Regulation of NKCC2 Phosphorylation by Hypokalemia through the PKA–DARPP-32–PP1 Pathway in the Renal Medulla

Phosphorylation of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) in the thick ascending limb (TAL) is essential for 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. NKCC2 phosphorylation is enhanced through inhibition of protein phosphatase 1 (PP1)-mediated dephosphorylation by inhibitor-1 (I-1), while DARPP-32 can similarly suppress PP1 when phosphorylated by PKA; however, the functional role of DARPP-32 in NKCC2 has not been elucidated. Furthermore, 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 during hypokalemia, focusing on the PKA-DARPP-32-PP1 dephosphorylation pathway.

C57BL/6 mice were fed normal-K+ (1%), low-K+ (LK; 0.05% or 0.2%), or high-K+ (5%) diets for 3 or 7 days. Half kidneys or renal medulla were collected for western blotting, immunofluorescence, and quantitative PCR, and blood was collected for plasma [K⁺] measurement. NKCC2 activity was confirmed by furosemide response test following thiazide pretreatment, 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-PP1 pathway effects on NKCC2 phosphorylation. In vitro, the effects of I-1 and DARPP-32 on PP1 activity were compared in HEK-293 cells co-transfected with plasmids encoding PP1A and either I-1 or DARPP-32.

Phosphorylated NKCC2 positively correlated with plasma [K⁺], in contrast to phosphorylated NCC, 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 preceded AQP2 protein and osmoregulatory gene downregulation, indicating impaired NKCC2 function and medullary hypertonicity. Phosphorylated PP1A was decreased along the apical membrane of medullary TALs in hypokalemic mice, while phosphorylated SPAK/OSR1 was upregulated, suggesting that impaired PKA-PP1 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. In vitro, forskolin treatment induced phosphorylation of DARPP-32 at Thr34 and I-1 at Thr35 to a similar extent; however, only DARPP-32, but not I-1, promoted PP1A phosphorylation. Importantly, pT34 DARPP-32 was more abundant in the renal medulla than pT35 I-1 and was mainly expressed in the TAL under basal conditions. In LK mice, DARPP-32 phosphorylation shifted from T34 to T75, then pT34 DARPP-32 was diminished along the apical membrane of medullary TALs, together with pPP1A, highlighting the importance of PKA-DARPP-32-PP1 pathway in regulating NKCC2.

Hypokalemia reduces ADH-PKA signaling and shifts DARPP-32 phosphorylation from T34 to T75, thereby promoting NKCC2 dephosphorylation via the PKA-DARPP-32-PP1 axis, which may underlie impaired urinary concentrating ability. This abstract was also submitted for the Kidney Week 2025 congress.