Hypokalemia-Induced NKCC2 Dephosphorylation via the PKA-DARPP-32-PP1 Pathway in the Renal Medulla

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Background: Phosphorylation of the Na+-K+-2Cl- cotransporter (NKCC2) in the thick ascending limb of Henle’s loop (TAL) is essential for its activation and contributes to medullary hypertonicity and urinary concentration. While NKCC2 phosphorylation is regulated by the vasopressin type 2 receptor (V2R)–cAMP–PKA pathway, the roles of downstream effectors remain unclear. Although hypokalemia enhances Na+-Cl- cotransporter (NCC) phosphorylation via the WNK4–SPAK/OSR1 pathway, its effect on NKCC2 is unknown. This study aimed to elucidate the mechanism of NKCC2 phosphorylation in hypokalemia, focusing on the DARPP-32–PP1 dephosphorylation pathway downstream of PKA. Methods: C57BL/6 mice were fed normal [1% K+], low K+ (LK) [0.05% or 0.2% K+], or high K+ [5% K+] potassium diets for 3 or 7 days. Kidneys and blood were collected for western blotting, immunofluorescence, and plasma [K+] measurement. NKCC2 activity was confirmed by furosemide response test with thiazide treatment, and medullary expression of osmoregulatory genes was analyzed. Ex vivo kidney slices were treated with DDAVP, forskolin, or calyculin A to assess V2R–cAMP–PKA pathway effects on NKCC2 phosphorylation. Results: Phosphorylated NKCC2 positively correlated with plasma [K+], unlike pNCC, which showed a negative correlation. In LK mice, pNKCC2 was reduced in the medulla but preserved in the cortex, and the furosemide response was attenuated. NKCC2 dephosphorylation occurred before AQP2 protein and osmoregulatory gene downregulation, indicating impaired NKCC2 function and medullary hypertonicity. Phosphorylated DARPP-32 was mainly expressed in the TAL in controls but was diminished along the apical membrane of medullary TALs in hypokalemic mice, along with PP1a. Meanwhile, phosphorylated SPAK/OSR1 was upregulated, suggesting that impaired DARPP-32–PP1a signaling, rather than lower WNK4–SPAK/OSR1 activity, plays a dominant role under hypokalemia. Ex vivo analyses revealed that DDAVP- and forskolin-induced phosphorylation of PP1a and NKCC2 was significantly suppressed in LK mice, while their responsiveness to calyculin A was preserved. Conclusion: Hypokalemia reduces V2R signaling and cAMP production, leading to NKCC2 dephosphorylation via the PKA–DARPP-32–PP1 pathway, which may contribute to impaired urinary concentration. Funding: Private Foundation Support, Government Support – Non-U.S.

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