Background: Dietary potassium intake inversely associates with blood pressure, with low dietary potassium intake increasing blood pressure and a high dietary potassium intake often resulting in a lower blood pressure (1). NCC phosphorylation (pseudo-marker of activity) and abundance are lower during higher dietary potassium intake, with the lower total NCC levels linked to greater ubiquitin-dependent NCC degradation (2). We have previously found that ubiquitylation of K890 in mouse NCC is important for modulation of NCC function in vitro (3). The aim of this study was to investigate the role of K890 ubiquitylation in the potassium-induced reduction of NCC in vivo.
Methods: A novel mouse model with a K890R mutation in NCC was generated using CRISPR/Cas9 technology. Male and female K890R and wildtype (wt) mice were fed a control (2% KCl, 0.74% NaCl) or high potassium (10% KCl, 0.74% NaCl) diet for 4 days after which their blood pressure was measured using tail cuff plethysmography and their kidney function assessed by balance studies in metabolic cages. After euthanasia, kidneys were collected and levels of NCC were examined by western blotting. Ex vivo kidney tubule suspensions from K890R and wt mice were incubated in control (4.2 mM KCl) or high potassium (8.0 mM KCl) media for 24 h and levels of NCC examined by western blotting.
Results: No significant differences in systolic blood pressure were apparent in male or female K890R and wt mice fed control or high potassium diets (males n=5-16, females n=5-8). No significant differences were observed in plasma potassium, chloride, or sodium levels, nor their urinary excretion rates, between genotypes fed either diet. On control diet, no differences between genotypes were detected in NCC or phosphorylated NCC abundances. In both male and female wt and K890R mice, high dietary potassium intake decreased total and phosphorylated NCC levels. In male K890R mice, the decrease of total NCC was greater than in wt mice, whereas phosphorylated NCC levels did not decrease to the same extent in female K890R mice relative to wt mice. Incubation of tubule suspensions from male or female wt mice in high potassium media significantly decreased total and phosphorylated NCC abundances. Similar reductions in total NCC (male and female) and phosphorylated NCC (females) were not detected in tubules from K890R mice.
Conclusion: These preliminary data suggest that ubiquitylation of NCC at K890, particularly in females, plays a role in the potassium-induced degradation of NCC. Further studies are required to clarify the role of K890 NCC ubiquitylation in potassium balance or blood pressure, and whether compensatory mechanisms exist in the K890R mice.