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Abstract: FR-PO599

The Molecular Chaperone GRP170 Regulates ENaC Biogenesis and Salt and Water Homeostasis in the Kidney

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Buck, Teresa M., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Mutchler, Stephanie, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Ray, Evan C., UPMC, Pittsburgh, Pennsylvania, United States
  • Marciszyn, Allison L., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Kleyman, Thomas R., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Brodsky, Jeffrey L., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background

The epithelial sodium channel (ENaC) is expressed in a variety of epithelial tissues. In the distal nephron, ENaC is responsible for Na+ reabsorption and regulates salt and water homeostasis and blood pressure. ENaC is a heterotrimeric channel composed of an α, β, and γ-subunit. Unassembled ENaC subunits are recognized by the ER associated degradation (ERAD) machinery and degraded. Using cellular models, we determined that the ER localized molecular chaperone, GRP170, regulates both ENaC quality control and trafficking. We determined that GRP170 is required for the degradation of the unassembled αENaC subunit, but not the β or γ-subunits. However, when all three ENaC subunits are present, GRP170 promotes trafficking of ENaC to the cell surface. Therefore, we hypothesized that GRP170 promotes ENaC surface expression in the mammalian kidney.

Methods

To test our hypothesis we generated a kidney tubule-specific, inducible, GRP170 knock-out (KO) mouse. A GRP170 allele flanked by LoxP sites (“floxed”) was generated, and crossed to a mouse that expresses Cre recombinase in kidney tubules upon doxycycline treatment. Adult mice were treated for 10 days with doxycycline to deplete GRP170 and given either a standard or high-salt diet. Mice were placed in metabolic cages and weight, water intake and urine output were monitored. Animals were sacrificed and blood, kidneys, lungs, and liver were harvested.

Results

GRP170 KO mice lose weight after doxycycline treatment (~20% body weight in three weeks), whereas control mice do not. Rapid weight loss is consistent with volume loss. Because Na+ reabsorption by ENaC promotes water reabsorption, loss of volume is consistent with decreased ENaC activity. Indeed, a significant increase in plasma aldosterone, a decrease in plasma Na+, and an increase in plasma K+ are observed in the GRP170 KO mouse. When experiments were repeated with a high-salt diet, changes in plasma Na+ and K+ levels were no longer observed. Because a high-salt diet partially rescues the GRP170 KO phenotype, these data are consistent with Na+ depletion and reduced ENaC activity.

Conclusion

The GRP170 chaperone regulates ENaC expression or activity in the mouse kidney, consistent with its effect in vitro. Ongoing experiments will examine whether GRP170 regulates other ion channels or transporters.

Funding

  • NIDDK Support