KS-WNK1 is Required to Translate the Response to Extreme Changes in Potassium Ingestion to NCC Activity and Expression.

HYPOTHESIS: KS-WNK1 a shorter isoform of WNK1 that is exclusively expressed in the kidney, with abundance in the distal convoluted tubule (DCT), but its physiological role remains elusive. KS-WNK1 stimulates NCC activity via WNK4-SPAK pathway. Under low potassium diet, a known stimuli for NCC activity, WNK bodies are formed in DCT cells, and this requires the presence of KS-WNK1. We recently shown that KS-WNNK1 is highly sensitive to the CUL3-KLHL3 complex (JCI 2020) and its expression under control conditions is negligible, but it is increased under low potassium diet (AJP Renal 2021). Thus, studies that have analyzed the role of KS-WNK1 in mice exposed to normal K+ diet may not be the adequate approach. In wildlife, carnivorous mammals are exposed to cycles of no food and thus, zero K+ ingestion, followed by a vast meal in a few hours and thus, acute and high K+ ingestion. Due to the high sensitivity of KS-WNK1 expression to K+ intake, we assessed NCC and KS-WNK1 using a model from low K+ to high K+ diet to imitate what occurs in wildlife.

METHODS: We exposed C57bl/6 (WT) and KS-WNK1-KO (KS-KO) mice to 10 days of zero K+ diet (0KD) or normal K+ diet (NKD), followed by high K+ ingestion (HKD) (5%). Groups of mice were sacrificed before HKD and at 12 or 24 hours after HKD. We evaluated plasma K+ and by western blot we assessed NCC, phospho-NCC, SPAK, phospho-SPAK and KS-WNK1 expression in kidney proteins.

RESULTS: In mice fed with NKD, there was no difference in plasma K+ or NCC phophorylation or expression between WT and KS-KO mice. After 10 days of 0KD plasma K+ was significantly lower in KS-KO mice than in WT mice (p<0.05) and NCC phosphorylation was higher in WT mice (p<0.05), consistently, KS-WNK1 expression in WT mice was only observed in 0KD, but not in NKD and it is not present in KS-KO mice. In WT mice pNCC/NCC dephosphorylation ratio at 12 hours after HKD was significantly higher in mice feed with 10 days of 0KD, than in mice fed with NKD, and accordingly, changes in plasma K+ were only observed in mice fed with 0KD and not in mice coming from NKD. The decrease in pNCC/tNCC dephosphorylation ratio after HKD, when mice were fed with 0KD was even lower in KS-KO than in WT mice. Additionally, KS-KO mice had higher plasma K+ after HKD coming from 0KD.

CONCLUSION: No differences were observed between WT and KS-KO in groups fed with NKD before the exposure to HKD. KS-WNK1 was not expressed during NKD. In contrast, in mice fed with 0KD, our data show that activation of NCC was higher in WT that KS-KO. Then, after HKD, down-regulation of pNCC was more effective in WT than in KS-KO mice. Therefore, our data suggest that KS-WNK1 is not present and has no role when mice were fed with NKD, but is up-regulated in 0KD and therefore, required to properly respond to extreme changes in potassium diet, as those that occurs in wildlife mammals.