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 βENaC 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 βENaC 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.
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We thank Olivier Bonny for critically reading the manuscript and Anne-Marie Mérillat for the excellent photographic work. Kidney protein lysate from SPAK KO mice was kindly provided by Olivier Staub. This work was supported by the Swiss National Science Foundation Grants FNRS 31003A_144198/1 and 31003A_163347 (to E.H.), the Leducq Foundation (to E.H.), Swiss National Center of Competence in Research (NCCR kidney.CH) and the networking support by the COST Action ADMIRE BM1301 (to E.H.).
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution. Protocols followed the Swiss federal guidelines and were approved by the veterinarian local authorities (« Service de la consommation et des affaires vétérinaires ») of the Canton de Vaud, Switzerland.
Electronic supplementary material
Schema of the experimental protocols. Representative setup used to determine physiological parameters in Scnn1bPax8/LC1 mice placed either in standard (thin line) or metabolic cages (bold line) and induced by doxycycline (Dox) under standard diet (A), a short term high Na+ and low K+ diet (B), a long-term high Na+ and low K+ diet (C), and a long-term low K+ diet (D), back to standard diet after one month under HNa+/LK+ diet (E), and back to LNa+ diet after 16 days under LK+ diet (F). Arrows indicate the end of the experiment, and the time point of serum and organ recovery. (PDF 1795 kb)
Physiological parameters as measured following induction under standard diet. Measurement of daily 24 hours (A) food (g/24 h/gBW), (B) water intake (ml/24 h/g BW), (C) feces (g/24 h/gBW), (D) urine volume output (ml/24 h/gBW), and measurement of (E) Na+ and (F) K+ intake (mmol/24/gBW) in Scnn1b control (n = 20) and experimental mice (n = 8), protocol A. Values were normalized to the body weight * P < 0.05, ** P < 0.01, ***P < 0.001 (PDF 208 kb).
Physiological parameters as measured following induction under acute High Na+ and low K+ diet. (A) Food intake (g/gBW), (B) water intake (ml//gBW), (C) feces output (g/gBW), (D) urine volume (ml/gBW), and measurement of daily urinary Na+ (E) and K+ (F) excretion (mmol/24 h/gBW), and (G) Na+ and K+ (H) intake (mmol/24 h/gBW), in Scnn1b control (n = 6) and experimental mice (n = 8) following doxycycline treatment upon standard diet (24-hour measurement) and high Na+/low K+ (6-hour measurement); protocol B. Values were normalized to the body weight. * P < 0.05, *** P < 0.001 (PDF 175 kb).
Long term high Na+ and low K+ diet normalizes all physiological parameters in Scnn1bPax8/LC1 mice. Measurement of (A) body weight changes (Δ body weight, BW) in percentage of initial BW, and (B) plasma Na+ and K+ concentration (mmol/l), (C) 24 hours urinary Na+ and (D) K+ excretion (mmol/24 h/gBW), and (E) Na+ and (F) K+ intake (mmol/24 h/gBW) in control (n = 8) and Scnn1bPax8/LC1 mice (n = 6), (protocol C). ** P < 0.01 (PDF 229 kb).
Long term high Na+ and low K+ diet normalizes food and water intake, feces weight and urinary volume excretion in Scnn1bPax8/LC1 mice. 24 hours (A) food (g/24 h/gBW) and (B) water intake (ml/24 h/gBW), (C) feces output (g/24 h/gBW), and (D) urine volume (ml/24 h/gBW) in control (n = 8) and Scnn1bPax8/LC1 mice (n = 6), (protocol C). Results are presented as mean ± SEM and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant. (PDF 232 kb).
α- and γ-ENaC protein expression is unchanged in Scnn1bPax8/LC1 mice. Representative Western blot analyses of α-, γ-ENaC and actin on kidney from Scnn1b control and experimental mice upon standard diet (protocol A) (A); controls, n = 6 and experimental Scnn1b mice: n = 6; (B) Acute high Na+ and low K+ diet (protocol B); control and experimental mice, each group, n = 6; (C) Long - term high Na+ and low K+ diet (protocol C); control (n = 8) and experimental mice (n = 6). Left panel, quantification of proteins from corresponding Western blots analyses. Protein levels were 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. (PDF 6735 kb).
High Na+ and low K+ diet normalizes pSPAK in Scnn1bPax8/LC1 mice. Representative Western blot analyses of phosphorylated SPAK and actin on kidney cortex extracts from Scnn1b control and experimental mice upon standard diet (protocol A) (A); controls, n = 6 and experimental Scnn1b mice: n = 5; (C) high Na+ and low K+ diet (protocol B); in control and experimental mice, each group, n = 5. (B and D) Quantification of proteins from corresponding Western blot analyses. Protein levels were normalized to actin and expressed in percentage of control. Results are presented as mean ± SEM and data were analyzed by unpaired t test. ** P < 0.01 (PDF 1032 kb).
Long-term low K+ diet and standard Na+ restores food and water intake, feces weight and urinary volume excretion in Scnn1bPax8/LC1 mice. Daily (A) food (g/24 h/gBW) and (B) water intake (ml/24 h/gBW), (C) feces output (g/24 h/gBW) and (D), urine volume (ml/24 h/gBW), in control (n = 6) and experimental mice (n = 6; protocol D). Results are presented as mean ± SEM and data were analyzed by unpaired t test. P values <0.05 were considered statistically significant. (PDF 196 kb).
Long-term low K+ diet normalizes pSPAK in Scnn1bPax8/LC1 mice. Representative Western blot analyses of phosphorylated SPAK and actin on kidney cortex extracts from Scnn1b control and experimental mice upon standard Na+ and low K+ diet (A); protocol D), each group, n = 5. (B) Quantification of proteins from corresponding Western blot analyses. Protein levels were 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. (PDF 569 kb).
Physiological parameters as measured under both standard Na+/low K+ and standard K+/low Na+ diet in Scnn1bPax8/LC1 mice. (A) Food intake (g/24h/gBW), (B) water intake (ml/24h/gBW), (C) feces output (g/24h/gBW), (D) urine volume (ml/24h/gBW), and measurement of (E) Na+ and K+ (F) intake (mmol/24h/gBW), in control (n = 6) and experimental mice (n = 4) under low K+ diet and following LNa+ diet (24-hour measurement); protocol F. Values were normalized to the body weight. * P<0.05, ** P <0.01 (PDF 121 kb).
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Boscardin, E., Perrier, R., Sergi, C. et al. Severe hyperkalemia is rescued by low-potassium diet in renal βENaC-deficient mice. Pflugers Arch - Eur J Physiol 469, 1387–1399 (2017). https://doi.org/10.1007/s00424-017-1990-2
- Epithelial sodium channel
- Pseudohypoaldosteronism type 1
- Thiazide-sensitive Na+/Cl− co-transporter
- STE20/SPS1-related proline-alanine-rich protein kinase