Kidney Week

Abstract: TH-OR005

Mg²⁺ Restriction Downregulates NCC Through NEDD4-2 and Prevents Its Activation by Hypokalemia

Session Information

• Advances in Fluid and Electrolyte Handling: Basic Physiology
  November 07, 2019 | Location: 146 C, Walter E. Washington Convention Center
  Abstract Time: 05:18 PM - 05:30 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• McCormick, James A., Oregon Health and Science University, Portland, Oregon, United States

James A. McCormick, PhD

• Ferdaus, Mohammed Zubaerul, Oregon Health and Science University, Portland, Oregon, United States

Mohammed Zubaerul Ferdaus,

• Blatt, Philip James, Oregon Health and Science University, Portland, Oregon, United States

Philip James Blatt,

• Nelson, Jonathan W., Oregon Health and Science University, Portland, Oregon, United States

Jonathan W. Nelson,

• Terker, Andrew, Vanderbilt University, Nashvile, Tennessee, United States

Andrew Terker,

• Staub, Olivier, University of Lausanne, Lausanne, Switzerland

Olivier Staub,

• Lin, Daohong, New York Medical College, Valhalla, New York, United States

Daohong Lin,

Background

Hypomagnesemia is associated with lower kidney function and life-threatening complications, and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg²⁺ excretion, and via activity of the Na⁺-Cl^- cotransporter (NCC) plays a key role in K⁺ homeostasis by metering Na⁺ delivery to distal segments. We previously showed that short-term (3 days) or long-term (14 days) Mg²⁺ restriction lowered abundances of total NCC (tNCC) and the active phosphorylated NCC (pNCC), but this did not involve the NCC-activating WNK-SPAK pathway.

Methods

We set out to further explore the mechanisms involved and determine interactions with K⁺ restriction, a strong activator of NCC, by performing dietary manipulations in mice then Western blotting.

Results

We confirmed a previous report that long-term Mg²⁺ restriction does not alter NCC mRNA abundance, and found the same with short-term Mg²⁺ restriction. The E3 ubiquitin-protein ligase neural precursor cell expressed developmentally downregulated gene 4-2 (NEDD4-2) is known to target NCC for proteasomal degradation. We found that short-term Mg²⁺ restriction did not lower tNCC abundance in inducible nephron-specific NEDD4-2 knockout mice. We next examined interactions with K⁺ restriction. tNCC and pNCC abundances were similar after short- or long-term Mg²⁺ or combined Mg²⁺-K⁺ restriction, but were dramatically lower compared with low K⁺ diet, suggesting that Mg²⁺ restriction overrides the effects of K⁺ restriction on NCC. After combined Mg²⁺-K⁺ restriction, adding back K⁺ alone to the diet had no effect on tNCC abundance, but adding back Mg²⁺ either at the same time or after K⁺ replenishment increased tNCC abundance. NEDD4-2 mediates degradation of the epithelial sodium channel (ENaC) during dietary K⁺ restriction so we next examined the effect of Mg²⁺ restriction on ENaC by performing amiloride response tests. Compared with normal diet the natriuretic effect of amiloride was strongly blunted after K⁺ restriction but not after Mg²⁺ restriction.

Conclusion

Together, these data suggest that NEDD4-2 mediates proteasomal degradation of NCC during Mg²⁺ restriction, Mg²⁺ restriction exerts differential effects on NCC and ENaC, and sustained NCC downregulation may enhance distal Na⁺ delivery during states of hypomagnesemia, maintaining hypokalemia.

Funding

• NIDDK Support