Kidney Week

Abstract: FR-PO523

α-Epithelial Sodium Channel (ENaC) Proteolysis Regulates Channel Function In Vivo in a Sex- and Tissue-Specific Manner

Session Information

• Fluid, Electrolyte, Acid-Base Disorders: Basic
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Nickerson, Andrew, University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States

Andrew Nickerson,

• Sheng, Shaohu, University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States

Shaohu Sheng,

• Ray, Evan C., UPMC, Pittsburgh, Pennsylvania, United States

Evan C. Ray,

• Carattino, Marcelo D., University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States

Marcelo D. Carattino,

• Marciszyn, Allison L., University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States

Allison L. Marciszyn,

• Lam, Tracey, University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States

Tracey Lam,

• Kleyman, Thomas R., UPMC, Pittsburgh, Pennsylvania, United States

Thomas R. Kleyman,

Background

Epithelial Na⁺ channels (ENaCs) facilitate Na⁺ and water reabsorption in the distal nephron and distal colon, while also controlling K⁺ secretion in the distal nephron. ENaC activity is regulated, in part, by proteolysis of the channel’s α and γ subunits. In vitro, α-ENaC is typically cleaved twice by furin proteases during synthesis. As a result, an autoinhibitory peptide sequence is excised, thereby increasing the channel’s open probability (P_O). However, the importance of α-ENaC cleavage with respect to Na⁺ and fluid handling in vivo is unknown.

Methods

We tested the hypothesis that α-ENaC cleavage is necessary for channel function in vivo by generating mice that lack a key furin cleavage site (²²⁹RSAR²³² > ²²⁹QSAQ²³²; α^Fm = α furin site mutant). We assessed ENaC function in α^Fm versus wild-type (WT) littermates via electrophysiological and systemic analyses under normal and Na⁺-restricted dietary conditions.

Results

At baseline, male α^Fm mice had elevated blood K⁺ versus WT littermates (α^Fm vs. WT: 4.7 ± 0.1 vs. 4.3 ± 0.1 mM; p=0.03), but this was not observed in females (α^Fm vs. WT: 4.8 ± 0.1 vs. 5.0 ± 0.2 mM; p=0.8). Otherwise, α^Fm mice of either sex exhibited no baseline phenotype regarding blood electrolyte or metabolic parameters. Western blot analysis showed no differences in ENaC subunit expression in the kidney or distal colon. Patch clamp experiments revealed no differences in ENaC activity (P_O) in isolated kidney tubules. However, short-circuit current (I_SC) measurements revealed that male α^FM mice had diminished ENaC activity in the distal colon (α^Fm vs. WT ΔI_SC(amiloride): -0.2 ± 2.3 vs. -17.7 ± 4.4 μA/cm²; p<0.01). Colonic ENaC activity in both genotypes was stimulated by dietary Na⁺ restriction (α^Fm vs. WT ΔI_SC(amiloride): -104 ± 38 vs. 64 ± 19 μA/cm²; p=0.4) and plasma aldosterone levels were also similar following treatment (α^Fm vs. WT: 2,910 ± 323 vs. 2,275 ± 584 pg/mL). Body composition analysis showed that during Na⁺-restriction, α^FM mice maintained body water content, but lost total body weight more rapidly than WT littermates.

Conclusion

α subunit cleavage is required for full ENaC function in male mice. However, loss of α-ENaC cleavage can be compensated by elevated aldosterone under Na⁺-restricted conditions to maitain salt and fluid balance.

Funding

• NIDDK Support