Kidney Week

Abstract: FR-PO529

Physiologic Importance of Proteolytic Cleavage of Epithelial Sodium Channel (ENaC)'s γ Subunit in Na+ and K+ Handling

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

• Ray, Evan C., University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Evan C. Ray,

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

Andrew Nickerson,

• Carrisoza-Gaytan, Rolando, Icahn School of Medicine at Mount Sinai, New York, New York, United States

Rolando Carrisoza-Gaytan,

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

Tracey Lam,

• Winfrey, Aaliyah, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Aaliyah Winfrey,

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

Allison L. Marciszyn,

• Satlin, Lisa M., Icahn School of Medicine at Mount Sinai, New York, New York, United States

Lisa M. Satlin,

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

Shaohu Sheng,

• Kleyman, Thomas R., University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Thomas R. Kleyman,

Background

Proteolytic removal of a peptide inhibitory tract (IT) from the epithelial Na⁺ channel (ENaC) γ subunit’s extracellular domain increases channel open probability (P_O) in vitro. IT removal requires cleavage at a furin recognition sequence (¹⁴⁰RKRR¹⁴³) proximal to the inhibitory tract and a site distal to the IT. We hypothesized that mice with impaired IT removal would exhibit diminished distal nephron ENaC P_O, decreased Na⁺ transport, and increased susceptibility to hyperkalemia and body fluid volume depletion.

Methods

We disrupted the γ subunit furin cleavage site in mice (¹⁴⁰QQQQ¹⁴³, or “Q4”), reducing furin cleaved / full length γ subunit in Q4 mouse kidneys compared to wild-type (WT) littermates.

Results

Q4 mice exhibited no significant difference in blood Na⁺, K⁺, tCO₂, BUN, Hb, and Ca²⁺ on a standard, low Na⁺, or high K⁺ diet and no difference in plasma aldosterone on a low Na⁺ diet.

Collecting duct patch clamp experiments revealed an 8 pS channel, consistent with canonical heterotrimeric ENaC, that exhibited no difference in P_O or NP_O. A 17pS ion channel also showed no change in P_O or NP_O.

In isolated, perfused collecting ducts from low Na⁺-diet fed mice, Na⁺ flux (J_Na) was not different between Q4 and WT animals at low or high luminal fluid flow rates (1 or 5 nl min^-1 mm^-1). K flux (J_K) was also not different at a low flow rate. At a high flow rate, the J_K magnitude was greater in WT than Q4 collecting ducts (6.0 ± 1.1 vs 3.8 ± 0.6 pmol min^-1 mm^-1, p ≤ 0.01).

In colonic epithelium, we observed no difference in amiloride-sensitive short-circuit currents (I_SC) in Q4 vs. WT.

Total body water (TBW), measured via quantitative magnetic resonance and normalized to starting TBW, declined more in Q4 than WT males. TBW change was not different in female Q4 mice compared with WT mice.

Conclusion

Impaired furin site cleavage in ENaC’s γ subunit did not influence blood electrolytes, colonic I_SC, or collecting duct channel P_O or J_Na, but diminished flow-induced stimulation of J_K in the collecting duct, and impaired body fluid conservation in males. Differences, where observed, were relatively subtle, indicating that additional factors influence ENaC activity and Na⁺ and K⁺ balance in live animals.

Funding

• NIDDK Support