Mg²⁺ restriction downregulates NCC through NEDD4-2 and prevents its activation by hypokalemia

Hypomagnesemia is associated with reduced 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), also plays a key role in K⁺ homeostasis by metering Na⁺ delivery to distal segments. Little is known about the mechanisms by which plasma Mg²⁺ concentration regulates NCC activity and how low-plasma Mg²⁺ concentration and K⁺ concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg²⁺ restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg²⁺ restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K⁺ restriction. Mg²⁺ restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg²⁺ or combined Mg²⁺-K⁺ restriction but were dramatically lower compared with a low-K⁺ diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na⁺ delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg²⁺-K⁺ restriction, but, surprisingly, NCC was not activated after long-term K⁺ restriction despite lower plasma K⁺ concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K⁺ restriction.