Sodium and potassium balance depends on αENaC expression in connecting tubule.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing

Inactivation of the CAP2/<i>Tmprss4</i> gene locus.

<p><b>(A)</b> Scheme of the wild-type allele, the targeting vector, and the targeted CAP2/<i>Tmprss4</i>^{<i>loxneo</i>} allele following homologous recombination, and the CAP2/<i>Tmprss4</i>^<i>lox</i> and the CAP2/<i>Tmprss4</i>^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 <i>frt</i> sites) are flanked by <i>lox</i>P sites. 5’ and 3’ probes as well as PCR primers used for ES cell screening and mouse genotyping are indicated. (<b>B</b>) Southern blot analyses of targeted ES cell clones using the external 5’probe (upper left panel) following digestion with <i>Spe</i>I and <i>Nhe</i>I, the neo probe (upper right panel) following <i>Eco</i>RI digestion, and the external 3’probe following digestion with <i>Bam</i>H1; 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. (<b>C</b>) Southern blot analysis of CAP2/<i>Tmprss4</i>^{<i>loxneo/+</i>}, CAP2/<i>Tmprss4</i>^{<i>lox/lox</i>} and/or CAP2/<i>Tmprss4</i>^<i>lox/+</i> and CAP2/<i>Tmprss</i>^Δ<i>/</i>Δ mice using the 5’ probe following <i>Spe</i>I/<i>Nhe</i>I digestion. (<b>D</b>) PCR-based genotyping of mice harbouring the wild type (<i>+</i>, 250bp, lane 1 and 3), <i>lox</i> alleles (<i>lox</i>, 350bp, lane 2) and knockout alleles (Δ, 500bp, lane 3 and 4).</p

ENaC mRNA transcript expression and activity in colon from CAP2/<i>Tmprss4</i> mice under sodium-deficient diet.

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

Histopathological analysis in ENaC-expressing organs from CAP2/<i>Tmprss4</i> knockout mice.

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

Distribution of wildtype CAP2/<i>Tmprss4</i> mRNA transcript expression.

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

Phenotype of CAP2/<i>Tmprss4</i>-deficient mice.

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

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

<p><b>(A-C)</b> Relative mRNA transcript and (<b>D-F</b>) protein expression of (<b>A</b>) <i>Scnn1a</i>, (<b>B</b>) <i>Scnn1b</i> and (<b>C)</b><i>Scnn1g</i> from CAP2/<i>Tmprss4</i> 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 (<b>D)</b> Scnn1a, (<b>E</b>) Scnn1b and (<b>F</b>) Scnn1g and its corresponding β-actin protein expression from CAP2/<i>Tmprss4</i> 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; * <i>P</i>< 0.05).</p

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK