The Role and Mechanism of WNK Condensates in Kinase Activation and in the Pathophysiology of Familial Hyperkalemic Hypertension

Introduction: WNK kinases phosphorylate and activate SPAK/OSR1, which phosphorylate and activate the Na + -Cl - cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. WNK4 participates in NCC regulation by plasma K + . Under elevated plasma K + , WNK4 activity is inhibited by Cl - binding. Under low plasma K + , [Cl - ] i decreases, relieving Cl - inhibition of WNK4. Additionally, WNK biomolecular condensates (WNK bodies) form in the DCT under low K + stress. In Familial hyperkalemic hypertension (FHHt), caused by NCC overactivation, WNK condensate formation in the DCT occurs due to mutations that impair WNK degradation and lead to WNK overexpression without directly increasing kinase activity. WNK condensates are also formed in cells that undergo osmotic stress. Whether WNK condensate formation drives kinase activation or merely correlates with catalytic activity, and the mechanism by which this occurs, remains unknown. We hypothesize that WNK condensates activate WNK kinases by forming Cl - -reduced microdomains, underpinning the pathophysiology of FHHt. Methods: Constructs of wild-type or chloride-insensitive WNK1, WNK3, and WNK4 fused to mCherry and Cry2clust enabled optogenetic condensate induction. The WNK1 kinase domain was fused to the intrinsically disordered region (IDR) of FUS, TDP-43, or DDX3X, and Cry2clust. A WNK4-ClopHensorN fusion for ratiometric Cl - measurement within WNK bodies was created. A novel pharmacogenetic model of phase separation was generated by fusing WNK4 to a tamoxifen-sensitive estrogen receptor ligand-binding domain. FHHt mice (homozygous KLHL3 R528H mutants) were subjected to a 5% K + diet to evaluate NCC phosphorylation, WNK body assembly, and K + homeostasis. Mice lacking KS-WNK1 in the KLHL3 R528H background were used to assess WNK body formation. Western Blot of total and phosphorylated forms of the proteins of interest was performed. Live-cell fluorescence imaging was done to assess the induction of biomolecular condensation and for Cl - measurement with ClopHensorN. Immunofluorescent staining of WNK body components was carried out along with that of NCC to mark DCT cells. Plasma electrolytes were measured using an iStat device. Statistical analyses included unpaired t-tests for two-group comparisons and two-way ANOVA for multiple groups, with an alpha value of 0.05. Results: Optogenetic and pharmacologic induction of WNK oligomerization promoted condensation and activated full-length WT WNK kinases (measured by SPAK phosphorylation), but not Cl - -insensitive WNK kinases, suggesting a Cl - -sensing-dependent activation mechanism. Autophosphorylation in the T-loop (S382) of WNK1 in condensates occurred with its native IDR, but not with FUS, TDP-43, or DDX3X fusions, suggesting that properties conferred by the WNK IDR are essential for condensation-induced activation. [Cl - ] in WNK bodies, measured with ClopHensorN, approximated R0Cl (fluorescence intensity ratio in 0 mM [Cl - ]). FHHt mice displayed WNK bodies and were unable to downregulate pNCC during a high-K + diet, even with severe hyperkalemia (mean plasma [K + ]: 8.8 mM vs. 5.2 mM in WT; p<0.0001 ). Surprisingly, KLHL3 R528H mice lacking KS-WNK1 still showed WNK4-positive condensates in their DCT, indicating that WNK4 overexpression suffices for condensate assembly in FHHt. Conclusion: WNK body formation drives kinase activation and represents a mechanism that regulates NCC and, very likely, K + homeostasis. Our experiments suggest that condensation-induced activation may be due to the formation of Cl - -reduced microdomains, and could be the cause of the FHHt phenotype by preventing kinase inactivation under the high intracellular Cl - conditions expected in this disease. CONAHCyT grant 737116 to HCC NIH R01 grant DK51496 to DE and GG Kidneycure TIG to MCB This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.