WNK4 Knockout Cell Line (HEK293)

The with-no-lysine (K) (WNK) kinases regulate processes such as cell volume and epithelial ion transport through the modulation of cation chloride cotransporters such as the NaCl cotransporter (NCC) present in the distal convoluted tubule (DCT) of the kidney. Recently, the interaction of WNKs with nuclear receptor binding protein 1 (NRBP1) and transforming growth factor-β-stimulated clone 22 domain (TSC22D) proteins was reported. Here, we explored the effect of NRBP1 and TSC22Ds on WNK signaling in vitro and in the DCT. TSC22D1.1, TSC22D2, and NRBP1 are localized in DCT WNK bodies, which are cytoplasmic biomolecular condensates associated with WNK activation. In HEK293 cells, long TSC22D isoforms and NRBP1 increase WNK4 activity. DCT-specific NRBP1-knockout mice have reduced NCC phosphorylation and activate a compensatory response. Thus, NRBP1 and long TSC22D proteins are positive modulators of WNK signaling and modulate Na reabsorption in the kidney. NRBP1 and TSC22Ds likely influence WNK signaling in other tissues, affecting various physiological processes.

WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif-binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.

BACKGROUND:The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS:We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS:HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS:Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

The With No lysine (WNK) kinases regulate processes such as cell volume and epithelial ion transport through the modulation of Cation Chloride Cotransporters such as the NaCl cotransporter, NCC, present in the distal convoluted tubule (DCT) of the kidney. Recently, the interaction of WNKs with Nuclear Receptor Binding Protein 1 (NRBP1) and Transforming Growth Factor β-Stimulated Clone 22 Domain (TSC22D) proteins was reported. Here we explored the effect of NRBP1 and TSC22Ds on WNK signaling in vitro and in the DCT. TSC22D1.1, TSC22D2, and NRBP1 are localized in DCT WNK bodies, which are cytoplasmic biomolecular condensates associated with WNK activation. In HEK293 cells, long TSC22D isoforms and NRBP1 increase WNK4 activity. DCT-specific NRBP1 knockout mice have reduced NCC phosphorylation and activate a compensatory response. Thus, NRBP1 and long TSC22D proteins are positive modulators of WNK signaling and modulate Na reabsorption in the kidney. NRBP1 and TSC22Ds likely influence WNK signaling in other tissues, impacting various physiological processes.