Plasma Potassium Determines NCC Abundance in Adult Kidney-Specific γ ENaC Knockout

Abstract

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 βΕΝaC, 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 βENaC 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.

Keywords: Na+/Cl- co-transporter; Pseudohypoaldosteronism; SPAK; epithelial sodium channel; thiazide-sensitive.

Graphical abstract

Figure 1.

Efficient recombination of γENaC in kidneys of inducible nephron-specific γENaC knockout mice. (A) Genotyping using Scnn1g-specific PCR primers on DNA extracted from kidney, liver, and heart from controls (lanes 1 and 2) and experimental (knockout, Scnn1g^Pax8/LC1) mice (lanes 3 and 4); note absence of recombination in controls (lanes 1 and 2) and the presence of recombination in the liver (lanes 3 and 4). (B) Western blot analyses of whole kidneys and (C) their quantification 2 days after induction in γENaC control and experimental mice. Protein levels were normalized to β-actin and expressed in percentage of control. (D) Immunofluorescence detection of γENaC and CaBP in consecutive kidney sections from control or knockout (Scnn1g^Pax8/LC1) mice after 2 days of standard diet; protocol A. Results are presented as mean±SEM and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant. ***P<0.001.

Figure 2.

Adult γENaC-deficient mice develop a severe PHA-1 phenotype. (A) Body weight changes (Δ body weight) in percentage of initial body weight monitored during three consecutive days after doxycycline induction on a standard diet (protocol A); Scnn1g: each group, n=5. (B) Measurement of plasma electrolytes, Na⁺ (left) and K⁺ (right, mmol/L) in γENaC control (n=7) and knockout (Scnn1g^Pax8/LC1, n=6) mice. Measurement of urinary Na⁺ (C) and K⁺ (D) excretion (mmol/24 h per gram of body weight) in γENaC control and knockout (each group, n=5) mice; protocol A. Values were normalized to the body weight. *P<0.05; ***P<0.001.

Figure 3.

Adult γENaC-deficient mice present severe hyperkalemia under HNa⁺/LK⁺ diet. (A) Body weight changes (Δ body weight) of γENaC control and experimental (Scnn1g^Pax8/LC1) mice (each group: n=8) in percentage of initial body weight on a standard and short-term HNa⁺/LK⁺ diet (protocol B). (B) Measurements of plasma Na⁺ (left) and K⁺ (right) concentrations (mmol/L) in γENaC control and knockout mice (each group, n=8) at 18 hours after diet switch. Measurement of (C) urinary Na⁺ and (D) K⁺ excretion (mmol/gram of body weight), and (E) Na⁺ and (F) K⁺ intake (mmol/gram of body weight) in γENaC control and knockout (each group, n=8) mice after doxycycline treatment on a standard diet (24-hour) and HNa⁺/LK⁺ diet (6-hour measurement); protocol B. Values were normalized to the body weight. *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

