Kidney Week

Abstract: PO1095

In Vivo Influence of a Protease-Resistant Epithelial Sodium Channel Gamma Subunit on Fluid Homeostasis

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  November 04, 2021 | Location: On-Demand, Virtual Only
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolyte, and Acid-Base Disorders

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

Authors

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

Evan C. Ray, MD, PhD, FASN

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

Tracey R. Lam,

• Jordahl, Alexa S., University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Alexa S. Jordahl,

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

Allison L. Marciszyn,

• Zhang, Lei, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Lei Zhang,

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

Shaohu Sheng,

• Shi, Shujie, University of Pittsburgh, Pittsburgh, Pennsylvania, United States

Shujie Shi,

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

Thomas R. Kleyman,

Group or Team Name

• University of Pittsburgh P30 O'Brien Center for Kidney Research

Background

Extracellular fluid depletion promotes proteolytic processing of ENaC’s γ subunit. Removal of the subunit’s inhibitory tract enhances channel open probability. Although several cleavage sites exist distal to the γ subunit’s inhibitory tract, only one known site resides proximal to the inhibitory tract: a furin cleavage site (RKRK¹⁴³). We hypothesized that a mouse expressing a protease-resistant ENaC γ subunit would exhibit signs consistent with reduced renal tubular ENaC activity, such as attenuated ability to adapt to dietary Na restriction.

Methods

We used TEV in Xenopus oocytes to confirm the ability of disruption of the γ subunit’s furin cleavage site (RKRK¹⁴³ to QQQQ¹⁴³, or "Q4") to reduce ENaC activity. In 129sv mice, we used CRISPR-Cas9 to introduce this amino acid substitution into ENaC's γ subunit. Altered cleavage of ENaC’s γ subunit was confirmed by Western blot of tissues from mice on a low (0.04%) Na diet (LSD). Lysates of kidneys from these animals were digested in PNGase to deglycosylate proteins, facilitating interpretation of molecular weights. Blood electrolytes were evaluated by iSTAT at sacrifice. Animal body fluid was assessed in live animals using quantitative magnetic resonance (Echo-MRI).

Results

Expression of the protease prostasin in oocytes, along with ENaC (N = 15), increased amiloride-sensitive currents, compared to oocytes with ENaC but no prostasin (N = 15; p < 0.0001). In oocytes expressing a Q4 gamma subunit (N = 15), prostasin no longer increased currents (N =15). Western blot of PNGase-digested tissue lysates revealed a full-length (~60 kDa) γ subunit and two shorter proteins, consistent with subunits either cleaved at the furin site (~53 kDa) or at a more distal site. Tissues from Q4 mice lacked the 53 kDa band, suggesting impaired Furin site cleavage. Blood K+ was normal in Q4 mice (N ≥ 7 for each sex and genotype). On a LSD, Q4 male mice exhibited greater loss of body water than control males (p = 0.04; N = 6-7), but females exhibited no difference in body water (N = 7-8).

Conclusion

These findings support a role for proteolytic activation of ENaC in male mice maintaining total body fluid in response to dietary Na depletion. Females did not show a impaired body fluid retention, suggesting additional compensatory mechanisms.

Funding

• NIDDK Support