No abstract

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl− cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

The distal convoluted tubule (DCT) NaCl cotransporter NCC is activated by phosphorylation, a process that is potassium-regulated and dependent on With-No-Lysine (WNK) kinases. KS-WNK1, a kidney-specific WNK1 isoform lacking the kinase domain, controls WNK signaling pathway localization in the DCT. Its role in NCC regulation, however, is unresolved: while early studies proposed that KS-WNK1 functions as an NCC inhibitor, recent work suggests that it activates NCC. To evaluate the role of KS-WNK1 on potassium-dependent NCC regulation, we studied KS-WNK1-KO mice across a wide range of plasma K+ (2.0-9.0 mmol/L), induced by dietary maneuvers and diuretic challenges. Potassium-deprived KS-WNK1-KO mice exhibited low WNK-dependent NCC phosphorylation compared to littermates, indicating that KS-WNK1 activates NCC during K+ deficiency. In contrast, relative NCC phosphorylation was high in potassium-loaded KS-WNK1-KO mice, consistent with KS-WNK1-mediated NCC inhibition during hyperkalemia. An integrated analysis revealed that KS-WNK1 expands the dynamic range of NCC responsiveness to potassium, steepening the linear inverse relationship between NCC phosphorylation and plasma [K+]. The effect of KS-WNK1 deletion was strongest in potassium-restricted females, as they developed exaggerated hypokalemia and thiazide insensitivity due to low NCC activity. Taken together, these findings indicate that KS-WNK1 is a potassium-responsive signaling amplifier that converts small changes in [K+] into large effects on NCC phosphorylation. This effect predominates in females during potassium deficiency, when high NCC activity is required to maintain salt reabsorption without exacerbating K+ losses. These observations define the role of KS-WNK1 in NCC regulation, and identify a novel mechanism that contributes to sexual dimorphism in the mammalian nephron.

Kidney‐Specific With‐No‐Lysine (KS‐WNK1) is a distal convoluted tubule (DCT) scaffold protein that serves as a docking site for the WNK‐SPAK/OSR1 kinase signaling pathway. During dietary K+ deprivation, KS‐WNK1 condenses with the active WNK‐SPAK/OSR1 cascade into large spherical membraneless structures in the DCT, termed WNK bodies. We propose that the assembly of these multikinase complexes promotes K+ conservation through the activation of the WNK‐SPAK/OSR1 pathway and its downstream target, the sodium‐chloride cotransporter (NCC). Mice lacking KS‐WNK1 are unable to form WNK bodies and have diminished activation of WNK‐SPAK/OSR1 pathway and NCC, resulting in a Gitelman‐like phenotype. A critical question is whether the blunted NCC activation in KS‐WNK1 KO mice is a direct consequence of the loss of WNK bodies, or a secondary effect from the generalized lack of KS‐WNK1. Here we address the issue by using CRISPR‐Cas technology to generate a mouse expressing a full‐length mutant version of KS‐WNK1 that does not form WNK bodies. The mutation was directed at a conserved N‐terminal hydrophobic motif (VFVIV ‐> QQQQQ) within exon 4A, and thus referred to as the “5Q” mouse. We confirmed in HEK293 cells that this mutation to KS‐WNK1 abolished WNK body formation. Next, we determiend the function of WNK bodies during 10 days of dietary K+ deprivation in the 5Q mouse versus littermates, assessing changes in WNK‐SPAK/OSR1 & NCC activation by immunostaining and western blot, and on electrolyte handling. We found that K+ restricted 5Q mice were unable to form spherical WNK bodies, and instead formed irregularly shaped aggregates around the nucleus and basolateral membrane. These malformed aggregates were enriched in phosphorylated SPAK/OSR1, which was nearly absent from the apical membrane. Consistent with intracellular sequestration of pSPAK/pOSR1, the abundance of pSPAK/pOSR1 was increased in immunoblots of kidney homogenates by 25% (males; p = 0.0007) & 30% (females; p = 0.0012), but phosphorylated NCC abundance (a surrogate for NCC activation) was decreased by 25% (males; p = 0.06) & 70% (females; p = 0.0005). This reduction in phospho‐NCC in the 5Q mutants resulted in a Gitelman‐like phenotype that was more pronounced in females. Female 5Q mice had a trend towards decreased plasma [K+] (0.4 meq/L reduction; p = 0.1), and significantly decreased [Cl‐] (4 meq/L reduction; p = 0.04) and increased [HCO3‐] (4 meq/L increase; p = 0.05). In conclusion, the KS‐WNK1 5Q mutation causes aberrant WNK body formation, resulting in mislocalization of the WNK‐SPAK/OSR1 pathway in K+ deprived mice. While the 5Q mice can activate SPAK, SPAK is unable to leave the dysfunctional aggregates to activate NCC at the apical membrane. Similar to KS‐WNK1 KO mice, this results in a Gitelman‐like phenotype that predominates in females. These findings demonstrate that KS‐WNK1 assembles WNK bodies to serve as functional protein scaffolds for multikinase complexes to enhance NCC activation during hypokalemia.