Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gαi proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gαi2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

Sepsis is one of the leading causes of death in critical patients worldwide and its occurrence is related to the excessive activation of macrophages. Chloride loss worsens the prognosis of patients with sepsis but the underlying mechanism is currently unclear. In this study, we founded that macrophages deficient in intracellular Cl- secrete more inflammatory cytokines such as IL-1β, IL-6 and TNF-α compared with control group. The intracellular chloride level decreased in WNK1 deficiency or activity inhibited macrophages with more severe inflammatory response after LPS treatment. Remimazolam, as classic GABAa receptor agonist, alleviates excessive inflammation cascade by promoting macrophage chloride influx during sepsis progression. Collectively, this study proves that macrophage WNK1 acts as a negative regulator of inflammatory response by sensing chloride to maintain intracellular chloride balance during sepsis coupled with hypochloremia.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

UNASSIGNED: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown.
UNASSIGNED: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs.
UNASSIGNED: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-β (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-β blockade using neutralizing anti-TGF-β antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals.
UNASSIGNED: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

Dietary potassium deficiency causes stimulation of sodium reabsorption leading to an increased risk in blood pressure elevation. The distal convoluted tubule (DCT) is the main rheostat linking plasma K+ levels to the activity of the Na-Cl cotransporter (NCC). This occurs through basolateral membrane potential sensing by inwardly rectifying K+ channels (Kir4.1/5.1); decrease in intracellular Cl-; activation of WNK4 and interaction and phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK); binding of calcium-binding protein 39 (cab39) adaptor protein to SPAK, leading to its trafficking to the apical membrane; and SPAK binding, phosphorylation, and activation of NCC. As kidney-specific with-no-lysine kinase 1 (WNK1) isoform (KS-WNK1) is another participant in this pathway, we examined its function in NCC regulation. We eliminated KS-WNK1 specifically in the DCT and demonstrated increased expression of WNK4 and long WNK1 (L-WNK1) and increased phosphorylation of NCC. As in other KS-WNK1 models, the mice were not hyperkalemic. Although wild-type mice under low-dietary K+ conditions demonstrated increased NCC phosphorylation, the phosphorylation levels of the transporter, already high in KS-WNK1, did not change under the low-K+ diet. Thus, in the absence of KS-WNK1, the transporter lost its sensitivity to low plasma K+. We also show that under low K+ conditions, in the absence of KS-WNK1, there was no formation of WNK bodies. These bodies were observed in adjacent segments, not affected by the targeting of KS-WNK1. As our data are overall consistent with those of the global KS-WNK1 knockout, they indicate that the DCT is the predominant segment affecting the salt transport regulated by KS-WNK1.NEW & NOTEWORTHY In this paper, we show that KS-WNK1 is a critical component of the distal convoluted tubule (DCT) K+ switch pathway. Its deletion results in an inability of the DCT to sense changes in plasma potassium. Absence of KS-WNK1 leads to abnormally high levels of WNK4 and L-WNK1 in the DCT, resulting in increased Na-Cl phosphorylation and function. Our data are consistent with KS-WNK1 targeting WNK4 and L-WNK1 to degradation.

Cellular oxidative stress plays a key role in the development and progression of hepatocellular carcinoma (HCC). A better understanding of the processes that regulate reactive oxygen species (ROS) homeostasis could uncover improved strategies for treating HCC. Herein, we identified protein kinase with-no-lysine kinase 1 (WNK1) as an antioxidative factor and therapeutic target in HCC. In human HCC, WNK1 expression was increased and correlated with poor patient prognosis. WNK1 knockdown significantly inhibited cell proliferation and xenograft tumor growth. Mechanistically, WNK1 competed with nuclear factor erythroid 2-related factor 2 (NRF2) for binding with the partial Kelch domain of Kelch-like ECH-associated protein 1 (KEAP1), reducing NRF2 ubiquitination and promoting NRF2 accumulation and nuclear translocation to increase antioxidant response. WNK1 silencing increased H2O2-induced apoptosis and inhibited cell growth by elevating ROS levels, which could be rescued by treatment with the antioxidant N-acetylcysteine and NRF2 activator tert-butylhydroquinone. Liver-specific WNK1 knockout mouse models of HCC substantiated that WNK1 promoted HCC development by regulating ROS levels. WNK463, an inhibitor of the WNK kinase family, suppressed HCC progression and altered the redox status. These findings suggest that WNK1 plays a critical role in HCC development and progression and that the WNK1-oxidative stress axis may be a promising therapeutic target for HCC. Significance: Inhibiting WNK1 induces NRF2 degradation and reduces the oxidative stress response to suppress hepatocellular carcinoma growth, indicating that targeting the WNK1-KEAP1-NRF2 axis is a potential strategy to treat liver cancer.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a \"Cl--sensitive kinase\", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

The purpose of this review is to highlight transformative advances that have been made in the field of biomolecular condensates, with special emphasis on condensate material properties, physiology, and kinases, using the With-No-Lysine (WNK) kinases as a prototypical example. To convey how WNK kinases illustrate important concepts for biomolecular condensates, we start with a brief history, focus on defining features of biomolecular condensates, and delve into some examples of how condensates are implicated in cellular physiology (and pathophysiology). We then highlight how WNK kinases, through the action of \"WNK droplets\" that ubiquitously regulate intracellular volume and kidney-specific \"WNK bodies\" that are implicated in distal tubule salt reabsorption and potassium homeostasis, exemplify many of the defining features of condensates. Finally, this review addresses the controversies within this emerging field and questions to address.

Oral poisoning can trigger diverse physiological reactions, determined by the toxic substance involved. One such consequence is hyperchloremia, characterized by an elevated level of chloride in the blood and leads to kidney damage and impairing chloride ion regulation. Here, we conducted a comprehensive genome-wide analysis to investigate genes or proteins linked to hyperchloremia. Our analysis included functional enrichment, protein-protein interactions, gene expression, exploration of molecular pathways, and the identification of potential shared genetic factors contributing to the development of hyperchloremia. Functional enrichment analysis revealed that oral poisoning owing hyperchloremia is associated with 4 proteins e.g. Kelch-like protein 3, Serine/threonine-protein kinase WNK4, Serine/threonine-protein kinase WNK1 and Cullin-3. The protein-protein interaction network revealed Cullin-3 as an exceptional protein, displaying a maximum connection of 18 nodes. Insufficient data from transcriptomic analysis indicates that there are lack of information having direct associations between these proteins and human-related functions to oral poisoning, hyperchloremia, or metabolic acidosis. The metabolic pathway of Cullin-3 protein revealed that the derivative is Sulfonamide which play role in, increasing urine output, and metabolic acidosis resulted in hypertension. Based on molecular docking results analysis it found that Cullin-3 proteins has the lowest binding energies score and being suitable proteins. Moreover, no major variations were observed in unbound Cullin-3 and all three peptide bound complexes shows that all systems remain compact during 50 ns simulations. The results of our study revealed Cullin-3 proteins be a strong foundation for the development of potential drug targets or biomarker for future studies.