WNK bodies cluster WNK4 and SPAK/OSR1 to promote NCC activation in hypokalemia

Abstract

K+ deficiency stimulates renal salt reuptake via the Na+-Cl− cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K+ losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K+ deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed “WNK bodies,” the significance of which is undetermined. By feeding diets of varying salt and K+ content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K+ deprivation, and it was promptly suppressed by K+ loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K+ channel-forming protein Kir4.1, which is required for the DCT to sense plasma K+ concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K+ deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K+ concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.

Kidney-Specific WNK1 Amplifies NCC Responsiveness to Potassium Imbalance

“…In agreement with a recent study [10], DCT WNK bodies contained phosphorylated (active) SPAK and OSR1 ( Fig 3A-C). Though the cytoplasmic punctate staining of phosphorylated SPAK and OSR1 was absent in KS-WNK1-KO mice, apical staining was still present (Fig 3A,B).…”

“…Antibodies. The following antibodies were used for immunoblot and/or immunofluorescence: Sodium chloride cotransporter (NCC; kindly provided by David Ellison [39]); Sodium chloride cotransporter (NCC; Millipore Ab3553 [40]); Phospho-NCC Thr 53 (pNCC; Phospho-solutions P1311-53 [41]); Phospho-NCC Ser 71 (pNCCSer71, kindly provided by Jan Loffing [3]); SPAK (SPAK; Cell Signaling [40]); Phospho-SPAK/ Phospho-OSR1 (pSPAK/pOSR1; Millipore 07-2273 [10]); With-no-lysine kinase 1 (WNK1; Atlas Antibodies HPA059157 [9]); With-no-lysine kinase 4, (WNK4 [9]); Gamma subunit epithelial sodium channel (gENaC; 83 kDa cleaved and 95 kDa uncleaved; Stressmarq SPC-405) [17]; ATP-sensitive inward rectifier potassium channel 10 (Kir4.1; Alomone APC-035 [27]); Renal outer medullary potassium channel (ROMK; 50-65 kDa complex glycosylated; R-80 was kindly provided by James Wade) [42].…”

“…KS-WNK1 controls WNK-SPAK/OSR1 pathway localization in the DCT. During hypokalemia, DCT-expressed WNK4, SPAK, and OSR1 condense into WNK bodies– signaling puncta whose appearance correlates with NCC activation [9, 10]. Potassium-restricted KS-WNK1 knockout mice fail to form WNK bodies, suggesting that KS-WNK1 functions as a DCT-specific scaffold for WNK signaling.…”

Calcium-Sensing Receptor and Regulation of WNK Kinases in the Kidney

“…It is also worth mentioning that CaSR exerts inhibitory effects on Kir4.1, which may result from physical or functional interactions between the receptors [55,56]. Kir4.1 is critical to the WNK4-SPAK activity and resulting activating NCC phosphorylation, especially in hypokalemic conditions [96,100,101]. From this perspective, basolateral CaSR activation may suppress the WNK-SPAK-NCC signaling due to Kir4.1 inhibition.…”

“…Future studies using isolated rodent nephron segments are mandatory for resolving this issue. WNK4 activity can be potentiated by an interaction with the truncated, kidney-specific WNK1 variant (KS-WNK1), which abrogates the inhibitory effects of [Cl − ] i [ 95 , 96 , 97 ]. In view of the fact that KS-WNK1 expression is much higher in DCT compared to the other nephron segment, this mechanism may play a role in distinct effects of CaSR-activation between TAL and DCT as well [ 95 , 98 , 99 ].…”