Cary R. Boyd-Shiwarski

To maintain potassium homeostasis, the kidney’s distal convoluted tubule (DCT) evolved to convert small changes in blood [K ⁺ ] into robust effects on salt reabsorption. This process requires NaCl cotransporter (NCC) activation by the with-no-lysine (WNK) kinases. During hypokalemia, the kidney-specific WNK1 isoform (KS-WNK1) scaffolds the DCT-expressed WNK signaling pathway within biomolecular condensates of unknown function termed WNK bodies. Here, we show that KS-WNK1 amplified kidney tubule reactivity to blood [K ⁺ ], in part via WNK bodies. In genetically modified mice, targeted condensate disruption trapped the WNK pathway, causing renal salt wasting that was more pronounced in females. In humans, WNK bodies accumulated as plasma potassium fell below 4.0 mmol/L, suggesting that the human DCT experiences the stress of potassium deficiency, even when [K ⁺ ] is in the low-to-normal range. These data identify WNK bodies as kinase signal amplifiers that mediate tubular [K ⁺ ] responsiveness, nephron sexual dimorphism, and BP salt sensitivity. Our results illustrate how biomolecular condensate specialization can optimize a mammalian physiologic stress response that impacts human health.

Background/Objectives: Hypo-hydration is a major health concern that affects performance and is associated with increasing morbidity and growing health care costs. There is an emerging interest in optimizing hydration and identifying how factors such as ingestion rate and beverage composition affect hydration. This study examined three beverages with varying ingestion rates and measured markers of hydration. Methods: Thirty healthy, active participants between the ages of 18 and 45 years were given three different beverages on three separate days. The beverages were of identical volumes (1 L), but differed in the rate of ingestion, carbohydrate content and electrolyte content. Beverage 1 and water alone were both consumed at a metered rate of one liter over four hours, whereas Beverage 2 was used as a positive control and was consumed at a bolus rate of one liter in 30 min. Results: After six hours, Beverage 1 significantly improved markers of hydration compared to water alone or Beverage 2. Beverage 1 decreased cumulative urine output vs. water alone by 32% (absolute difference −0.33 L; CI ± −0.16 to −0.51) and vs. Beverage 2 by 26% (absolute difference −0.26 L; CI ± −0.13 to −0.38). Beverage 1 increased the beverage hydration index vs. water alone by 64% (absolute difference +0.64 L; CI ± 0.36 to 0.92) and vs. Beverage 2 by 48% (absolute difference +0.53 L; CI ± 0.30 to 0.76). Conclusions: Beverage 1 is superior to water alone at improving hydration when it is ingested at similar rates. Moreover, metered ingestion of Beverage 1 improved hydration compared to a bolus ingestion of Beverage 2, this could be due to the dissimilar ingestion rates and/or beverage composition.

Background: Hypo-hydration is a major health concern that affects performance and is associated with increasing morbidity and growing health care costs. There is an emerging interest in optimum hydration and identifying how factors such as ingestion rate and beverage composition affect hydration. This study examined three beverages with varying ingestion rates and measured markers of hydration. Methods: Thirty healthy, active participants between ages 18-45 years were given three different beverages on three separate days. The beverages were of identical volumes (1 Liter), but differed in the rate of ingestion, carbohydrate content and electrolyte content. Beverage 1 (Buoy, San Diego, CA) and water-alone were both consumed at a metered rate of one liter over four hours, whereas Beverage 2 was used as a positive control and consumed at a bolus rate of one liter in 30 minutes. Findings: After six hours Beverage 1 significantly improved markers of hydration compared to water-alone or Beverage 2. Beverage 1 decreased cumulative urine output vs water-alone by 32% (absolute difference -0.33L; CI ± -0.16 to -0.51) and vs Beverage 2 by 26% (absolute difference - 0.26L; CI ± -0.13 to -0.38). Beverage 1 increased the beverage hydration index vs water-alone by 64% (absolute difference +0.64L; CI ± 0.36 to 0.92) and vs Beverage 2 by 48% (absolute difference +0.53L; CI ± 0.30 to 0.76) Interpretation: Beverage 1 is superior to water-alone at improving hydration when ingested at similar rates. Moreover, metered ingestion of Beverage 1 improved hydration compared to a bolus ingestion of Beverage 2, this could be due to the dissimilar ingestion rates and/or beverage composition. RESEARCH IN CONTEXT: Despite the overwhelming number of commercial hydration beverages on the market, there are only a very limited number of studies that address whether these beverages are actually effective at improving hydration. Using PubMed and Google Scholar using the search term “Beverage Hydration Index” with the search date from 2016-2024 (2016 was when the Beverage Hydration Index was established) we found less than 10 articles on this topic that used the beverage hydration index to assess the efficacy of popular beverages and supplements, and none of them have previously evaluated the efficacy of Beverage 1 (Buoy). Additionally, only one other study assessed how that rate of beverage ingestion can influence the beverage hydration index. This current study has found Beverage 1 increased the beverage hydration index vs water-alone by 64% (absolute difference +0.64L; CI ± 0.36 to 0.92). We propose that Beverage 1 increases the beverage hydration index due to its abundance of electrolytes including sodium and chloride, as it does not contain carbohydrates, protein, or artificial sweeteners that are common in other commercial hydration beverages. Identifying beverages that improve hydration compared to water-alone can play an important role in preventing severe hypohydration and dehydration, including renal failure, seizures, arrythmia, and altered mental status. It has been estimated that over half a million hospitalizations per year are due to dehydration with a cost of over 5.5 billion United States dollars(1). Thus, there are both clinical and economic reasons to identify simple, cost-effective methods to promote euhydration.

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.

The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism.

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 multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes. NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT‐specific changes that are directly consequent to knockout of each component.

To maintain potassium homeostasis, the kidney’s distal convoluted tubule (DCT) converts small changes in blood [K ⁺ ] into robust effects on salt reabsorption. This process requires NaCl cotransporter (NCC) activation by WNK kinases. During hypokalemia, the Kidney-Specific WNK1 isoform (KS-WNK1) scaffolds the DCT-expressed WNK signaling pathway within biomolecular condensates of unknown function termed WNK bodies. Here, we show that KS-WNK1 amplifies the dynamic range of NCC activity in response to potassium imbalance, in part via WNK bodies. Targeted condensate disruption traps the WNK pathway, causing renal salt-wasting that is more pronounced in females. In humans, WNK bodies accumulate as plasma potassium falls below 4.0mmol/L, suggesting avid condensate-mediated salt reabsorption even when [K ⁺ ] is low-normal. These data identify WNK bodies as signal amplifiers that mediate tubular potassium responsiveness, nephron sexual dimorphism, and blood pressure salt-sensitivity. Our results illustrate how condensate specialization can optimize a mammalian physiologic stress response that impacts human health.

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.