mice. (C) Relative CAP2/Tmprss4 mRNA transcript expression in colon from CAP2/Tmprss4 ^WT, CAP2/Tmprss4 ^HET and CAP2/Tmprss4 ^KO mice (n = 6 mice per group); β-actin is used as internal control. (D) Representative immunoblot showing the presence of a 70kDa CAP2/Tmprss4-specific band in colon extracts from CAP2/Tmprss4 ^WT (lane 1 and 2), CAP2/Tmprss4 ^HET (lane 3–5) mice and absence in CAP2/Tmprss4 ^KO (lane 6–8) mice; arrow indicates the size of the expected but absent CAP2/Tmprss4-specific band in knockouts.

Fig 4. Histopathological analysis in ENaC-expressing organs from CAP2/Tmprss4 knockout mice.

Representative H&E stained section of colon, lung, kidney and skin from CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice; n = 2 females and 2 males for each group and genotype; bar indicates 100μm.

Fig 5. ENaC mRNA transcript and protein expression in kidneys from CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice under regular sodium diet.

(A-C) Relative mRNA transcript and (D-F) ENaC and β-actin protein expression in kidneys of (A) Scnn1a in CAP2/Tmprss4 wildtype (WT, n = 6), heterozygous mutant (HET, n = 7) and knockout (KO, n = 5) mice, (B) Scnn1b in CAP2/Tmprss4 wildtype (WT, n = 6), heterozygous mutant (HET, n = 7) and knockout (KO, n = 5) mice, and (C) Scnn1g in CAP2/Tmprss4 wildtype (WT, n = 6), heterozygous mutant (HET, n = 5) and knockout (KO, n = 5) mice; β-actin was used as internal control. Representative immunoblots of (D) Scnn1a, (E) Scnn1b and (F) Scnn1g and its corresponding β-actin protein expression in CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice; kidney extracts from Scnn1 wildtype (WT) and knockout (KO) mice were used as positive and negative control respectively; arrows indicate the full-length and the corresponding cleaved ENaC fragments.

Fig 6. ENaC mRNA transcript and protein expression in kidneys from CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice under sodium-deficient diet.

(A-C) Relative mRNA transcript and (D-F) protein expression of (A) Scnn1a, (B) Scnn1b and (C) Scnn1g from CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET), and knockout (KO) mice; n = 4 for each group and genotype; β-actin was used as internal control. Representative immunoblots of (D) Scnn1a, (E) Scnn1b and (F) Scnn1g and its corresponding β-actin protein expression from CAP2/Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice (n = 5 for each group and genotype); kidney extracts from Scnn1 wildtype (WT) and knockout (KO) mice were used as positive and negative control respectively; arrows indicate the full-length and the corresponding cleaved ENaC fragments; * P< 0.05).

Fig 7. ENaC mRNA transcript expression and activity in colon from CAP2/Tmprss4 mice under sodium-deficient diet.

(A-C) Relative mRNA transcript expression of (A) Scnn1a, (B) Scnn1b and (C) Scnn1g from CAP2/Tmprss4 wildtype (WT, n = 4), heterozygous mutant (HET, n = 5), and knockout (KO, n = 4) mice; *P< 0.05); β-actin was used as internal control. (D) Morning and afternoon amiloride-sensitive rectal potential difference (PD) measurements at 10-12am and 4-6pm of two consecutive days in Tmprss4 wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice; n = 4 for each group and genotype.