Short-term LK⁺ diet fails to normalize the body weight and the plasma electrolytes of γENaC-deficient mice. (A) Body weight changes (Δ body weight) in percentage of initial body weight, and (B) plasma Na⁺ (left) and K⁺ (right) concentrations (mmol/L) under low potassium diet (protocol C) in control and knockout mice (Scnn1g^Pax8/LC1) (each group, n=6). Twenty-four-hour urinary (C) Na⁺ and (D) K⁺ excretion (mmol/24 h per gram of body weight), and (E) 24 hours Na⁺ and (F) K⁺ intake (mmol/24 h per gram of body weight) in controls and knockout mice (each group, n=6); values (A) and (C–F) are normalized to body weight. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Low potassium in combination with standard or high sodium diet does not normalize total NCC and pNCC in γENaC-deficient mice. (A) Representative Western blot analysis of total NCC, pNCC, and actin on kidney cortex extracts from control and knockout (Scnn1g^Pax8/LC1) mice on a standard diet; gENaC control, n=6 and knockout mice, n=5 (protocol A). (B) Short-term HNa⁺/LK⁺ diet (protocol B) in γENaC control and knockout mice; each group: n=5. (C) Short-term low potassium diet (1 day) in γENaC control and knockout group; each group: n=4 (protocol C). (D–F) Quantification of proteins and (G–I) ratio of pNCC to total NCC abundance from corresponding Western blot analyses. Protein levels are normalized to actin and expressed in percentage of control. Results are presented as mean±SEM and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant; *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Low potassium in combination with standard or high sodium diet does not normalize pSPAK in γENaC-deficient mice. (A) Representative Western blot analyses of pSPAK and actin on kidney cortex extracts from control and knockout (Scnn1g^Pax8/LC1) mice on a standard diet; γENaC control, n=6 and knockout mice. n=5 (protocol A). (B) Short-term HNa⁺/LK⁺ diet (protocol B) in γENaC control and knockout mice; each group, n=5. (C) Short-term LK⁺ diet (1 day) in γENaC control and knockout group; each group, n=4 (protocol C). (D–F) Quantification of proteins and (G–I) ratio of pSPAK to total SPAK abundance from corresponding Western blot analyses. Kidneys from SPAK knockout mice were used as negative controls. Protein levels are normalized to actin and expressed in percentage of control. C−, kidney protein lysate from SPAK-deficient (negative control); C+, from SPAK wildtype mice (positive control). Results are presented as mean±SEM and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant; *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

The βENaC subunit expression is reduced on a standard diet in γENaC-deficient mice. (A–C) Representative Western blot analyses for α and βENaC on whole kidney protein extracts from γENaC control and knockout mice on (A) standard diet (control, n=6; knockout, Scnn1g^Pax8/LC1, n=4), (B) HNa⁺/LK⁺ (control, n=8; knockout, n=6), and (C) K⁺-deficient diet (control, n=4) plus kayexalate (control, n=4; knockout, n=5; left panels, and quantification of proteins from corresponding Western blot analyses; right panels, protocol D). Protein expression was normalized to the amount of β-actin and reported relative to control values. Results are presented as mean±SEM, and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant. *P<0.05.

Figure 8.

Loss of apical βENaC subunit expression within the DCT2 in γENaC-deficient mice. Immunofluorescence detection of βENaC and CaBP in consecutive kidney sections from control (left) and knockout (Scnn1g^Pax8/LC1, right) mice after 2 days of standard diet; protocol A. CN, CNT; D2, DCT2.

Figure 9.

γENaC-deficient mice do not differ from control mice under K⁺-deficient diet. (A) Body weight changes (Δ body weight) of γENaC control and knockout (Scnn1g^Pax8/LC1) mice (each group: n=9) in percentage of initial body weight on an LK⁺ diet (protocol E). (B) Measurements of plasma Na⁺ (left) and K⁺ (right) concentrations (mmol/L) in γENaC control and knockout mice (each group, n=9) at day 6 of diet application. (C) Representative Western blot analyses of total NCC, pNCC, and actin on kidney cortex extracts from control and knockout (Scnn1g^Pax8/LC1) mice on a K⁺-deficient diet. (D) Quantification of proteins and (E) ratio of pNCC to total NCC abundance from corresponding Western blot analyses. (F) Representative Western blot analyses of total SPAK, pSPAK, and actin on kidney cortex extracts from control and knockout (Scnn1g^Pax8/LC1) mice on a K⁺-deficient diet. (G) Quantification of proteins and (H) ratio of pSPAK to total SPAK abundance from corresponding Western blot analyses. Protein levels are normalized to actin and expressed in percentage of control. C−, kidney protein lysate from SPAK-deficient (negative control); C+, from SPAK wildtype mice (positive control). Results are presented as mean±SEM and data (B, D, E, G, H) were analyzed by unpaired t test. P values <0.05 were considered statistically significant.