ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

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

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
  • Nickerson, Andrew, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Carrisoza-Gaytan, Rolando, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Lam, Tracey, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Winfrey, Aaliyah, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Marciszyn, Allison L., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Satlin, Lisa M., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Sheng, Shaohu, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Kleyman, Thomas R., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

Proteolytic removal of a peptide inhibitory tract (IT) from the epithelial Na+ channel (ENaC) γ subunit’s extracellular domain increases channel open probability (PO) 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 PO, 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 PO or NPO. A 17pS ion channel also showed no change in PO or NPO.

In isolated, perfused collecting ducts from low Na+-diet fed mice, Na+ flux (JNa) 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 (JK) was also not different at a low flow rate. At a high flow rate, the JK 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 (ISC) 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 ISC, or collecting duct channel PO or JNa, but diminished flow-induced stimulation of JK 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